CN120152717A - Combination of T cell therapy and continuous or intermittent DGK inhibitor administration - Google Patents

Abstract

Translated fromChinese

本文提供涉及T细胞疗法(诸如过继T细胞疗法)以及二酰基甘油激酶(DGK)抑制剂的方法、组合物和用途。所提供的方法、组合物和用途包括涉及施用或使用一种或多种DGK抑制剂(DGKi)与T细胞疗法(诸如涉及用重组受体(诸如嵌合抗原受体(CAR)或T细胞受体(TCR))工程化的细胞的基因工程化T细胞疗法)的组合疗法的那些。Provided herein are methods, compositions and uses involving T cell therapy (such as adoptive T cell therapy) and diacylglycerol kinase (DGK) inhibitors. Provided methods, compositions and uses include those involving administering or using one or more DGK inhibitors (DGKi) and T cell therapy (such as genetically engineered T cell therapy involving cells engineered with recombinant receptors (such as chimeric antigen receptors (CAR) or T cell receptors (TCR)) combined therapies.

Description

Combination of T cell therapy and continuous or intermittent DGK inhibitor administration

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/404,955, filed on 8, 9, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

The sequence listing is incorporated by reference

The present application is presented with a sequence listing in electronic format. The sequence listing was provided under the name 735042026540SeqList. Xml, created at month 8 of 2023, 23, and a 358 kilobyte document. The information in electronic format of the sequence listing is incorporated by reference in its entirety.

Technical Field

The present disclosure relates in certain aspects to methods, compositions, and uses involving T cell therapies, such as adoptive T cell therapies (adoptive T CELL THERAPY), and diacylglycerol kinase (DGK) inhibitors. Methods, compositions, and uses provided include those involving administration or use of a combination therapy of one or more DGK inhibitors (DGKi) with T cell therapies, such as genetically engineered T cell therapies involving cells engineered with recombinant receptors, such as Chimeric Antigen Receptor (CAR) or T Cell Receptor (TCR).

Background

There are various strategies for T cell therapy, such as administering engineered T cells for adoptive therapy. For example, strategies can be used to engineer T cells expressing genetically engineered antigen receptors (such as CARs or TCRs) and administer compositions containing such cells to a subject. There is a need for improved strategies to increase the efficacy of T cells in cell therapies, such as improving the persistence, activity and/or proliferation of cells upon or after administration to a subject. The methods, compositions, uses, and kits provided herein meet these needs.

Disclosure of Invention

In some embodiments, provided herein are methods of treatment comprising administering to a subject suffering from cancer a combination therapy comprising (a) a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered on day 1 of the combination therapy, and (b) a dgkα and/or dgkζ inhibitor, wherein administration of the inhibitor begins on any of days 1-8 (including days 1 and 8) of the combination therapy, and the inhibitor is administered continuously, at least until the number of engineered T cells of the T cell therapy reaches a peak in the subject after administration of the T cell therapy.

In some of any of the embodiments, the administration of the inhibitor begins after administration of the T cell therapy.

In some embodiments, provided herein are also methods of treatment comprising administering a DGK alpha and/or DGK zeta inhibitor to a subject suffering from cancer, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the subject has previously administered the T cell therapy prior to beginning administration of the inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy, administration of the inhibitor begins on any of days 1-8 of the combination therapy (including days 1 and 8), and the inhibitor is administered continuously, at least until the number of engineered T cells of the T cell therapy peaks in the subject after administration of the T cell therapy.

In some of any of the embodiments, the administration of the inhibitor begins on day 1 of the combination therapy prior to the administration of the T cell therapy.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by cancer cells, wherein the T cell therapy is administered as part of a combination therapy comprising T cell therapy and a dgkα and/or dgkζ inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy, administration of the inhibitor begins on day 1 of the combination therapy before administration of the T cell therapy, and the inhibitor is administered continuously, at least until the number of engineered T cells of the T cell therapy in the subject peaks after administration of the T cell therapy.

In some of any of the embodiments, the inhibitor is administered daily, once every two days, or once every three days. In some of any of the embodiments, the inhibitor is administered daily.

In some of any of the embodiments, the inhibitor is administered once per day of administration of the inhibitor.

In some of any of the embodiments, the inhibitor is administered until the number of engineered T cells of the T cell therapy peaks in the subject after administration of the T cell therapy.

In some of any of the embodiments, the inhibitor is administered at or over a period of time between about 21 and 42 consecutive days (including 21 and 42 days).

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a combination therapy comprising (a) a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered on day 1 of the combination therapy, and (b) a dgkα and/or dgkζ inhibitor, wherein administration of the inhibitor begins on any of days 1-8 (including days 1 and 8) of the combination therapy, and the inhibitor is administered at a therapeutically effective dose for a period of time between 21 and 42 days or between about 21 and 42 days.

In some of any of the embodiments, the administration of the inhibitor begins after administration of the T cell therapy.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a DGK alpha and/or DGK zeta inhibitor, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cells of the cancer, wherein the subject has previously administered the T cell therapy prior to beginning administration of the inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy, administration of the inhibitor begins on any of days 1-8 of the combination therapy (including days 1 and 8), and the inhibitor is administered at a therapeutically effective dose for a period of time between about 21 and 42 days.

In some of any of the embodiments, the administration of the inhibitor begins on day 1 of the combination therapy prior to the administration of the T cell therapy.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by cancer cells, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy, administration of the inhibitor begins on day 1 of the combination therapy prior to administration of the T cell therapy, and the inhibitor is administered at a therapeutically effective dose at or for a period of time between about 21 and 42 days.

In some of any of the embodiments, the therapeutically effective dose provides a continuous therapeutically effective inhibitor dose over a period of time of between about 21 and 42 days.

In some of any of the embodiments, the inhibitor is administered every 2 days or 3 days.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a combination therapy comprising (a) a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered on day 1 of the combination therapy, and (b) a dgkα and/or dgkζ inhibitor, wherein administration of the inhibitor begins on any of days 1-8 (including days 1 and 8) of the combination therapy, and the inhibitor is administered in a plurality of cycles, each cycle comprising an administration period for administration of the inhibitor followed by a rest period for no administration of the inhibitor.

In some of any of the embodiments, the administration of the inhibitor begins after administration of the cell therapy.

In some embodiments, provided herein are methods of treatment comprising administering a DGK alpha and/or DGK zeta inhibitor to a subject suffering from cancer, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the subject has previously administered the T cell therapy prior to beginning administration of the inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy, administration of the inhibitor begins on any of days 1-8 of the combination therapy (including days 1 and 8), and the inhibitor is administered in a plurality of cycles, each cycle comprising an administration period for administration of the inhibitor, followed by a rest period in which the inhibitor is not administered.

In some of any of the embodiments, the inhibitor is administered daily, every two days, or every three days during the administration period. In some of any of the embodiments, the inhibitor is administered daily during the administration period.

In some of any of the embodiments, the inhibitor is administered once per day during the administration period.

In some of any of the embodiments, the inhibitor is administered in a plurality of cycles until the number of engineered T cells of the T cell therapy peaks in the subject after administration of the T cell therapy.

In some of any of the embodiments, the inhibitor is administered at or over a period of between about 21 and 42 days (including 21 and 42 days) over multiple cycles.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a combination therapy comprising (a) a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered on day 1 of the combination therapy, and (b) a dgkα and/or dgkζ inhibitor, wherein administration of the inhibitor begins on any of days 1-8 (including days 1 and 8) of the combination therapy, and the inhibitor is administered once per day, or between about 21 and 42 consecutive days (including days 21 and 42).

In some of any of the embodiments, the administration of the inhibitor begins after administration of the T cell therapy.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a DGK alpha and/or DGK zeta inhibitor, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising living engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the subject has previously administered the T cell therapy prior to beginning administration of the inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy, administration of the inhibitor begins on any of days 1-8 of the combination therapy (including days 1 and 8), and the inhibitor is administered once daily for between or about 21 and 42 consecutive days (including days 21 and 42).

In some of any of the embodiments, the administration of the inhibitor begins on any of days 1-6 (including days 1 and 6) of the combination therapy. In some of any of the embodiments, the administration of the inhibitor begins on any of days 1-4 (including days 1 and 4) of the combination therapy. In some of any of the embodiments, the administration of the inhibitor begins on day 1 or day 2 of the combination therapy. In some of any of the embodiments, the administration of the inhibitor begins on day 1 of the combination therapy.

In some of any of the embodiments, the administration of the inhibitor begins on day 1 of the combination therapy prior to the administration of the T cell therapy.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy, administration of the inhibitor begins on day 1 of the combination therapy prior to administration of the T cell therapy, and the inhibitor is administered once daily for between or about 21 and 42 consecutive days (including 21 and 42 days).

In some of any of the embodiments, the inhibitor is administered at or over a period of between about 21 and 35 days (including 21 and 35 days). In some of any of the embodiments, the inhibitor is administered at or over a period of between about 21 and 28 days (including 21 and 28 days). In some of any of the embodiments, the inhibitor is administered at or over a period of time between about 28 and 42 days (including 28 and 42 days). In some of any of the embodiments, the inhibitor is administered at or over a period of between about 28 and 35 days (including 28 and 35 days). In some of any of the embodiments, the inhibitor is administered over a period of time of at or about 28 days. In some of any of the embodiments, the inhibitor is administered over a period of time of at or about 30 days.

In some of any of the embodiments, the inhibitor is administered at or over a period of between about 21 and 35 days (including 21 and 35 days) over multiple cycles. In some of any of the embodiments, the inhibitor is administered at or over a period of between about 21 and 28 days (including 21 and 28 days) over multiple cycles. In some of any of the embodiments, the inhibitor is administered at or over a period of between about 28 and 42 days (including 28 and 42 days) over multiple cycles. In some of any of the embodiments, the inhibitor is administered at or over a period of between about 28 and 35 days (including 28 and 35 days) over multiple cycles. In some of any of the embodiments, the inhibitor is administered over a period of time of at or about 28 days in multiple cycles. In some of any of the embodiments, the inhibitor is administered over a period of at or about 30 days in multiple cycles.

In some of any of the embodiments, the inhibitor is administered once daily at or between about 21 and 35 consecutive days (including 21 and 35 days). In some of any of the embodiments, the inhibitor is administered once daily at or between about 21 and 28 consecutive days (including 21 and 28 days). In some of any of the embodiments, the inhibitor is administered once daily at or between about 28 and 42 consecutive days (including 28 and 42 days). In some of any of the embodiments, the inhibitor is administered once daily at or between about 28 and 35 consecutive days (including 28 and 35 days). In some of any of the embodiments, the inhibitor is administered once daily for or for about 28 consecutive days.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a combination therapy comprising (a) a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered on day 1 of the combination therapy, and (b) a dgkα and/or dgkζ inhibitor, wherein administration of the inhibitor begins on day 1 of the combination therapy, and the inhibitor is administered once daily on days 1-28 (including days 1 and 28) of the combination therapy.

In some of any of the embodiments, the administration of the inhibitor begins after administration of the cell therapy.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a dgkα and/or dgkζ inhibitor, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the subject has previously administered the T cell therapy prior to beginning administration of the inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy, administration of the inhibitor begins on day 1 of the combination therapy, and the inhibitor is administered once daily on days 1-28 (including days 1 and 28) of the combination therapy.

In some of any of the embodiments, the administration of the inhibitor is initiated prior to administration of the T cell therapy.

In some embodiments, provided herein are also methods of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy, administration of the inhibitor begins on day 1 of the combination therapy prior to administration of the T cell therapy, and the inhibitor is administered once daily on days 1-28 (including days 1 and 28) of the combination therapy.

In some of any of the embodiments, the inhibitor is administered once daily on days 1-30 (including days 1 and 30) of the combination therapy.

In some of any of the embodiments, the administration of the inhibitor begins within 12 hours or about 12 hours of administration of the T cell therapy. In some of any of the embodiments, the administration of the inhibitor begins within 6 hours or about 6 hours of administration of the T cell therapy. In some of any of the embodiments, the administration of the inhibitor begins within 4 hours or about 4 hours of administration of the T cell therapy. In some of any of the embodiments, the administration of the inhibitor begins within 2 hours or about 2 hours of administration of the T cell therapy. In some of any of the embodiments, the administration of the inhibitor begins within 1 hour or about 1 hour of administration of the T cell therapy.

In some of any of the embodiments, the administration of the inhibitor begins on day 1 of the combination therapy concurrently with the administration of the T cell therapy. In some of any of the embodiments, the administration of the inhibitor begins during administration of the T cell therapy on day 1 of the combination therapy.

In some of any of the embodiments, the subject has been pretreated with a lymphocyte removal therapy comprising administration of fludarabine and/or cyclophosphamide prior to administration of the T cell therapy. In some of any of the embodiments, the subject has been pretreated with a lymphocyte removal therapy comprising administration of fludarabine and cyclophosphamide prior to administration of the T cell therapy. In some of any of the embodiments, the method further comprises administering the lymphocyte removal therapy to the subject.

In some of any of the embodiments, the lymphocyte removal therapy comprises administration of cyclophosphamide at or between about 200 and 400mg/m² (including 200 and 400mg/m²), daily for or between about 2 and 4 days (including 2 and 4 days), and/or fludarabine at or between about 20 and 40mg/m² (including 20 and 40mg/m²), daily for or between about 2 and 4 days (including 2 and 4 days). In some of any of the embodiments, the lymphocyte removal therapy comprises administration of cyclophosphamide at or between about 200 and 400mg/m² (including 200 and 400mg/m²) for or between about 2 and 4 days (including 2 and 4 days) per day. In some of any of the embodiments, the lymphocyte removal therapy comprises administration of fludarabine at or between about 20 and 40mg/m² (including 20 and 40mg/m²) for a duration of at or between about 2 and 4 days (including 2 and 4 days) per day. In some of any of the embodiments, the lymphocyte removal therapy comprises administering cyclophosphamide at or about 300mg/m² and fludarabine at or about 30mg/m² for or for about 3 days per day.

In some of any of the embodiments, the T cell therapy is administered at or between about 2 and 7 days (including days 2 and 7) after administration of the lymphocyte removal therapy.

In some of any of the embodiments, the inhibitor is a dgkα inhibitor, but not a significant inhibitor of dgkζ. In some of any of the embodiments, the inhibitor is a dgkζ inhibitor, but not a significant inhibitor of dgkα. In some of any of the embodiments, the inhibitor is a dgkα and dgkζ inhibitor.

In some of any of the embodiments, the inhibitor is not a significant inhibitor of other DGKs.

In some of any of the embodiments, the inhibitor is a compound of formula (I):

Or a pharmaceutically acceptable salt thereof, wherein:

R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, C_3–4 cycloalkyl substituted with 0 to 4R_1a, C_1–3 alkoxy substituted with 0 to 4R_1a, -NR_aR_a、–S(O)_nR_e or-P (O) R_eR_e;

Each R_1a is independently F, cl, -CN, -OH, -OCH₃, or-NR_aR_a;

Each R_a is independently H or C_1–3 alkyl;

Each R_e is independently C_3–4 cycloalkyl or C_1–3 alkyl substituted with 0 to 4R_1a;

R₂ is H, C_1–3 alkyl substituted with 0 to 4R_2a, or C_3–4 cycloalkyl substituted with 0 to 4R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), C_3–4 cycloalkyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₃ is H, F, cl, br, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_3–4 cycloalkyl, C_3–4 fluorocycloalkyl or-NO₂;

R₄ is –CH₂R_4a、–CH₂CH₂R_4a、–CH₂CHR_4aR_4d、–CHR_4aR_4b or-CR_4aR_4bR_4c;

R_4a and R_4b are independently:

(i) C_1–6 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy, -NR_aR_a、–S(O)₂R_e, or-NR_aS(O)₂R_e;

(ii) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH)_1–3O(C_1–3 alkyl), C_1–3 Fluoroalkoxy 、–O(CH)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl, -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–6 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or

(Iii) C_1–4 alkyl substituted with one cyclic group selected from the group consisting of C_3–6 cycloalkyl, heterocyclyl, aryl and heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from the group consisting of F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–6 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f;

Each R_f is independently F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–6 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

R_4d is-OCH₃;

Each R_c is independently H or C_1–2 alkyl;

R_d is phenyl substituted with 0 to 1 substituents selected from F, cl, -CN, -CH₃, and-OCH₃;

Each R₅ is independently-CN, C_1–6 alkyl substituted with 0 to 4R_g, C_2–4 alkenyl substituted with 0 to 4R_g, C_2–4 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 4R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 4R_g, - (CH₂)_1–2 (heterocyclyl substituted with 0 to 4R_g)), a compound of formula (i), - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

Each R_g is independently F, cl, -CN, -OH, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -O (CH₂)_1–2O(C_1–2 alkyl), or-NR_cR_c;

m is 0, 1,2 or 3, and

N is 0, 1 or 2.

In some of any of the embodiments, the inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, cyclopropyl substituted with 0 to 3R_1a, C_1–3 alkoxy substituted with 0 to 3R_1a, -NR_aR_a、–S(O)_nCH₃ or-P (O) (CH₃)₂;

Each R_1a is independently F, cl or-CN;

Each R_a is independently H or C_1–3 alkyl;

R₂ is H or C_1–2 alkyl substituted with 0 to 2R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), cyclopropyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₃ is H, F, cl, br, -CN, C_1–2 alkyl, -CF₃, cyclopropyl or-NO₂;

R_4a and R_4b are independently:

(i) C_1–4 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy and-NR_aR_a;

(ii) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, -CH₂OH、–(CH₂)_1–2O(C_1–2 alkyl), 0 to 4, C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy, -O (CH)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl, -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–4 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or

(Iii) C_1–3 alkyl substituted with one cyclic group selected from the group consisting of C_3–6 cycloalkyl, heterocyclyl, phenyl and heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from the group consisting of F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–4 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f;

each R_f is independently F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–4 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

And each R₅ is independently-CN, C_1–5 alkyl substituted with 0 to 4R_g, C_2–3 alkenyl substituted with 0 to 4R_g, c_2–3 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 3R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 3R_g, - (CH₂)_1–2 (heterocyclyl substituted with 0 to 4R_g)), a compound of formula (i), - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl).

In some of any of the embodiments, the inhibitor is a compound of formula (I), or a pharmaceutically acceptable salt thereof, having the structure:

Wherein:

R₁ is-CN;

R₂ is-CH₃;

R₃ is H, F or-CN;

r₄ is:

in some of any of the embodiments, the inhibitor is a compound of formula (I), or a pharmaceutically acceptable salt thereof, having the structure:

Or (b)

In some of any of the embodiments, the inhibitor is a compound of formula (II):

Or a salt thereof, wherein:

R₁ is H, F, cl, br, -CN, -OH, C_1–3 alkyl substituted with 0 to 4R_1a, C_3–4 cycloalkyl substituted with 0 to 4R_1a, C_1–3 alkoxy substituted with 0 to 4R_1a, -NR_aR_a、–S(O)_nR_e, or-P (O) R_eR_e;

Each R_1a is independently F, cl, -CN, -OH, -OCH₃, or-NR_aR_a;

Each R_a is independently H or C_1–3 alkyl;

Each R_e is independently C_3–4 cycloalkyl or C_1–3 alkyl substituted with 0 to 4R_1a;

R₂ is H, C_1–3 alkyl substituted with 0 to 4R_2a, or C_3–4 cycloalkyl substituted with 0 to 4R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), C_3–4 cycloalkyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₄ is –CH₂R_4a、–CH₂CH₂R_4a、–CH₂CHR_4aR_4d、–CHR_4aR_4b or-CR_4aR_4bR_4c;

R_4a and R_4b are independently:

(i) -CN or C_1–6 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy, -NR_aR_a、–S(O)₂R_e, or-NR_aS(O)₂R_e;

(ii) C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, phenyl or 5-to 10-membered heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, through 0 to 4, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy, -O (C_1–4 hydroxyalkyl), a, -O (CR_xR_x)_1–3O(C_1–3 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–CH₂NR_aR_a、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -CR_xR_x)_0–2NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl, - (CR_xR_x)_1–2(C_3–4 cycloalkyl), - (CR_xR_x)_1–2 (morpholinyl), - (CR_xR_x)_1–2 (difluoromethyl), - (CR_xR_x)_1–2 (dimethylmorpholinyl)) and the like, - (CR_xR_x)_1–2 (oxaazabicyclo [2.2.1] heptane), (CR_xR_x)_1–2 (oxaazaspiro [3.3] heptane), - (CR_xR_x)_1–2 (methylpiperazinonyl), - (CR_xR_x)_1–2 (acetylpiperazinyl)) and the like, - (CR_xR_x)_1–2 (piperidinyl), - (CR_xR_x)_1–2 (difluoropiperidinyl), - (CR_xR_x)_1–2 (methoxypiperidinyl), - (CR_xR_x)_1–2 (hydroxypiperidinyl)), and the like, -O (CR_xR_x)_0–2(C_3–6 cycloalkyl), -O (CR_xR_x)_0–2 (methylcyclopropyl), -O (CR_xR_x)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CR_xR_x)_0–2 (oxetanyl)), -O (CR_xR_x)_0–2 (methylazetidinyl)), -O (CR_xR_x)_0–2 (tetrahydropyranyl) -O (CR_xR_x)_1–2 (morpholinyl)), -O (CR_xR_x)_0–2 (thiazolyl), Cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, dioxolanyl, pyrrolidone group and R_d, or

(Iii) C_1–4 alkyl substituted with one cyclic group selected from C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, mono-or bicyclic aryl, or 5-to 10-membered heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), and C_3–6 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl group or a 3-to 6-membered heterocyclyl group, each substituted with 0 to 3R_f;

Each R_f is independently F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–6 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

R_4d is-OCH₃;

Each R_c is independently H or C_1–2 alkyl;

R_d is phenyl substituted with 0 to 1 substituents selected from F, cl, -CN, -CH₃, and-OCH₃;

Each R₅ is independently-CN, C_1–6 alkyl substituted with 0 to 4R_g, C_2–4 alkenyl substituted with 0 to 4R_g, C_2–4 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 4R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 4R_g, - (CH₂)_1–2 (4-to 10-membered heterocyclyl substituted with 0 to 4R_g), and, - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

Each R_g is independently F, cl, -CN, -OH, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -O (CH₂)_1–2O(C_1–2 alkyl), or-NR_cR_c;

m is 0, 1,2 or 3, and

N is 0, 1 or 2.

In some of any of the embodiments, the inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein:

R₁ is H, F, cl, br, -CN, -OH, C_1–3 alkyl substituted with 0 to 4R_1a, cyclopropyl substituted with 0 to 3R_1a, C_1–3 alkoxy substituted with 0 to 3R_1a, -NR_aR_a、–S(O)_nCH₃, or-P (O) (CH₃)₂;

R₂ is H or C_1–2 alkyl substituted with 0 to 2R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), cyclopropyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R_4a and R_4b are independently:

(i) -CN or C_1–4 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy and-NR_aR_a;

(ii) C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, phenyl or 5-to 10-membered heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, through 0 to 4, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1-2 hydroxyalkyl, -CH₂NR_aR_a、–(CH₂)_1–2O(C_1–2 alkyl, - (CH₂)_1–2NR_xC(O)O(C_1-2 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CR_xR_x)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy 、–O(CH₂)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl, -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl), - (CH₂)_1–2(C_3–4 cycloalkyl), -CR_xR_x (morpholinyl), -CR_xR_x (difluoromethyl), -CR_xR_x (dimethylmorpholinyl), -CR_xR_x (oxaazabicyclo [2.2.1] heptanyl), -CR_xR_x (oxaazaspiro [3.3] heptanyl), -CR_xR_x (methylpiperazinonyl), -CR_xR_x (acetylpiperazinyl), and-, -CR_xR_x (piperidinyl), -CR_xR_x (difluoropiperidinyl), -CR_xR_x (methoxypiperidinyl), -CR_xR_x (hydroxypiperidinyl), -O (CH₂)_0–2(C_3–4 cycloalkyl), -O (CH₂)_0–2 (methylcyclopropyl), -O (CH₂)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CH₂)_0–2 (oxetanyl)), -O (CH₂)_0–2 (methylazetidinyl), -O (CH₂)_1–2 (morpholinyl), -O (CH₂)_0–2 (tetrahydropyranyl), -O (CH₂)_0–2 (thiazolyl)), Cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, dioxolanyl, pyrrolidone group, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or

(Iii) C_1–3 alkyl substituted with one cyclic group selected from C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, mono-or bicyclic aryl, or 5-to 10-membered heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), and C_3–4 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl group or a 3-to 6-membered heterocyclyl group, each substituted with 0 to 3R_f;

Each R_f is independently F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–4 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

Each R₅ is independently-CN, C_1–5 alkyl substituted with 0 to 4R_g, C_2–3 alkenyl substituted with 0 to 4R_g, c_2–3 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, phenyl substituted with 0 to 3R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 3R_g, - (CH₂)_1–2 (4-to 10-membered heterocyclyl substituted with 0 to 4R_g), and, - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

Each R_x is independently H or-CH₃, and

M is 1, 2 or 3.

In some of any of the embodiments, the inhibitor is a compound of formula (II), or a pharmaceutically acceptable salt thereof, having the structure:

R₁ is-CN;

R₂ is-CH₃;

r_5a is-CH₃ or-CH₂CH₃, and

R_5c is-CH₃、–CH₂CH₃ or-CH₂CH₂CH₃.

In some of any of the embodiments, the inhibitor is a compound of formula (II), or a pharmaceutically acceptable salt thereof, having the structure:

in some of any of the embodiments, the inhibitor is a compound of formula (II), or a pharmaceutically acceptable salt thereof, having the structure:

in some of any of the embodiments, the inhibitor is administered in a therapeutically effective amount. In some of any of the embodiments, the therapeutically effective dose provides a dose of the inhibitor that is therapeutically effective for a period of time during which the inhibitor is administered.

In some of any of the embodiments, the inhibitor is administered in an amount of between or about 0.25 and 250mg (including 0.25 and 250 mg) per day. In some of any of the embodiments, the inhibitor is administered in an amount of between or about 0.5 and 100mg (including 0.5 and 100 mg) per day.

In some of any of the embodiments, the inhibitor is administered in an amount of between 0.25 and 250mg or between about 0.25 and 250mg (including 0.25 and 250 mg) once a day. In some of any of the embodiments, the inhibitor is administered in an amount of between or about 0.5 and 100mg (including 0.5 and 100 mg) once daily.

In some of any of the embodiments, the inhibitor is administered orally.

In some of any of the embodiments, the recombinant receptor is an engineered T cell receptor (eTCR).

In some of any embodiment, the recombinant receptor is a Chimeric Antigen Receptor (CAR).

In some of any of the embodiments, the antigen is CD19 and the CAR is an anti-CD 19 CAR. In some of any embodiment, the CAR is(Li Jimai, lisocabtagene maraleucel), TECARTUS^TM (buckyolol, brexucabtagene autoleucel), KYMRIAH^TM (s Li Fuming, tisagenlecleucel), or YESCARTA^TM (alzerland, axicabtagene ciloleucel). In some of any of the embodiments, the T cell therapy is(Li Jimai, galenical), TECARTUS^TM (brucellosis), KYMRIAH^TM (s Li Fuming) or YESCARTA^TM (alopecies).

In some of any of the embodiments, the antigen is BCMA and the CAR is an anti-BCMA CAR. In some of any embodiment, the CAR is(Ai Dika barren, idecabtagene vicleucel) or CARVYKTI^TM (sidaky olanexide, ciltacabtagene autoleucel). In some of any of the embodiments, the T cell therapy is(Ai Dika Bajin, idecabtagene vicleucel) or CARVYKTI^TM (Sida-based Orthoxel, ciltacabtagene autoleucel).

In some of any of the embodiments, the cancer is a solid tumor. In some of any of the embodiments, the cancer is a hematological (liquid) tumor.

In some of any of the embodiments, the cancer is a B cell malignancy.

In some of any embodiment, the cancer is leukemia. In some of any of the embodiments, the cancer is a lymphoma. In some of any of the embodiments, the cancer is myeloma. In some of any of the embodiments, the myeloma is multiple myeloma.

In some of any of the embodiments, the cancer is recurrent or refractory.

In some of any of the embodiments, the T cell therapy comprises between or about 0.1x10⁶ and 1,000x10⁶ (including 0.1x10⁶ and 1,000x10⁶) total number of total recombinant receptor expressing T cells. In some of any of the embodiments, the T cell therapy comprises between or about 10x10⁶ and 1,000x10⁶ (including 10x10⁶ and 1,000x10⁶) total number of total group receptor expressing T cells. In some of any of the embodiments, the T cell therapy comprises between or about 10x10⁶ and 500x10⁶ (including 10x10⁶ and 500x10⁶) total number of total recombinant receptor-expressing T cells.

In some of any of the embodiments, the recombinant receptor expressing T cell is a live recombinant receptor expressing T cell.

In some of any of the embodiments, the T cell therapy is administered intravenously.

In some of any of the embodiments, the T cells of the T cell therapy are autologous to the subject. In some of any of the embodiments, the T cells of the T cell therapy are allogeneic to the subject.

In some of any of the embodiments, the T cells of the T cell therapy are human T cells.

In some embodiments, provided herein is also a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells for use as part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein the T cell therapy and the inhibitor are administered to a subject suffering from cancer according to any of the described methods.

In some embodiments, provided herein are also dgkα and/or dgkζ inhibitors for use as part of a combination therapy comprising the inhibitors and T cell therapies comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapies and the inhibitors are administered to a subject suffering from cancer according to any of the described methods.

In some embodiments, provided herein is also a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells and a dgkα and/or dgkζ inhibitor for use as part of a combination therapy comprising the T cell therapy and the inhibitor, wherein the T cell therapy and the inhibitor are administered to a subject suffering from cancer according to any of the described methods.

In some embodiments, also provided herein is the use of a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells as part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein the T cell therapy and the inhibitor are administered to a subject suffering from cancer according to any of the described methods.

In some embodiments, also provided herein is the use of a dgkα and/or dgkζ inhibitor as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy and the inhibitor are administered to a subject suffering from cancer according to any of the described methods.

In some embodiments, also provided herein is the use of a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells and a dgkα and/or dgkζ inhibitor as part of a combination therapy comprising the T cell therapy and the inhibitor, wherein the T cell therapy and the inhibitor are administered to a subject suffering from cancer according to any of the described methods.

In some embodiments, also provided herein is the use of a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells in the manufacture of a medicament that is part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein the T cell therapy and the inhibitor are administered to a subject suffering from cancer according to any of the described methods.

In some embodiments, also provided herein is the use of a dgkα and/or dgkζ inhibitor in the manufacture of a medicament that is part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy and the inhibitor are administered to a subject suffering from cancer according to any of the described methods.

In some embodiments, also provided herein is the use of a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells and a dgkα and/or dgkζ inhibitor in the manufacture of a medicament, the medicament being a combination therapy comprising the T cell therapy and the inhibitor, wherein the T cell therapy and the inhibitor are administered to a subject suffering from cancer according to any of the described methods.

In some of any embodiment, the T cell therapy is any T cell therapy as described herein. In some embodiments, the inhibitor is any formulation as described herein. In some embodiments, the subject is any subject as described herein. In some embodiments, the cancer is any cancer as described herein.

Drawings

Figures 1A and 1B show the expression of activation and differentiation markers in CAR-expressing T cells following stimulation with CAR-specific anti-idiotype antibodies in the presence of an exemplary DGK inhibitor (compound 17). Figure 1A shows the mean of log₂ fold changes in% positive cells of various activation and differentiation markers in donors (presence of compound 17 vs. absence) after stimulation with 3 μg/mL (left panel) or 30 μg/mL (right panel) CAR-specific anti-idiotype antibody. Figure 1B shows the mean of log₂ fold changes in MFI of cells in donors (presence vs. absence of compound 17) after stimulation with 3 μg/mL (left panel) or 30 μg/mL (right panel) CAR-specific anti-idiotype antibody. In fig. 1A and 1B, positive values are indicated by circles.

Figure 2 shows the growth curve (percent cell fusion in wells, as monitored using an Incucyte imaging system) of CAR-expressing cells stimulated with CAR-specific anti-idiotype antibodies in the presence of compound 17 or control.

FIG. 3 shows the percentage of specific lysis achieved by co-culturing CAR expressing T cells with CD19 antigen expressing target cells in the presence of compound 17 (or control), K562.CD19 cells (high CD19 expression; left panel), granta-519 cells (low CD19 expression; middle panel) and Raji cells (middle/high CD19 expression; right panel).

FIG. 4 shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNF α (right panel) after co-culturing CAR expressing T cells with K562.CD19 cells (circles), granta-519 cells (squares) and Raji cells (triangles) in the presence of compound 17.

Figures 5A-5B and 6A-6B show cytolytic activity and cytokine production during re-challenge of long-term stimulated CAR-expressing T cells in the presence of compound 17 (simultaneous treatment). Figure 5A shows tumor cell numbers of k562.cd19 (left panel), granta-519 (middle panel) and Raji cells (right panel) during 2D co-culture with CAR T cells that have been stimulated for long periods of time in the presence of compound 17. FIG. 5B shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNF α (right panel) after 2D co-cultivation of CAR T cells with K562.CD19 cells (circles), granta-519 cells (squares) and Raji cells (triangles) for 48 hours. Figure 6A shows normalized tumor volumes of a549.cd19 (left panel) and Granta-519 (right panel) tumor spheres on day 9 co-culture with CAR T cells that had been treated with compound 17 simultaneously during long term stimulation. FIG. 6B shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNF α (right panel) after 3D co-culture of CAR T cells with A549.CD19 (circular) and Granta-519 (square) tumor spheres for 5 days.

Figures 7A-7B and 8A-8B show cytolytic activity and cytokine production during re-challenge of long-term stimulated CAR-expressing T cells in the presence of compound 17 (rescue treatment). Figure 7A shows tumor cell numbers of k562.cd19 (left panel), granta-519 (middle panel) and Raji cells (right panel) during co-culture with CAR T cells 2D in the presence of compound 17. FIG. 7B shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNFα (right panel) after 2D co-cultivation of CAR T cells with K562.CD19 cells (circles), granta-519 cells (squares) and Raji cells (triangles) for 48 hours in the presence of compound 17. Figure 8A shows normalized tumor volumes of a549.cd19 (left panel) and Granta-519 (right panel) tumor spheres on day 9 co-culture with CAR T cells in the presence of compound 17. FIG. 8B shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNF α (right panel) after 3D co-cultivation of CAR T cells with A549.CD19 (circular) and Granta-519 (square) tumor spheres for 5 days in the presence of compound 17.

Figures 9 and 10 show cytolytic activity and cell proliferation of T cells expressing engineered T cell receptor T (eTCR) after rescue treatment with compound 17 after long term stimulation. FIG. 9 shows the number of tumor cells of CaSki cells monitored at different times during co-culture with long-term stimulated eTCR T cells in the presence of compound 17 (left panel, eTCR T cells long-term stimulated with 10 μg/mL anti-Vbreta; right panel, eTCR T cells long-term stimulated with 20 μg/mL anti-Vbreta). FIG. 10 shows cell counts of eTCR T cells from two donors, as shown from left to right, (1) long-term stimulated eTCR T cells without compound treatment (control), (2) long-term stimulated eTCR T cells with compound 17 treatment, or (3) fresh eTCR T cells without long-term stimulation.

Figure 11 shows the tumor burden of Raji cell-transplanted mice receiving three different dosing regimens ("early", "delayed" or "sustained") of DGKi compound 17 in combination with anti-CD 19 CAR T cells. In fig. 11, the shaded area indicates the period of time that compound 17 was orally administered once per day after CAR T cell administration in an early, delayed or sustained regimen.

Fig. 12 shows survival of Raji cell-transplanted mice following early, delayed or sustained compound 17 regimen in combination with anti-CD 19 CAR T cells (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001).

Fig. 13 shows tumor control index values (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001) for Raji cell-transplanted mice following early, delayed or sustained compound 17 regimen in combination with anti-CD 19 CAR T cells.

Fig. 14 shows ANOVA results of tumor control index values and tumor control index values of Raji cell-transplanted mice (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001) after receiving early, delayed or sustained compound 17 regimen in combination with anti-CD 19 CAR T cells. In fig. 14, results from all vehicle-treated mice groups were pooled into one group for statistical comparison for ANOVA testing.

Fig. 15 shows the number of circulating CAR expressing cells and the CD4 to CD8 ratio of circulating T cells on day 15 (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001) in Raji cell transplanted mice receiving a delayed or sustained regimen.

Figure 16 shows the total number of circulating CAR expressing cells on day 29 in Raji cell-transplanted mice receiving early, delayed or sustained compound 17 regimen in combination with anti-CD 19 CAR T cells.

Figure 17 shows the tumor burden of Raji cell-transplanted mice receiving sustained dosing DGKi of compound 17 in combination with anti-CD 19 CAR T cells. Compound 17 was orally administered once daily on days 1-30 following administration of CAR T cells on day 1.

Figure 18 shows survival of Raji cell transplanted mice receiving a sustained compound 17 regimen in combination with anti-CD 19 CAR T cells.

Fig. 19 shows tumor control index values (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001) for Raji cell-transplanted mice receiving the sustained compound 17 regimen in combination with anti-CD 19 CAR T cells.

Fig. 20 shows the number of circulating CAR expressing cells following administration of CAR T cells (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001) in Raji cell transplanted mice receiving the persistence regimen.

Figure 21 shows the number of circulating CAR expressing cells from four healthy human donors following CAR T cell administration in Raji cell-transplanted mice receiving administration DGKi on days 1-7.

Figure 22 shows tumor burden of Raji cell transplanted mice receiving sustained dosing DGKi of compound 17 in combination with anti-CD 19 CAR T cells or receiving dgkα/dgkζ single or double knockout of anti-CD 19 CAR T cells.

Fig. 23 shows survival of Raji cell-transplanted mice receiving either the combination of sustained compound 17 regimen with anti-CD 19 CAR T cells or dgkα/dgkζ single or double knockout anti-CD 19 CAR T cells (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001).

Fig. 24 shows tumor control index values (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001) for Raji cell-transplanted mice receiving either the combination of persistent compound 17 regimen with anti-CD 19 CAR T cells or dgkα/dgkζ single or double knockout anti-CD 19 CAR T cells.

Fig. 25 shows the number of circulating CAR expressing cells following administration of CAR T cells in Raji cell-transplanted mice receiving a persistence regimen or Raji cell-transplanted mice receiving dgkα/dgkζ single or double knockout CAR T cells (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001).

FIGS. 26A-26B show the tumor burden of Nalm6 cell transplanted mice. Figure 26A shows the tumor burden of Nalm6 cell-transplanted mice receiving sustained dosing DGKi of compound 17 in combination with anti-CD 19 CAR T cells. Fig. 26B shows historical tumor burden data for Nalm6 cell-transplanted mice receiving anti-CD 19 CAR T cells.

Fig. 27 shows survival of Nalm6 cell transplanted mice receiving a combination of sustained compound 17 regimen with anti-CD 19 CAR T cells (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001).

Fig. 28 shows tumor control index values (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001) for Nalm6 cell-transplanted mice receiving a combination of sustained compound 17 regimen with anti-CD 19 CAR T cells.

Fig. 29 shows the number of circulating CAR expressing cells following CAR T cell transplantation in Nalm6 cell-mice receiving the persistence regimen (n.s. [ not significant ]; p <0.05; p <0.01; p <0.001; p < 0.0001).

Figure 30 shows the tumor burden after a previous re-challenge of Nalm6 cell-transplanted mice that had received sustained administration DGKi of a combination of compound 17 and anti-CD 19 CAR T cells.

Fig. 31 shows survival (n.s. [ insignificant ]; p <0.05; p <0.01; p <0.001; p < 0.0001) of Nalm6 cell-transplanted mice that had previously received a combination of sustained compound 17 regimen with anti-CD 19 CAR T cells.

Detailed Description

Provided herein are combination therapies for treating a disease or disorder that involve administering a T cell therapy, such as Chimeric Antigen Receptor (CAR) -T cell therapy or engineered T cell receptor (eTCR) -T cell therapy, and a DGK inhibitor to an antigen associated with the disease or disorder. In some embodiments, the T cell therapy is a composition comprising T cells for adoptive cell therapy, wherein the cells are engineered with a recombinant receptor (e.g., CAR or eTCR) that targets the antigen. In some embodiments, the disease or condition may be cancer, an infectious disease, or an autoimmune disease. In some embodiments, the DGKi is an inhibitor of dgkα, dgkζ, or both dgkα and dgkζ, such as a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34, or a pharmaceutically acceptable salt thereof. In some aspects, the provided methods and uses enhance or modulate proliferation and/or activity of T cells (e.g., CAR-expressing T cells or eTCR-expressing T cells) associated with administration of the T cell therapy.

T cell-based therapies, such as adoptive T cell therapies, including those involving administration of cells expressing recombinant antigen receptors specific for a disease or disorder of interest (such as CAR or eTCR), can be effective in treating cancer and other diseases and disorders. Engineered expression of a recombinant receptor (such as CAR or eTCR) on the surface of T cells redirects T cells specifically. In clinical studies, CAR-T cells (e.g., anti-CD 19 CAR-T cells) have produced a durable, complete response in both leukemia and lymphoma patients (Porter et al (2015) SCI TRANSL Med.,7:303ra139; kochenderfer (2015) J.Clin. Oncol.,33:540-9; lee et al (2015) Lancet,385:517-28; maude et al (2014) N Engl J Med, 371:1507-17).

In some cases, the methods of adoptive cell therapy available may not always be entirely satisfactory. For example, in some cases, although persistence of CAR T cells can be detected in many subjects with lymphoma, fewer Complete Responses (CRs) are observed in subjects with NHL than in subjects with ALL. More specifically, while a higher total response rate of up to 80% has been reported after CAR T cell infusion (CR rate 47% to 60%), the response in some subjects was transient and subjects showed relapse in the presence of sustained CAR T cells (Neelapu, 58, american Society of Hematoma (ASH): 2016;San Diego,CA,USA. Abstract No. lba-6.2016;Abramson,Blood.2016Dec 01;128 (22): 4192). Another study reported a long-term CR rate of 40% (Schuster, ann Hematol.2016Oct;95 (11): 1805-10).

In some aspects, an explanation for this is immune depletion of circulating T cells (e.g., CAR expressing T cells) and/or a change in T lymphocyte populations of T cell therapies. In some cases, the optimal therapeutic effect may depend on the ability of the administered cells to recognize and bind to a target (e.g., target antigen), transport, locate, and successfully enter the appropriate location within the subject, tumor, and its environment. In some cases, optimal efficacy may depend on the ability of the administered cells to activate, expand, exert various effector functions (including cytotoxic killing and secretion of various factors such as cytokines), persist (including long term), differentiate, transition or participate in reprogramming into certain phenotypic states (such as long-life memory, low differentiation and effector states), avoid or reduce immunosuppressive conditions in the local microenvironment of the disease, provide an effective and powerful recall response upon clearance and re-exposure to the target ligand or antigen, and avoid or reduce depletion, disability, peripheral tolerance, terminal differentiation and/or differentiation into an inhibited state.

In some embodiments, the engineered cell exposure and persistence of the T cell therapy is reduced or decreased after administration to the subject. However, observations indicate that in some cases, increased exposure of the subject to the administered cells expressing the recombinant receptor (e.g., increased cell number or duration) can improve the efficacy and therapeutic outcome in adoptive cell therapy. In a number of clinical trials, preliminary analysis after administration of different CD 19-targeted CAR-expressing T cells to subjects with various CD 19-expressing cancers showed a correlation between greater and/or longer exposure to CAR-expressing cells and treatment outcome. These results include survival and remission of the patient, even in individuals with severe or significant tumor burden.

In some embodiments, after prolonged stimulation or exposure to an antigen and/or exposure to conditions of the tumor microenvironment, T cells may become dysfunctional over time and/or exhibit characteristics associated with a depleted state. In some aspects, this reduces the persistence and effectiveness of T cells against an antigen and limits their effective capacity. There is a need for methods of improving the effectiveness and function of T cells (e.g., CAR-expressing T cells or eTCR-expressing T cells) in T cell therapies, particularly to minimize, reduce, prevent, or reverse a low-functioning or depleted state.

Diacylglycerol kinase (DGK) is a lipid kinase that mediates the conversion of diacylglycerol to phosphatidic acid, thereby terminating T cell function transmitted through the TCR signaling pathway. Thus, DGK acts as an intracellular checkpoint and inhibits DGK, hopefully enhancing T cell signaling pathways and T cell activation. Supportive evidence includes knockout mouse models of DGK alpha or DGK zeta that exhibit a highly reactive T cell phenotype and improved anti-tumor immune activity (Riese M.J. et al, journal of Biological Chemistry, (2011) 7:5254-5265; zha Y et al, nature Immunology, (2006) 12:1343;Olenchock B.A. Et al, (2006) 11:1174-81). Furthermore, over-expression of DGK alpha was observed in tumor-infiltrating lymphocytes isolated from human renal cell carcinoma patients, which resulted in inhibited T cell function (Prinz, P.U. et al, JImmunology (2012) 12:5990-6000). Thus, DGKα and DGKζ are considered targets for tumor immunotherapy (Riese M.J. et al, front Cell Dev biol. (2016) 4:108; chen, S.S. et al, front Cell Dev biol. (2016) 4:130; avila-Flores, A.et al, immunology and Cell Biology (2017) 95:549-563; noessner, E., front Cell Dev biol. (2017) 5:16; krishna, S., et al, front Immunology (2013) 4:178; jing, W.et al, CANCER RESEARCH (2017) 77:5676-5686).

The provided methods are based on the observation that DGKi (such as the described exemplary compound 17) improves T cell function (including functions related to the production of one or more cytokines, cytotoxicity, expansion, proliferation, and persistence of T cells). In some aspects, provided methods enhance or modulate proliferation and/or activity of T cells associated with administration of T cell therapies (e.g., CAR expressing T cells). These methods and uses have been found to provide or achieve improved or stronger T cell function and thereby improve antitumor efficacy.

It has also been found herein that in addition to enhancing T cell function, such DGKi (e.g., compound 17) exhibits an effect of reversing, delaying, or preventing T cell depletion, including one or more genes regulated by increasing T cell signaling and/or altering differences following chronic (long term) stimulation. Thus, while agents that increase or enhance T cell activity may, in some cases, drive the cells to a depleted state, it was found herein that this DGKi (e.g., compound 17) activity that exerts an enhancing effect on T cell activity is separate from T cell depletion. In some embodiments, the provided methods involve administering such DGKi (e.g., compound 17) to enhance T cell activity and delay, limit, reduce, inhibit, or prevent depletion.

Furthermore, observations herein indicate that DGKi (e.g., compound 17) exhibits activity to rescue T cells from T cell depletion, such as by restoring or partially restoring one or more T cell activities after the cells exhibit depletion characteristics. Notably, the results herein demonstrate that exposure of T cells that have been chronically stimulated and that exhibit depleted T cell characteristics to DGKi (such as compound 17) described herein is capable of restoring or partially restoring activity. The observations herein support that the provided methods can also achieve improved or more durable responses, such as in a particular treatment subject group, as compared to certain alternative methods.

These observations were obtained using a long-term stimulation assay that resulted in reduced T cell (e.g., CAR T cell) function (e.g., reduced cytolysis and IL-2 secretion). Using this model, engineered T cells (e.g., CAR T cells or eTCR T cells) were examined to evaluate the effect of DGKi (such as compound 17) on the function of the engineered T cells, when present, during (simultaneous) or after (rescue) exposure to conditions that result in a low-functioning, depleted state. Upon re-challenge with antigen, the results of the studies provided herein demonstrate that simultaneous treatment of engineered T cells (e.g., CAR T cells or eTCR T cells) during the conditions reverses the activity and phenotype associated with reduced T cell function and retains more of the effector functions. Also, the results indicate that subsequent exposure to DGKi (e.g., compound 17) can rescue or restore T cell function, including depletion of T cell cytokine production and cytolytic activity. In addition, different recombinant antigen receptors (including CARs and eTCR) also have different results.

It is also shown herein that DGKi (e.g., compound 17) administered in a continuous dosing regimen (e.g., at least once daily for a plurality of consecutive days, such as 28 consecutive days) improves the in vivo anti-tumor effect following administration of engineered T cells (e.g., CAR T cells or eTCR T cells) for T cell therapy. As indicated herein, the sustained presence of DGKi after administration of a T cell therapy, such as starting on the same day as administration of a T cell therapy, can improve expansion and/or persistence of CAR T cells, particularly cd4+ CAR T cells. Without wishing to be limited by a particular mechanism of action, the results presented herein are consistent with the discovery that the continued presence of DGKi following administration of a T cell therapy can prevent or reduce T cell depletion of the T cell therapy, thereby increasing the anti-tumor activity and proliferation capacity of the T cells following administration.

The provided findings indicate that combination therapies of DGKi (i.e., dgkα, dgkζ, or inhibitors of both dgkα and dgkζ, such as compounds of formula (I) or (II), such as compounds selected from compounds 1-34, or pharmaceutically acceptable salts thereof) achieve functional improvements in T cell therapies in methods involving T cell therapies, such as involving administration of compositions of engineered T cells, such as by enhancing T cell activity and reducing, preventing or delaying T cell depletion, or rescue of T cells from T cell depletion. In some embodiments, the combination therapy involves administration or use of T cell therapy (e.g., CAR T cells or eTCR T cells) and DGKi of the compound of formula (II). In some embodiments, the combination therapy involves the administration or use of T cell therapies (e.g., CAR T cells or eTCR T cells) and DGKi (compound 4- ((2 s,5 r) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, or a stereoisomer thereof). In some embodiments, the combination therapy involves the administration or use of T cell therapies (e.g., CAR T cells or eTCR T cells) and DGKi (compound 4- ((2S, 5 r) -2, 5-diethyl-4- ((S) -1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (compound 17)). In some embodiments, the combination of the T cell therapy (e.g., administration of engineered T cells) with the DGKi (e.g., compound 17) improves or enhances one or more functions and/or effects of the T cell therapy, such as persistence, expansion, toxicity, and/or therapeutic outcome (e.g., ability to kill or reduce the burden of a tumor or other disease or target cell).

All publications, including patent documents, scientific documents, and databases, mentioned in this specification are incorporated by reference in their entirety for any purpose to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. If a definition set forth herein is inconsistent or inconsistent with a definition in a patent, application, published application and other publication, the definition set forth herein is incorporated by reference in the public place where the definition set forth herein takes precedence over the definition set forth herein.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. Combination therapy

In some embodiments, provided herein are methods of treatment comprising administering a cell therapy and a DGK inhibitor to a subject suffering from a disease or disorder. In some embodiments, the DGK inhibitor is a dgkα and/or dgkζ inhibitor. In some embodiments, the cell therapy is T cell therapy. In some embodiments, the T cell therapy comprises an engineered T cell. In some embodiments, the engineered T cell expresses a recombinant receptor. In some embodiments, the recombinant receptor is a Chimeric Antigen Receptor (CAR). In some embodiments, the recombinant receptor is an engineered T Cell Receptor (TCR).

In some embodiments, the DGK inhibitor (e.g., dgkα and/or dgkζ inhibitor) is administered prior to the T cell therapy, e.g., the onset of administration of the inhibitor occurs, is performed, or occurs prior to the onset of administration of the T cell therapy. In some embodiments, the DGK inhibitor (e.g., dgkα and/or dgkζ inhibitor) is administered concurrently with the T cell therapy, e.g., the onset of administration of the inhibitor is concurrent, performed, or occurs with the administration of the T cell therapy. In some embodiments, the DGK inhibitor (e.g., dgkα and/or dgkζ inhibitor) is administered after the T cell therapy, e.g., the onset of administration of the inhibitor occurs, is performed, or occurs after the onset of administration of the T cell therapy.

In some embodiments, provided herein are also methods of treatment comprising administering a DGK inhibitor to a subject suffering from a disease or condition. In some embodiments, also provided herein are methods of rescuing engineered cells of a cell therapy from depletion, e.g., rescuing engineered T cells of a T cell therapy from depletion, comprising administering a DGK inhibitor to a subject suffering from a disease or disorder. In some embodiments, provided herein are also methods of reducing or delaying the onset of T cell depletion of T cells of a T cell therapy, the methods comprising administering a DGK inhibitor to a subject suffering from a disease or disorder. In some embodiments, the subject has previously been administered a cell therapy (e.g., T cell therapy) for treating the disease or condition.

The combination therapies (e.g., combination therapies comprising an engineered cell expressing a recombinant receptor such as a Chimeric Antigen Receptor (CAR) and the DGK inhibitor, or combination therapies comprising an engineered cell and/or DGK inhibitor as described herein) can be used for a variety of therapeutic, diagnostic, and prophylactic indications. For example, the combination may be used to treat various diseases and disorders in a subject. These methods and uses include therapeutic methods and uses, for example, involving administering the engineered cells, DGK inhibitors, and/or compositions containing one or both to a subject suffering from a disease, condition, or disorder (such as a tumor or cancer). In some embodiments, the engineered cells, the DGK inhibitor, and/or a composition containing one or both are administered in an effective amount to effectively treat the disease or disorder. Uses include the use of the engineered cells, the DGK inhibitors and/or compositions containing one or both in the methods and treatments, and in the manufacture of medicaments for carrying out these methods of treatment. In some embodiments, the method is by administering the engineered cells, the DGK inhibitor, and/or a composition containing one or both to a subject suffering from or suspected of suffering from the disease or disorder. In some embodiments, the method thus treats a disease, condition, or disorder in the subject. In some embodiments, the DGK inhibitor is any one as described in section I-B. In some embodiments, the engineered cell is any engineered cell as described in section I and II.

The disease or condition being treated may be one in which expression of the antigen is associated with or implicated in the etiology of the disease or condition, e.g., antigen expression results in, exacerbates or is otherwise implicated in the disease or condition. Exemplary diseases and conditions include diseases or conditions associated with transformation of malignant tumors or cells (e.g., cancer), autoimmune or inflammatory diseases or infectious diseases (e.g., caused by bacteria, viruses, or other pathogens). Exemplary antigens are described herein, including antigens associated with various treatable diseases and conditions. In certain embodiments, the antigen binding domain of the recombinant receptor (e.g., CAR or TCR) expressed by the cell therapy (e.g., T cell therapy) is administered as part of the methods provided herein, specifically binds to an antigen associated with the disease or condition.

In some embodiments, the disease or condition includes tumors (including solid tumors, hematological malignancies, and melanomas, including local and metastatic tumors), infectious diseases, such as viral or other pathogen (e.g., HIV, HCV, HBV, CMV, HPV) infections, and parasitic diseases, as well as autoimmune and inflammatory diseases. In some embodiments, the disease or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease or disorder. These diseases include, but are not limited to, leukemia, lymphoma, such as acute myelogenous (or myelogenous) leukemia (AML), chronic myelogenous (or myelogenous) leukemia (CML), acute lymphoblastic (or lymphoblastic) leukemia (ALL), chronic Lymphocytic Leukemia (CLL), hairy Cell Leukemia (HCL), small Lymphocytic Lymphoma (SLL), mantle Cell Lymphoma (MCL), marginal zone lymphoma, burkitt lymphoma (Burkittlymphoma), hodgkin lymphoma (Hodgkinlymphoma, HL), non-hodgkin lymphoma (NHL), acellular Large Cell Lymphoma (ALCL), follicular lymphoma, refractory follicular lymphoma, diffuse Large B Cell Lymphoma (DLBCL), and Multiple Myeloma (MM).

In some embodiments, the disease or condition is a B cell malignancy. In some embodiments, the B cell malignancy is selected from Acute Lymphoblastic Leukemia (ALL), adult ALL, chronic Lymphoblastic Leukemia (CLL), non-hodgkin's lymphoma (NHL), and Diffuse Large B Cell Lymphoma (DLBCL). In some embodiments, the disease or condition is NHL. In some embodiments, the NHL is selected from the group consisting of invasive NHL, diffuse large B-cell lymphoma (DLBCL), NOS (primary and transformed from indolent), primary mediastinal large B-cell lymphoma (PMBCL), T-cell/histiocyte large B-cell lymphoma (TCHRBCL), burkitt's lymphoma, mantle Cell Lymphoma (MCL), and/or Follicular Lymphoma (FL), optionally grade 3B follicular lymphoma (FL 3B).

In some embodiments, the disease or disorder is a B cell related disorder. In some of any of the embodiments provided by the methods provided, the disease or disorder is an autoimmune disease or disorder. In some of any of the embodiments provided by the methods provided, the autoimmune disease or disorder is Systemic Lupus Erythematosus (SLE), lupus nephritis, inflammatory bowel disease, rheumatoid arthritis, ANCA-related small vessel inflammation, idiopathic Thrombocytopenic Purpura (ITP), thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, chagas ' disease, grave's disease (Grave's disease), wegener's granulomatosis (Wegener ' sgranulomatosis), polyarteritis nodosa, sjogren's syndrome (Sjogren's syndrome), herpes vulgaris, scleroderma, multiple sclerosis, psoriasis, igA nephropathy, igM polyneuropathy, vasculitis, diabetes mellitus, reynolds syndrome (Reynaud's syndrome), antiphospholipid syndrome, goodpasture's syndrome (Goodpasture's disease), hemolytic anemia, sjogren's disease, or renal hypermyopathy.

In some embodiments, the disease or disorder is an infectious disease or disorder, such as, but not limited to, viral, retroviral, bacterial and protozoal infections, immunodeficiency, cytomegalovirus (CMV), epstein-Barr virus (EBV), adenovirus, BK polyomavirus infectious disease or disorder. In some embodiments, the disease or disorder is an autoimmune or infectious disease or disorder, such as arthritis (e.g., rheumatoid Arthritis (RA)), type I diabetes, systemic Lupus Erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroiditis, lattice Lei Fusi disease, crohn's disease, multiple sclerosis, asthma, and/or a disease or disorder associated with transplantation.

In some embodiments, the disease or disorder is a solid tumor or a cancer associated with a non-hematologic tumor. In some embodiments, the disease or disorder is a solid tumor or a cancer associated with a solid tumor. In some embodiments, the disease or disorder is pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumor, bone cancer, or soft tissue sarcoma. In some embodiments, the disease or disorder is bladder cancer, lung cancer, brain cancer, melanoma (e.g., small cell lung cancer, melanoma), breast cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, endometrial cancer, esophageal cancer, renal cancer, liver cancer, prostate cancer, skin cancer, thyroid cancer, or uterine cancer. In some embodiments, the disease or disorder is pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumor, bone cancer, or soft tissue sarcoma.

In some embodiments, the antigen associated with the disease or condition is or includes αvβ6 integrin (avb 6 integrin), B cell activator receptor (BAFF-R), B Cell Maturation Antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA 9, also known as CAIX or G250), cancer testosterone antigen, cancer/testosterone antigen 1B (CTAG, also known as NY-ESO-1 and rage-2), carcinoembryonic antigen (CEA), cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v6, CD44v7/8, CD70, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG 4), delta-like ligand 3 (DLL 3), epidermal growth factor protein (EGFR), EGFR III mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa), and, estrogen receptor, fc-like receptor 5 (FCRL 5; also known as Fc receptor homolog 5 or FCRH 5), fetal acetylcholine receptor (fetal AchR), folic acid binding protein (FBP), folic acid receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD 2), ganglioside GD3, glycoprotein 100 (gp 100), phosphatidylinositol glycan-3 (GPC 3), G protein coupled receptor group C5 member D (GPRC 5D), her2/neu (erb-B2 receptor tyrosine kinase), her3 (erb-B3), her4 (erb-B4), her2, G-C (GPRC 5D), erbB dimer, human high molecular weight melanomA-Associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLa-A1), human leukocyte antigen A2 (HLa-A2), IL-22 receptor alpha (IL-22 Rα), IL-13 receptor alpha 2 (IL-13 Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine rich repeat 8 family member A (LRRC 8A), lewis Y, melanomA-Associated antigen (MAGE) -A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine Cytomegalovirus (CMV), mucin 1 (MUC 1), MUC16, natural killer group 2 member D (NKG 2D) ligand, melanin A (MART-1), neural Cell Adhesion Molecule (NCAM), placenta antigen, melanoma preferential expression antigen (PRAME), progesterone receptor, prostate specific antigen, prostate Stem Cell Antigen (PSCA), prostate Specific Membrane Antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR 1), and, Survivin, trophoblastic glycoprotein (TPBG also known as 5T 4), tumor-associated glycoprotein 72 (TAG 72), tyrosinase-associated protein 1 (TRP 1, also known as TYRP1 or gp 75), tyrosine-associated protein 2 (TRP 2, also known as dopachrome tautomerase, dopachrome delta isomerase, or DCT), vascular Endothelial Growth Factor Receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR 2), wilms tumor 1 (WT-1), pathogen-specific or pathogen-expressing antigens or antigens associated with universal TAGs and/or biotinylated molecules, and/or by HIV, the methods of treating cancer, HCV, HBV, or other pathogen expressed molecules.

In some embodiments, the disease or disorder is a B cell malignancy. In some embodiments, the disease or disorder is a large B cell lymphoma. In some embodiments, the B cell malignancy is selected from Acute Lymphoblastic Leukemia (ALL), adult ALL, chronic Lymphoblastic Leukemia (CLL), non-hodgkin's lymphoma (NHL), and Diffuse Large B Cell Lymphoma (DLBCL). In some embodiments, the disease or disorder is NHL. In some embodiments, the NHL is selected from invasive NHL, diffuse large B-cell lymphoma (DLBCL), NOS (primary and transformed from indolent), primary mediastinal large B-cell lymphoma (PMBCL), T-cell/histiocyte large B-cell lymphoma (TCHRBCL), burkitt's lymphoma, mantle Cell Lymphoma (MCL), and/or Follicular Lymphoma (FL), optionally grade 3B follicular lymphoma (FL 3B). In any of the embodiments, the disease or disorder recurs or is refractory to one or more previous treatments (recurrent or refractory disease; R/R). In some aspects, the recombinant receptor (e.g., CAR or TCR) specifically binds to an antigen associated with a disease or disorder or is expressed in a cell in the context of a pathology associated with a B-cell malignancy. In some embodiments, the antigen targeted by the receptor includes antigens associated with B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is BAFF-R, CD, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30, or a combination thereof. For example, in some embodiments, the antigen is BAFF-R and the CAR is as described in Qin et al Science TranslationalMedicine (511): eaaw9414 (2019). In some embodiments, the antigen targeted by the receptor is CD19. For example, the recombinant receptor can be a CAR that is a CAR of alemtuquor (Yescarta), span Li Fuming (Kymriah), or licarbazepine (Breyanzi). In some embodiments, the recombinant receptor can be a CAR that is a CAR of TECARTUS^TM (brix). In some embodiments, the T cell therapy (e.g., anti-CD 19 CAR T cell therapy) is alemtuquor (Yescarta), se Li Fuming (Kymriah), li Jimai alemtuquor (Breyanzi), or TECARTUS^TM (bristol).

In some embodiments, the disease or disorder is a large B cell lymphoma. In some embodiments, the large B cell lymphoma is a relapsed or refractory large B cell lymphoma. In some embodiments, the large B-cell lymphoma is diffuse large B-cell lymphoma (DLBCL) not explicitly specified (including DLBCL caused by indolent lymphoma), advanced B-cell lymphoma, primary mediastinum large B-cell lymphoma, or grade 3B follicular lymphoma. In some embodiments, the target antigen that binds through the recombinant receptor and is associated with large B cell lymphomas is CD19. In some embodiments, the recombinant receptor is an anti-CD 19 CAR. In some embodiments, the anti-CD 19 CAR is a CAR of Li Jimai th am (Breyanzi). In some embodiments, the T cell therapy is an anti-CD 19 CAR T cell therapy. In some embodiments, the anti-CD 19 CAR T cell therapy is Li Jimai am (Breyanzi, see Sehgal et al, 2020,Journal of Clinical Oncology 38:15_suppl,8040;Teoh et al, 2019, blood134 (supplement_1): 593, and Abramson et al 2020,The Lancet 396 (10254): 839-852).

In some embodiments, the disease or disorder is mantle cell lymphoma or B cell precursor Acute Lymphoblastic Leukemia (ALL). In some embodiments, the disease or disorder is relapsed or refractory mantle cell lymphoma or relapsed or refractory B-cell precursor Acute Lymphoblastic Leukemia (ALL). In some embodiments, the target antigen that binds through the recombinant receptor and is associated with the disease or disorder is CD19. In some embodiments, the recombinant receptor is an anti-CD 19 CAR. In some embodiments, the anti-CD 19 CAR is a CAR of TECARTUS^TM (brix). In some embodiments, the T cell therapy is an anti-CD 19 CAR T cell therapy. In some embodiments, the anti-CD 19 CAR T cell therapy is TECARTUS^TM (Bristolonite, see Mian and Hill,2021,Expert Opin Biol Ther;21 (4): 435-441; and Wang et al 2021, blood138 (Supplement 1): 744).

In some embodiments, the disease or disorder is diffuse large B-cell lymphoma (DLBCL) or Acute Lymphoblastic Leukemia (ALL). In some embodiments, the disease or disorder is relapsed or refractory diffuse large B-cell lymphoma (DLBCL) or relapsed or refractory Acute Lymphoblastic Leukemia (ALL). In some embodiments, the target antigen that binds through the recombinant receptor and is associated with the disease or disorder is CD19. In some embodiments, the recombinant receptor is an anti-CD 19 CAR. In some embodiments, the anti-CD 19 CAR is a CAR of Li Fuming (Kymriah). In some embodiments, the T cell therapy is an anti-CD 19 CAR T cell therapy. In some embodiments, the anti-CD 19 CAR T cell therapy is Se Li Fuming (Kymriah, see Bishop et al, 2022,N Engl J Med 386:629:639;Schuster et al, 2019,N Engl J Med 380:45-56; halford et al, 2021,Ann Pharmacother 55 (4): 466-479; mueller et al, 2021, blood adv.5 (23): 4980-4991; and Fowler et al, 2022,Nature Medicine 28:325-332).

In some embodiments, the disease or disorder is B-cell lymphoma. In some embodiments, the B cell lymphoma is relapsed or refractory B cell lymphoma. In some embodiments, the B-cell lymphoma is diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, advanced B-cell lymphoma, DLBCL caused by follicular lymphoma, or follicular lymphoma. In some embodiments, the target antigen that binds to and is associated with B cell lymphoma via the recombinant receptor is CD19. In some embodiments, the recombinant receptor is an anti-CD 19CAR. In some embodiments, the anti-CD 19CAR is a CAR of aliskiren (Yescarta). In some embodiments, the T cell therapy is an anti-CD 19CAR T cell therapy. In some embodiments, the anti-CD 19CAR T cell therapy is Alcalamine (Yescarta, see Neelapu et al, 2017,N Engl J Med377 (26): 2531-2544; jacobson et al, 2021,The Lancet 23 (1): P91-103; and Locke et al, 2022,N Engl J Med 386:640-654).

In some embodiments, the disease or disorder is myeloma, such as multiple myeloma. In some embodiments, the antigen targeted by the recombinant receptor comprises an antigen associated with multiple myeloma. In some aspects, the antigen is expressed on multiple myeloma, such as B Cell Maturation Antigen (BCMA), G protein coupled receptor group C5 member D (GPRC 5D), CD38 (cyclic ADP-ribose hydrolase), CD138 (syndecan-1, synecan, SYN-1), CS-1 (CS 1, CD2 subclass 1, CRACC, SLAMF7, CD319 and 19a 24), BAFF-R, TACI, and/or FcRH5. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, CD44, CD20, CD40, CD74, CD200, EGFR, beta 2-microglobulin, HM1.24, IGF-1R, IL-6R, TRAIL-R1, and type IIA activin receptor (actRIA). See Benson and Byd, J.Clin. Oncol. (2012) 30 (16): 2013-15; tao and Anderson, bone Marrow Research (2011): 924058; chu et al, leukemia (2013) 28 (4): 917-27; garpal et al, discover Med. (2014) 17 (91): 37-46). In some embodiments, the antigens include those present on lymphoid tumors, myeloma, AIDS-related lymphomas, and/or post-transplant lymphoproliferation, such as CD38. Antibodies or antigen binding fragments to these antigens are known and include, for example, those described in U.S. Pat. nos. 8,153,765, 8,603477,8,008,450, U.S. publication nos. US20120189622 or US20100260748, and/or international PCT publication nos. WO2006099875, WO2009080829 or WO2012092612 or WO 2014210064. In some embodiments, these antibodies or antigen binding fragments thereof (e.g., scFv) are contained in multispecific antibodies, multispecific chimeric receptors (such as multispecific CARs), and/or multispecific cells.

In some embodiments, the disease or disorder is Multiple Myeloma (MM). In some embodiments, the disease or disorder is associated with expression of G protein coupled receptor group C5 member D (GPRC 5D) and/or expression of B Cell Maturation Antigen (BCMA). In some embodiments, the subject suffers from or is suspected of suffering from MM associated with expression of a tumor associated antigen, such as B Cell Maturation Antigen (BCMA), G protein coupled receptor group C5 member D (GPRC 5D), or Fc-like receptor 5 (FCRL 5; also referred to as Fc receptor homolog 5 or FCRH 5). In some aspects, the recombinant receptor (e.g., CAR or TCR) specifically binds to an antigen that binds to MM, such as specifically binds BCMA, GPRC5D, or FCRL5. In some embodiments, the target antigen associated with the disease or disorder (e.g., with MM) is BCMA. In some embodiments, the recombinant receptor is(Ai Dika barks) or CARVYKTI^TM (cetostensite), e.g., anti BCMACAR. In some embodiments, the T cell therapy (e.g., anti-BCMACAR T cell therapy) is(Ai Dika Bajin) or CARVYKTI^TM (Sida base Orthoxel).

In some embodiments, the disease or disorder is multiple myeloma. In some embodiments, the multiple myeloma is relapsed or refractory multiple myeloma. In some embodiments, the target antigen bound by the recombinant receptor and associated with multiple myeloma is BCMA. In some embodiments, the recombinant receptor is an antibody BCMACAR. In some embodiments, the anti BCMACAR is a CAR of CARVYKTI^TM (sidaorensai). In some embodiments, the T cell therapy is an anti-BCMACAR T cell therapy. In some embodiments, the anti-BCMACAR T cell therapy is CARVYKTI^TM (Sida-based Orthomson, see Berdeja et al, lancet.2021, 7 months, 24; 398 (10297): 314-324; and Martin, abstract #549[ dictation ], published in the American Society of Hematology (ASH) and Exposure at 2021)).

In some embodiments, the disease or disorder is multiple myeloma. In some embodiments, the multiple myeloma is relapsed or refractory multiple myeloma. In some embodiments, the target antigen bound by the recombinant receptor and associated with multiple myeloma is BCMA. In some embodiments, the recombinant receptor is an antibody BCMACAR. In some embodiments, the anti BCMACAR is(Ai Dika Bajin). In some embodiments, the T cell therapy is an anti-BCMACAR T cell therapy. In some embodiments, the anti-BCMACAR T cell therapy is(Ai Dika Bajin, see Raje et al, 2019,N Engl J Med 380:1726-1737; and Munshi et al, 2021,N Engl J Med 384:705-716).

In some embodiments, the disease or disorder is Chronic Lymphocytic Leukemia (CLL). In some embodiments, the subject suffers from or is suspected of suffering from CLL associated with expression of a tumor-associated antigen, such as receptor tyrosine kinase-like orphan receptor 1 (ROR 1). In some embodiments, the antigen is ROR1 and the disease or disorder is CLL. In some aspects, the recombinant receptor (e.g., CAR or TCR) specifically binds an antigen associated with CLL, such as specifically binds ROR1.

In some embodiments, the disease or disorder is non-small cell lung cancer (NSCLC). In some embodiments, the subject suffers from or is suspected of suffering from NSCLC associated with expression of a tumor-associated antigen, such as receptor tyrosine kinase-like orphan receptor 1 (ROR 1). In some embodiments, the antigen is ROR1 and the disease or disorder is NSCLC. In some aspects, the recombinant receptor (e.g., CAR or TCR) specifically binds an antigen associated with NSCLC, such as specifically binds ROR1. In some embodiments, the CAR is as described in Specht et al, CANCER RES 79:79:4 appendix, abstract P2-09-13.

In some embodiments, the disease or disorder is Triple Negative Breast Cancer (TNBC). In some embodiments, the subject suffers from or is suspected of suffering from TNBC associated with expression of a tumor-associated antigen, such as receptor tyrosine kinase-like orphan receptor 1 (ROR 1). In some embodiments, the antigen is ROR1 and the disease or disorder is TNBC. In some aspects, the recombinant receptor (e.g., CAR or TCR) specifically binds to an antigen associated with TNBC, such as specifically binds ROR1. In some embodiments, the CAR is as described in Specht et al, CANCER RES 79:79:4 appendix, abstract P2-09-13.

In some embodiments, the disease or disorder is Small Cell Lung Cancer (SCLC), optionally relapsed/refractory SCLC. In some embodiments, the subject suffers from or is suspected of suffering from SCLC associated with expression of a tumor-associated antigen (such as DLL 3). In some embodiments, the antigen is DLL3 and the disease or disorder is SCLC. In some aspects, the recombinant receptor (e.g., CAR or TCR) specifically binds an antigen associated with SCLC, such as specifically binds DLL3. In some embodiments, the CAR is as described in Byers et al, journal of Clinical Oncology, no.15_suppl (2019).

In some embodiments, the disease or disorder is Renal Cell Carcinoma (RCC). In some embodiments, the subject suffers from or is suspected of suffering from RCC associated with expression of a tumor-associated antigen (such as CD 70). In some embodiments, the antigen is CD70 and the disease or disorder is RCC. In some aspects, the recombinant receptor (e.g., CAR or TCR) specifically binds an antigen associated with RCC, such as specifically binds CD70. In some embodiments, the CAR is as described in Panowski et al, CANCER RES 79 (13 appendix) 2326 (2019).

In some embodiments, the disease or disorder is Acute Myelogenous Leukemia (AML). In some embodiments, the subject suffers from or is suspected of suffering from AML associated with expression of a tumor-associated antigen (such as CD 70). In some embodiments, the antigen is CD70 and the disease or disorder is AML. In some aspects, the recombinant receptor (e.g., CAR or TCR) specifically binds an antigen associated with AML, such as specifically binds CD70. In some embodiments, the CAR is as described in Sauer et al, blood 134 (appendix_1): 1932 (2019).

In some embodiments, the antigen is or includes a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and/or a parasitic antigen.

A.T administration of cell therapies

In some aspects, the methods provided herein include combination therapies by administering a cell therapy (e.g., T cell therapy) in combination with a DGK inhibitor to a subject suffering from a disease or disorder (such as any of the diseases or disorders described above). In some aspects, methods provided herein comprise administering a DGK inhibitor to a subject in combination with (e.g., before, simultaneously with, or after) administration of a cell therapy (e.g., T cell therapy). In some embodiments, provided methods include administering a DGK inhibitor to a subject who has previously been administered a cell therapy (e.g., T cell therapy) for treating a disease or disorder. In some embodiments, the cell therapy (e.g., T cell therapy) is administered to the subject prior to the DGKi being administered to the subject. In some embodiments, the cell therapy (e.g., T cell therapy) and the DGKi are administered simultaneously.

In some embodiments, the cell therapy is T cell therapy. The T cell therapy may include T cells engineered with recombinant receptors, such as CARs or engineered TCRs (e.g., eTCR). In some embodiments, the T cell therapy comprises T cells (e.g., cd4+ and/or cd8+ T cells) that express a CAR that targets or binds to an antigen associated with the disease or disorder. In some embodiments, the T cell therapy comprises T cells (e.g., cd4+ and/or cd8+ T cells) that express eTCR that target or bind to an antigen associated with the disease or disorder. T cell therapies used in the provided methods include any of a variety of CAR expressing or eTCR expressing T cells. It is within the skill level of the skilled artisan to select an appropriate recombinant receptor (e.g., CAR or eTCR) expressing T cell therapy for treating the disease or disorder. Examples of T cell therapies (including CAR-expressing T cells and eTCR-expressing T cells) are described in section II. In some embodiments, the T cells of the T cell therapy are allogeneic to the subject being treated. In some embodiments, the T cells of the T cell therapy are autologous to the subject being treated.

Methods of cell administration for adoptive cell therapy are known and may be used with the provided methods, compositions and articles of manufacture and kits. Adoptive cell therapies are described, for example, in Gruenberg et al, U.S. patent application publication No. 2003/0170238, rosenberg, U.S. patent No. 4,690,915, rosenberg (2011) NAT REV CLIN Oncol.8 (10): 577-85). See, for example, themeli et al (2013) Nat Biotechnol.31 (10): 928-933; tsukahara et al (2013) Biochem Biophys Res Commun (1): 84-9; davila et al (2013) PLoS ONE 8 (4): e 61338).

In some embodiments, the cell therapy (e.g., adoptive T cell therapy) is performed by autologous transfer, wherein cells are isolated and/or otherwise prepared from a subject to be subjected to the cell therapy or from a sample derived from the subject. Thus, in some aspects, the cells are derived from a subject (e.g., patient) in need of treatment, and the cells are administered to the same subject after isolation and treatment.

In some embodiments, the cell therapy (e.g., adoptive T cell therapy) is performed by allogeneic transfer, wherein the cells are isolated and/or otherwise prepared from a subject other than the subject (e.g., first subject) that is to receive or ultimately receives the cell therapy. In such embodiments, the cells are then administered to a different subject (e.g., a second subject) of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

T cells of a T cell therapy may be administered in a composition formulated for administration, or alternatively, in more than one composition (e.g., two compositions) formulated for separate administration. The dose of cells may include a specific number or relative number of cells or engineered cells, and/or a defined ratio or composition of two or more subclasses in the composition, such as CD4 vs CD 8T cells.

The cells may be administered by any suitable means, for example, by bolus infusion, by injection (e.g., intravenous or subcutaneous injection, intraocular injection, peribulbar injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intracoronary injection, intracameral injection, subconjunctival injection (subconjectval injection), subconjunctival injection (subconjuntival injection), sub-Tenon's injection, retrobulbar injection, or retroscleral delivery. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, and intralesionally, if topical treatment is desired. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, the given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus injections of the cells (e.g., over a period of no more than 3 days) or by continuous infusion of the cells. In some embodiments, administration of the cell dose or any additional therapy (e.g., lymphocyte removal therapy, intervention therapy, and/or combination therapy) is performed via an outpatient delivery.

For the treatment of a disease, the appropriate dosage may depend on the type of disease to be treated, the type of cell or recombinant receptor, the severity and course of the disease, previous therapies, the clinical history and response of the subject to the cell, and the discretion of the attending physician. In some embodiments, the composition and cells are suitably administered to the subject at one time or in a series of treatments.

After administration of the cells, the biological activity of the engineered cell population is measured in some embodiments, for example, by any of a number of known methods. Parameters evaluated include specific binding of engineered or native T cells or other immune cells to antigen in vivo (e.g., by imaging) or in vitro (e.g., by ELISA or flow cytometry). In certain embodiments, the ability of the engineered cells to destroy target cells may be measured using any suitable known method, such as toxicity assays described, for example, in Kochenderfer et al, J.Immunotherapy,32 (7): 689-702 (2009) and Herman et al, J.Immunologic methods,285 (1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and/or secretion of one or more cytokines, such as CD107a, ifnγ, IL-2, and TNF.

In some embodiments, the cell dose of the T cell therapy, such as a T cell therapy comprising cells engineered with a recombinant antigen receptor (e.g., CAR or TCR), is provided as a composition or formulation, such as a pharmaceutical composition or formulation. These compositions may be used according to the provided methods, such as for the prevention or treatment of diseases, conditions, and disorders.

In some embodiments, the T cell therapy, such as an engineered T cell (e.g., CAR or TCR T cell), is formulated with a pharmaceutically acceptable carrier. In some aspects, the selected portion of the vector is determined by the particular cell or agent and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of from about 0.0001% to about 2% by weight of the total composition. Vectors are described, for example, by Remington' sPharmaceutical Sciences, 16 th edition, osol, A.J. (1980). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to, buffers (such as phosphates, citrates and other organic acid salts), antioxidants (such as ascorbic acid and methionine), preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenols, butanols or benzyl alcohols, alkyl para-hydroxybenzoates such as methyl or propyl para-hydroxybenzoates, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins (such as serum albumin, gelatin or immunoglobulins), hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrins), chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt forming counterions such as sodium, metal complexes (e.g., zn-protein complexes), and/or nonionic surfactants such as PEG(s).

In some aspects, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example ,Remington:The Science and Practice of Pharmacy,Lippincott Williams&Wilkins;21st ed.(May 1,2005).

The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient for the particular indication, disease or condition being prevented or treated with the cell or agent, wherein the respective activities do not adversely affect each other. These active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunomycin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

In some embodiments, the pharmaceutical composition contains an amount of cells effective to treat or prevent the disease or disorder, such as a therapeutically effective or prophylactically effective amount. In some embodiments, the therapeutic or prophylactic efficacy is monitored by periodic assessment of the subject being treated. For repeated administrations of several days or longer, depending on the condition, the treatment is repeated until the desired inhibition of the disease symptoms occurs. However, other dosing regimens may be useful and determinable. The desired dose may be delivered by a single bolus administration of the composition, by multiple bolus administration of the composition, or by continuous infusion administration of the composition.

Cells may be administered using standard administration techniques, formulations, and/or devices. Formulations and devices, such as syringes and vials, for storing and administering the compositions are provided. In the case of cells, administration may be autologous or allogenic. For example, an immune response cell or ancestor may be obtained from one subject and administered to the same subject or a different compatible subject. The peripheral blood-derived immune response cells or progeny thereof (e.g., in vivo, ex vivo, or in vitro derived) may be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immune response cells) is administered, it is typically formulated in unit dose injectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration. In some embodiments, the agent or cell population is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the agent or cell population is administered to the subject by intravenous, intraperitoneal, or subcutaneous injection using peripheral system delivery.

In some embodiments, the composition is provided as a sterile liquid formulation (e.g., isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition), which may be buffered in some aspects to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, the liquid composition is somewhat more convenient to administer, in particular by injection. On the other hand, adhesive compositions may be formulated within an appropriate adhesive range to provide longer contact times with specific tissues. The liquid or viscous composition may comprise a carrier, which may be a solvent or dispersion medium, containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

Sterile injectable solutions may be prepared by incorporating the cells in a solvent, such as with a suitable carrier, diluent or excipient (such as sterile water, physiological saline, dextrose, or the like). The composition may also be lyophilized. The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gel or viscosity enhancing additives, preservatives, flavoring agents, coloring agents and the like, depending upon the route of administration and the desired formulation. In some aspects, reference may be made to standard text for preparing suitable formulations.

Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms is ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). Absorption of the injectable pharmaceutical form may be prolonged by the use of delayed absorbers (e.g., aluminum monostearate and gelatin).

Formulations for in vivo administration are typically sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

For the prevention or treatment of a disease, the appropriate dosage may depend on the type of disease to be treated, the type of one or more agents, the type of cell or recombinant receptor, the severity and course of the disease, the administration of the agents or cells for prophylactic or therapeutic purposes, previous therapies, the clinical history of the subject and the response to the agents or cells, and the discretion of the attendant physician. In some embodiments, the composition is suitably administered to the subject at one time or in a series of treatments.

In some cases, the cell therapy is administered as a single pharmaceutical composition comprising the cells. In some embodiments, a given dose is administered by a single bolus administration of the cell or agent. In some embodiments, it is administered by multiple bolus administration of the cell or agent (e.g., for a period of no more than 3 days) or by continuous infusion administration of the cell or agent.

In some embodiments, a cell dose is administered to a subject according to the provided combination therapy methods. In some embodiments, the size or time of the dose is determined as a function of the particular disease or condition of the subject. Based on the description provided, one can empirically determine the size or time of the dose for a particular disease.

In certain embodiments, the cells (or individual populations of cell subtypes) are isolated in a range of about 10 to about 1000 hundred million cells and/or in a cell amount per kilogram body weight of the subject, such as, for example, 10 to about 500 hundred million cells (e.g., about 500 ten thousand cells, about 2500 ten thousand cells, about 5 hundred million cells, about 10 hundred million cells, about 50 hundred million cells, about 200 hundred million cells, about 300 hundred million cells, about 400 hundred million cells, or a range defined by any two of the foregoing), 100 to about 500 hundred million cells (e.g., about 500 ten thousand cells, about 2500 ten thousand cells, about 5 cells, about 10 hundred million cells, about 50 cells, about 200 hundred million cells, about 300 hundred million cells, about 400 hundred million cells, or a range defined by any two of the foregoing), such as about 1000 to about 1000 cells (e.g., about 2000 tens of thousands of cells, about 3000 tens of thousands of cells, about 4000 tens of thousands of cells, about 6000 tens of thousands of cells, about 7000 tens of thousands of cells, about 8000 tens of thousands of cells, about 9000 tens of thousands of cells, about 100 hundreds of millions of cells, about 250 hundreds of millions of cells, about 500 hundreds of millions of cells, about 750 hundreds of millions of cells, about 900 hundreds of millions of cells, or a range defined by any two of the foregoing values), and in some cases about 1 billion cells to about 500 billion of cells (e.g., about 1.2 billion cells, about 2.5 billion cells, about 3.5 billion cells, about 4.5 billion cells, about 6.5 billion cells, about 8 billion cells, about 9 billion cells, about 30 billion cells, about 300 billion cells, about 450 billion cells), about 1000 tens of thousands to about 6 billion cells, about 1 billion to about 6 billion cells, about 1 to about 4.5 billion cells, about 1.5 billion to about 3 or about 4.5 billion cells, or any value between these values and/or the subject's weight per kilogram is administered to the subject. The dosage may vary depending on the particular nature of the disease or disorder and/or the patient and/or other treatment. In some embodiments, these values refer to the number of recombinant receptor expressing cells (e.g., CAR or TCR expressing cells), and in other embodiments they refer to the number of T cells or PBMCs or total cells administered.

In some embodiments, the cell therapy comprises administering a dose comprising an amount of cells from or from about 1x10⁵ to 1x10⁸ total recombinant receptor expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs), from or from about 5x10⁵ to 1x10⁷ total recombinant receptor expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs), or from about 1x10⁶ to 1x10⁷ total recombinant receptor expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs), each including an end value. In some embodiments, the cell therapy comprises administering a cell dose comprising at least or about at least 1x10⁵ total recombinant receptor expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs) of cells, such as at least or at least 1x10⁶, at least or about at least 1x10⁷, at least or about at least 1x10⁸ of the cells.

In some embodiments, for example, when the subject is a human, the dose comprises less than about 5x10⁸ total recombinant receptor (e.g., TCR or CAR) expressing cells, T cells, or Peripheral Blood Mononuclear Cells (PBMCs), e.g., in the range of about 1x10⁶ to 5x10⁸, such as 2x10⁶、5x10⁶、1x10⁷、5x10⁷、1x10⁸ or 5x10⁸ total of the cells or a range between any two of the foregoing values.

In some embodiments, the amount is related to the total number of cd3+ or cd8+, and in some cases also to recombinant receptor expressing (e.g., tcr+ or car+) cells. In some embodiments, the cell therapy comprises administering a dose comprising a number of cells from or from about 1x10⁵ to 1x10⁸ cd3+ or cd8+ total T cells or cd3+ or cd8+ recombinant receptor expressing cells from or from about 5x10⁵ to 1x10⁷ cd3+ or cd8+ total T cells or cd3+ or cd8+ recombinant receptor expressing cells, or from about 1x10⁶ to 1x10⁷ cd3+ or cd8+ total T cells or cd3+ or cd8+ recombinant receptor expressing cells (each including the endpoints). In some embodiments, the cell therapy comprises administering a dose comprising a number of cells from or from about 1x10⁵ to 1x10⁸ total cd3+/car+ or cd8+/car+ cells from or from about 5x10⁵ to 1x10⁷ total cd3+/car+ or cd8+/car+ cells, or from about 1x10⁶ to 1x10⁷ total cd3+/car+ or cd8+/car+ cells (each including the endpoints).

In some embodiments, the dose of genetically engineered cells comprises from or from about 1x10⁵ to 5x10⁸ total weight of total recombinant receptor expressing T cells, 1x10⁵ to 2.5x10⁸ total weight of total recombinant receptor expressing T cells, 1x10⁵ to 1x10⁸ total number of receptor-expressing T cells, 1x10⁵ to 5x10⁷ total number of receptor-expressing T cells, 1x10⁵ to 2.5x10⁷ total weight group receptor expressing T cells, 1x10⁵ to 1x10⁷ total weight group receptor expressing T cells, 1x10⁵ to 5x10⁶ total recombinant receptor expressing T cells, 1x10⁵ to 2.5x10⁶ total recombinant receptor expressing T cells, 1x10⁵ to 1x10⁶ total number of receptor-expressing T cells, 1x10⁶ to 5x10⁸ total number of receptor-expressing T cells, 1x10⁶ to 2.5x10⁸ total weight group receptor expressing T cells, 1x10⁶ to 1x10⁸ total weight group receptor expressing T cells, 1x10⁶ to 5x10⁷ total recombinant receptor expressing T cells, 1x10⁶ to 2.5x10⁷ total recombinant receptor expressing T cells, 1x10⁶ to 1x10⁷ total number of receptor-expressing T cells, 1x10⁶ to 5x10⁶ total number of receptor-expressing T cells, 1x10⁶ to 2.5x10⁶ total weight group receptor expressing T cells, 2.5x10⁶ to 5x10⁸ total weight group receptor expressing T cells, 2.5x10⁶ to 2.5x10⁸ total recombinant receptor expressing T cells, 2.5x10⁶ to 1x10⁸ total recombinant receptor expressing T cells, 2.5x10⁶ to 5x10⁷ total recombinant receptor expressing T cells, 2.5x10⁶ to 2.5x10⁷ total recombinant receptor expressing T cells, 2.5x10⁶ to 1x10⁷ total weight group receptor expressing T cells, 2.5x10⁶ to 5x10⁶ total weight group receptor expressing T cells, 5x10⁶ to 5x10⁸ total recombinant receptor expressing T cells, 5x10⁶ to 2.5x10⁸ total recombinant receptor expressing T cells, 5x10⁶ to 1x10⁸ total weight of total group receptor-expressing T cells, 5x10⁶ to 5x10⁷ total weight of total group receptor-expressing T cells, 5x10⁶ to 2.5x10⁷ total weight of receptor-expressing T cells, 5x10⁶ to 1x10⁷ total weight of receptor-expressing T cells, 1x10⁷ to 5x10⁸ total recombinant receptor expressing T cells, 1x10⁷ to 2.5x10⁸ total recombinant receptor expressing T cells, 1x10⁷ to 1x10⁸ total number of receptor-expressing T cells, 1x10⁷ to 5x10⁷ total number of receptor-expressing T cells, 1x10⁷ to 2.5x10⁷ total weight group receptor expressing T cells, 2.5x10⁷ to 5x10⁸ total weight group receptor expressing T cells, 2.5x10⁷ to 2.5x10⁸ total recombinant receptor expressing T cells, 2.5x10⁷ to 1x10⁸ total recombinant receptor expressing T cells, 2.5x10⁷ to 5x10⁷ total weight group receptor expressing T cells, 5x10⁷ to 5x10⁸ total weight group receptor expressing T cells, 5x10⁷ to 2.5x10⁸ total weight of receptor-expressing T cells, 5x10⁷ to 1x10⁸ total weight of receptor-expressing T cells, 1x10⁸ to 5x10⁸ total recombinant receptor expressing T cells, 1x10⁸ to 2.5x10⁸ total recombinant receptor expressing T cells or 2.5x10⁸ to 5x10⁸ total recombinant receptor expressing T cells.

In some embodiments, the dose of genetically engineered cells comprises at least or at least about 1x10⁵ TCR or CAR-expressing cells, at least or at least about 2.5x10⁵ TCR or CAR-expressing cells, at least or at least about 5x10⁵ TCR or CAR-expressing cells, at least or at least about 1x10⁶ TCR-or CAR-expressing cells, at least or at least about 2.5x10⁶ TCR or CAR-expressing cells, at least or at least about 5x10⁶ TCR or CAR-expressing cells, at least or at least about 1x10⁷ TCR or CAR-expressing cells, at least or at least about 2.5x10⁷ TCR or CAR-expressing cells, at least or at least about 5x10⁷ TCR or CAR-expressing cells, at least or at least about 1x10⁸ TCR or CAR-expressing cells, at least or at least about 2.5x10⁸ TCR or CAR-expressing cells, or at least about 5x10⁸ TCR or CAR-expressing cells.

In some embodiments, the dose of genetically engineered cells comprises from or from about 1500 to 10 million total recombinant receptor expressing T cells. In some embodiments, the dose of genetically engineered cells comprises from or from about 1500 to 6 million total recombinant receptor expressing T cells. In some embodiments, the dose of genetically engineered cells comprises from or from about 1.5 to 6 hundred million total recombinant receptor expressing T cells. In some embodiments, the dose of genetically engineered cells comprises from or from about 1.5 to 4.5 hundred million total recombinant receptor expressing T cells.

In some embodiments, the dose of genetically engineered cells comprises at least or at least about 1x10⁵ total weight receptor-expressing cells, at least or at least about 2.5x10⁵ total weight receptor-expressing cells, at least or at least about 5x10⁵ total weight receptor-expressing cells, at least or at least about 1x10⁶ total weight receptor-expressing cells, at least or at least about 2.5x10⁶ total weight receptor-expressing cells, at least or at least about 5x10⁶ total weight receptor-expressing cells, at least or at least about 1x10⁷ total weight receptor-expressing cells, at least or at least about 2.5x10⁷ total weight receptor-expressing cells, at least or at least about 5x10⁷ total weight receptor-expressing cells, at least or at least about 1x10⁸ total weight receptor-expressing cells, at least or at least about 2.5x10⁸ total weight receptor-expressing cells, or at least about 5x10⁸ total weight receptor-expressing cells.

In some embodiments, the cell therapy comprises administering a dose comprising an amount of cells from or from about 1x10⁵ to 1x10⁸ total packed receptor expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs), from or from about 5x10⁵ to 1x10⁷ total packed receptor expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs), or from about 1x10⁶ to 1x10⁷ total packed receptor expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs), each including an end value. In some embodiments, the cell therapy comprises administering a cell dose comprising at least or about at least 1x10⁵ total recombinant receptor expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs), such as a cell number of at least or at least 1x10⁶, at least or about at least 1x10⁷, at least or about at least 1x10⁸ of the cells.

In some embodiments, for example, when the subject is a human, the dose comprises less than about 5x10⁸ total recombinant receptor (e.g., CAR or TCR) expressing cells, T cells, or Peripheral Blood Mononuclear Cells (PBMCs), e.g., in the range of about 1x10⁶ to 5x10⁸, such as 2x10⁶、5x10⁶、1x10⁷、5x10⁷、1x10⁸ or 5x10⁸ total of the cells, or a range between any two of the foregoing values.

In some embodiments, the amount is related to the total number of cd3+ or cd8+, and in some cases also to recombinant receptor expressing (e.g., CAR or TCR expressing) cells. In some embodiments, the cell therapy comprises administering a dose comprising a number of cells from or from about 1x10⁵ to 1x10⁸ cd3+ or cd8+ total T cells or cd3+ or cd8+ recombinant receptor expressing cells from or from about 5x10⁵ to 1x10⁷ cd3+ or cd8+ total T cells or cd3+ or cd8+ recombinant receptor expressing cells, or from about 1x10⁶ to 1x10⁷ cd3+ or cd8+ total T cells or cd3+ or cd8+ recombinant receptor expressing cells (each including the endpoints). In some embodiments, the cell therapy comprises administering a dose comprising a number of cells from or from about 1x10⁵ to 1x10⁸ total cd3+/recombinant receptor+ or cd8+/recombinant receptor+ cells from or from about 5x10⁵ to 1x10⁷ total cd3+/recombinant receptor+ or cd8+/recombinant receptor+ cells, or from about 1x10⁶ to 1x10⁷ total cd3+/recombinant receptor+ or cd8+/recombinant receptor+ cells (each including the endpoints).

In some embodiments, the cell therapy comprises administering a composition comprising from or from about 1x10⁵ to 5x10⁸ total recombinant receptor expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs), from or from about 5x10⁵ to 1x10⁷ total recombinant receptor expressing cells, A dose of total T cells or total Peripheral Blood Mononuclear Cells (PBMCs) or cells from or from about 1x10⁶ to 1x10⁷ total numbers of recombinant receptor expressing cells, total T cells or total Peripheral Blood Mononuclear Cells (PBMCs), each including an end value. In some embodiments, the cell therapy comprises administering a composition comprising at least or at least about 1x10⁵ total recombinant receptor-expressing cells, total T cells, or total Peripheral Blood Mononuclear Cells (PBMCs) (such as at least or at least about 1x10⁶, at least or at least about 1x10⁷, at least or at least about 1x10⁸ of said cells). In some embodiments, the amount is related to the total number of cd3+ or cd8+, and in some cases also to recombinant receptor expressing (e.g., car+ or tcr+) cells. In some embodiments, the cell therapy comprises administering a dose comprising a number of cells from or from about 1x10⁵ to 5x10⁸ cd3+ or cd8+ total T cells or cd3+ or cd8+ recombinant receptor expressing cells from or from about 5x10⁵ to 1x10⁷ cd3+ or cd8+ total T cells or cd3+ or cd8+ recombinant receptor expressing cells, or from about 1x10⁶ to 1x10⁷ cd3+ or cd8+ total T cells or cd3+ or cd8+ recombinant receptor expressing cells (each including the endpoints). In some embodiments, the cell therapy comprises administering a dose comprising a number of cells from or from about 1x10⁵ to 5x10⁸ total cd3+/recombinant receptor+ or cd8+/recombinant receptor+ cells from or from about 5x10⁵ to 1x10⁷ total cd3+/recombinant receptor+ or cd8+/recombinant receptor+ cells, or from about 1x10⁶ to 1x10⁷ total cd3+/recombinant receptor+ or cd8+/recombinant receptor+ cells (each including the endpoints).

In some embodiments, the dose of T cells comprises cd4+ T cells, cd8+ T cells, or cd4+ and cd8+ T cells.

In some embodiments, for example, when the subject is a human, the dose of cd8+ T cells (including in the dose comprising cd4+ and cd8+ T cells) comprises between about 1x10⁶ and 5x10⁸ total recombinant receptor (e.g., CAR or TCR) expressing cd8+ cells, e.g., in the range of about 5x10⁶ to 1x10⁸ of the cells, the total number of cells being 1x10⁷、2.5x10⁷、5x10⁷、7.5x10⁷、1x10⁸ or 5x10⁸, or any two of the above values. In some embodiments, multiple doses are administered to the patient, and each dose or total dose may be within the range of any of the above values. In some embodiments, the cell dose comprises administering cd8+ T cells expressing from or from about 1x10⁷ to 0.75x10⁸ total recombinant receptors, cd8+ T cells expressing from 1x10⁷ to 2.5x10⁷ total recombinant receptors, cd8+ T cells expressing from or from about 1x10⁷ to 0.75x10⁸ total recombinant receptors (each including the endpoints). In some embodiments, the cell dose comprises administering cd8+ T cells expressing at or about 1x10⁷、2.5x10⁷、5x10⁷、7.5x10⁷、1x10⁸ or 5x10⁸ total recombinant receptors.

In some embodiments, the cell dose (e.g., recombinant receptor expressing T cells) is administered to the subject only once as a single dose or over a period of two weeks, one month, three months, six months, 1 year, or more.

In some embodiments, the cell therapy comprises administering a dose comprising an amount of cells of at least or at least about or at or about 0.1x10⁶ cells/kg of the subject body weight, 0.2x10⁶ cells/kg, 0.3x10⁶ cells/kg, 0.4x10⁶ cells/kg, 0.5x10⁶ cells/kg, 1x10⁶ cells/kg, 2.0x10⁶ cells/kg, 3x10⁶ cells/kg, or 5x10⁶ cells/kg.

In some embodiments, the cell therapy comprises administering a composition comprising or between about 0.1x10⁶ cells/kg body weight of a subject and 1.0x10⁷ cells/kg body weight of a subject, between or between about 0.5x10⁶ cells/kg and 5x10⁶ cells/kg, Between or about 0.5X10⁶ cells/kg and 3X10⁶ cells/kg, between or about 0.5X10⁶ cells/kg and 2X10⁶ cells/kg, Between or about 0.5x10⁶ cells/kg and 1x10⁶ cells/kg, between or about 1.0x10⁶ cells/kg body weight and 5x10⁶ cells/kg body weight, Between or in about 1.0x10⁶ cells/kg and 3x10⁶ cells/kg, between or in about 1.0x10⁶ cells/kg and 2x10⁶ cells/kg, At or between about 2.0x10⁶ cells/kg subject body weight and 5x10⁶ cells/kg subject body weight, Cell doses of cells in amounts of between or about 2.0x10⁶ cells/kg and 3x10⁶ cells/kg or between or about 3.0x10⁶ cells/kg subject body weight and 5x10⁶ cells/kg subject body weight, each inclusive.

In some embodiments, the cell dose is comprised between or about 2x10⁵ cells/kg and or about 2x10⁶ cells/kg, such as between or about 4x10⁵ cells/kg and or about 1x10⁶ cells/kg or between or about 6x10⁵ cells/kg and or about 8x10⁵ cells/kg. In some embodiments, the cell dose comprises no more than 2x10⁵ cells (e.g., antigen-expressing such as CAR-expressing cells or TCR-expressing cells) per kilogram of the subject's body weight (cells/kg), such as no more than or about 3x10⁵ cells/kg, no more than or about 4x10⁵ cells/kg, No more than or about 5x10⁵ cells/kg, no more than or about 6x10⁵ cells/kg, no more than or about 7x10⁵ cells/kg, no more than or about 8x10⁵ cells/kg, no more than or about 9x10⁵ cells/kg, no more than or about 1x10⁶ cells/kg, or no more than or about 2x10⁶ cells/kg. In some embodiments, the cell dose comprises at least or at least about or about 2x10⁵ of the cells (e.g., antigen-expressing cells such as CAR-expressing cells or TCR-expressing cells) per kilogram of the subject's body weight (cells/kg), such as at least or at least about or about 3x10⁵ cells/kg, at least or at least about or about 4x10⁵ cells/kg, at least or at least about or about 5x10⁵ cells/kg, at least or at least about or about 6x10⁵ cells/kg at least or at least about or about 7x10⁵ cells/kg, at least or at least about or about 8x10⁵ cells/kg, At least or at least about or at or about 9x10⁵ cells/kg, at least or at least about or at or about 1x10⁶ cells/kg, or at least about or at or about 2x10⁶ cells/kg.

In the case of adoptive cell therapy, administration of a given "dose" of cells encompasses administration of a given amount or number of cells as a single composition and/or single uninterrupted administration (e.g., as a single injection or continuous infusion), and also encompasses administration of a given amount or number of cells provided as divided doses in multiple separate compositions or infusions for a particular period of time (no more than 3 days). Thus, in some cases, the dose is administered in a single administration or a specific number of cells to be administered or started at a single time point are administered consecutively. In some cases, however, the dose is administered as multiple injections or infusions over a period of no more than three days, such as once a day for three or two days, or multiple infusions over a period of a day.

Thus, in some aspects, the cell dose is administered in a single pharmaceutical composition. In some embodiments, the cell dose is administered in a plurality of compositions that generally contain the cell dose.

The term "divided dose" refers to a dose that is divided so as to be administered over a period of more than one day. This type of dose is encompassed in the present method and is considered a single dose. In some embodiments, a divided dose of cells is administered in a plurality of compositions generally comprising the dose of cells over a period of no more than three days.

Thus, the cell dose may be administered as a divided dose. For example, in some embodiments, the dose may be administered to the subject over 2 days or over 3 days. An exemplary method of split dosing includes administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the dose may be administered on the first day and the remaining 67% on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the divided doses should not be dispersed for more than 3 days.

In some embodiments, the cell dose is generally large enough to effectively reduce disease burden.

In some embodiments, the cells are administered at a desired dose, which in some aspects includes a desired dose or number of cells or cell types and/or a desired ratio of cell types. Thus, in some embodiments, the cell dose is based on the total number of cells (or number per kg body weight) and a desired ratio of individual populations or subtypes, such as the cd4+ to cd8+ ratio. In some embodiments, the cell dose is based on the desired total number of cells (or number per kg body weight) of the individual population or individual cell type. In some embodiments, the dose is based on a combination of such features, such as a desired total cell number, a desired ratio, and a desired total number of cells in an individual population.

In some embodiments, the population or subtype of cells (such as CD8⁺ and CD4⁺ T cells) is administered at a desired dose of total cells (such as a desired dose of T cells) or within tolerance differences of the desired dose. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit body weight (e.g., cells/kg) of the subject to whom the cells are administered. In some aspects, the required dose is at or above a minimum cell number or minimum cell number per unit body weight. In some aspects, in total cells administered at a desired dose, the individual populations or subtypes are present at or near a desired output ratio (such as the CD4⁺ to CD8⁺ ratio), e.g., within a certain tolerance or error range of the ratio.

In some embodiments, the cells are administered within tolerance differences in or at the required dose of one or more of the cell individual populations or subtypes, such as the required dose of cd4+ cells and/or the required dose of cd8+ cells. In some aspects, the desired dose is a desired number of cells of the subtype or population or a desired number of cells per unit body weight (e.g., cells/kg) of the subject to whom the cells are administered. In some aspects, the required dose is at or above the minimum number of cells of the population or subtype or the minimum number of cells of the population or subtype per unit body weight.

Thus, in some embodiments, the dose is based on a desired fixed dose and a desired ratio of total cells, and/or based on a desired fixed dose of one or more (e.g., each) of the individual subtypes or subpopulations. Thus, in some embodiments, the dose is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4⁺ to CD8⁺ cells, and/or a desired fixed or minimum dose based on CD4⁺ and/or CD8⁺ cells.

In some embodiments, the cells are administered at or within the tolerance range of a desired output ratio of multiple cell populations or subtypes (such as cd4+ and cd8+ cells or subtypes). In some aspects, the desired ratio may be a particular ratio or may be a range of ratios. For example, in some embodiments, the desired ratio (e.g., the ratio of CD4⁺ to CD8⁺ cells) is between or about 1:5 and or about 5:1 (or above about 1:5 and below about 5:1), or between or about 1:3 and or about 3:1 (or above about 1:3 and below about 3:1), such as between or about 2:1 and or about 1:5 (or above about 1:5 and below about 2:1, such as or about 5:1、4.5:1、4:1、3.5:1、3:1、2.5:1、2:1、1.9:1、1.8:1、1.7:1、1.6:1、1.5:1、1.4:1、1.3:1、1.2:1、1.1:1、1:1、1:1.1、1:1.2、1:1.3、1:1.4、1:1.5、1:1.6、1:1.7、1:1.8、1:1.9、1:2、1:2.5、1:3、1:3.5、1:4、1:4.5 or 1:5). In some aspects, the tolerance difference is within a range of about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

In particular embodiments, the number and/or concentration of cells refers to the number of recombinant receptor (e.g., CAR or TCR) expressing cells. In other embodiments, the number and/or concentration of cells refers to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells administered.

In some aspects, the size of the dose is determined according to one or more criteria, such as the likelihood or incidence of a subject's response to a previous treatment (e.g., chemotherapy), the subject's disease burden (such as tumor burden, volume, size, or extent, range, or type of metastasis), the stage, and/or the subject's occurrence of a toxic outcome (e.g., CRS, macrophage activation syndrome, oncolytic syndrome, neurotoxicity, and/or host immune response to the administered cells and/or recombinant receptors).

In some embodiments, administration of a DGK inhibitor in combination with the cells is capable of significantly increasing expansion or proliferation of the cells, and thus lower cell doses may be administered to a subject. In some cases, the provided methods allow for administration of lower doses of the cells to achieve the same or better therapeutic effect as in methods of administering cell therapies that do not administer the DGK inhibitor, such as at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold lower than in methods of administering cell therapies that do not administer the DGK inhibitor.

In some embodiments, for example, the dose contains between or in the range of about 5.0x10⁶ and 2.25x10⁷, 5.0x10⁶ and 2.0x10⁷, Between 5.0x10⁶ and 1.5x10⁷, between 5.0x10⁶ and 1.0x10⁷, Between 5.0x10⁶ and 7.5x10⁶, between 7.5x10⁶ and 2.25x10⁷, Between 7.5x10⁶ and 2.0x10⁷, between 7.5x10⁶ and 1.5x10⁷, Between 7.5x10⁶ and 1.0x10⁷, between 1.0x10⁷ and 2.25x10⁷, Between 1.0x10⁷ and 2.0x10⁷, between 1.0x10⁷ and 1.5x10⁷, between 1.5x10⁷ and 2.25x10⁷, between 1.5x10⁷ and 2.0x10⁷, Between 2.0x10⁷ and 2.25x10⁷. in some embodiments, the cell dose contains a number of cells between at least or at least about 5x10⁶、6x10⁶、7x10⁶、8x10⁶、9x10⁶、10x10⁶ and about 15x10⁶ recombinant receptor expressing cells (such as cd8+ recombinant receptor expressing cells). In some embodiments, the dose (such as the target number of cells) refers to the total number of recombinant receptor expressing cells in the composition administered.

In some embodiments, for example, the low dose contains less than about 5x10⁶ cells, recombinant receptor (e.g., TCR or CAR) expressing cells, T cells, and/or PBMCs per kilogram body weight of the subject, such as less than about 4.5x10⁶、4x10⁶、3.5x10⁶、3x10⁶、2.5x10⁶、2x10⁶、1.5x10⁶、1x10⁶、5x10⁵、2.5x10⁵ or 1x10⁵ of the cells per kilogram body weight of the subject. In some embodiments, the low dose contains less than about 1x10⁵、2x10⁵、5x10⁵ or 1x10⁶ of the cells per kilogram of subject body weight, or a range between any two of the above values. In some embodiments, these values refer to the number of recombinant receptor expressing cells, and in other embodiments they refer to the number of T cells or PBMC or total cells administered.

In some embodiments, the subject receives multiple doses, e.g., two or more doses of the cells or multiple consecutive doses. In some embodiments, two doses are administered to a subject. In some embodiments, the subject receives a continuous dose, e.g., the second dose is administered about 4,5, 6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the first dose. In some embodiments, a plurality of consecutive doses is administered after the first dose, such that additional one or more doses are administered after administration of the consecutive doses. In some aspects, the number of cells administered to the subject in the additional dose is the same or similar to that in the first dose and/or the consecutive doses. In some embodiments, the additional dose or doses are greater than the previous dose. In some embodiments, one or more subsequent cell doses may be administered to the subject. In some embodiments, the subsequent cell dose is administered greater than or greater than about 7 days, 14 days, 21 days, 28 days, or 35 days after the beginning of administration of the first cell dose. The subsequent cell dose may be greater than, about equal to, or less than the first dose. In some embodiments, the T cell therapy (such as administration of the first and/or second cell dose) may be repeatedly administered.

In some embodiments, the beginning of administration of the cell therapy (e.g., the first dose of the cell dose or fractionated cell dose) is administered before (prior to), concurrently with, or after (subsequent to or subsequent to) administration of the dgkα and/or dgkζ inhibitor. In some embodiments, the onset of administration of the cell therapy is correlated with the onset of administration of the dgkα and/or dgkζ inhibitor, as described in any of the embodiments described in section I-B-2.

In some embodiments, the methods involve evaluating one or more functions of T cells, such as expansion or persistence of cells, e.g., as determined by levels or amounts in blood, or other phenotypes or desired results, of a sample from the subject after administration of a cell dose of the T cell therapy (e.g., adoptive T cell therapy), but prior to administration of the DGK inhibitor, as described herein, e.g., such as those described in section III. In some embodiments, the methods involve evaluating expression of one or more depletion markers in a sample from the subject after administration of a cell dose of the T cell therapy (e.g., adoptive T cell therapy), but prior to administration of the DGK inhibitor. Various parameters for determining or evaluating the combination therapy regimen are described in section III.

B. Administration of DGK alpha and/or DGK zeta inhibitors

In some aspects, the methods provided herein comprise combination therapies by administering a DGK inhibitor in combination with a cell therapy (e.g., T cell therapy) to a subject suffering from a disease or disorder, such as any of the diseases or disorders described above. The DGKi can be administered prior to, concurrently with, or after administration of a cell therapy (e.g., T cell therapy). In some aspects, the methods provided herein comprise administering a cell therapy (e.g., T cell therapy) to a subject suffering from a disease or disorder, wherein the subject has previously been administered a DGK inhibitor. In some embodiments, the DGK inhibitor and a cell therapy (e.g., T cell therapy) are administered to the subject simultaneously.

In some embodiments, the DGK inhibitor is a dgkα and/or dgkζ inhibitor. In some embodiments, the inhibitor of dgkα and/or dgkζ is a dgkα inhibitor. In certain embodiments, the inhibitor of dgkα and/or dgkζ is a dgkζ inhibitor. In certain embodiments, inhibitors of dgkα and/or dgkζ inhibit both enzymes. The level of enzyme inhibition can be measured as described further herein. In certain embodiments, the inhibitor of dgkα and/or dgkζ is not a significant inhibitor of other DGK enzymes.

In certain embodiments, the dgkα and/or dgkζ inhibitors increase immune response, such as by increasing T cell activity. For example, dgkα and/or dgkζ inhibitors may increase primary T cell signaling, as demonstrated, for example, by an increase in pERK/pPKC signaling, which may be measured as described further herein. In some embodiments, the DGK alpha and/or DGK zeta inhibitor has one or more of (i) it reduces the threshold of antigen stimulation, (ii) increases CTL effector function, and (iii) enhances tumor cell killing. When dgkα and/or dgkζ inhibitors enhance tumor killing, this activity may depend on cd8+ T cells, as shown for example in the CT26 animal model. When dgkα and/or dgkζ inhibitors enhance tumor killing, the activity may depend on NK cells, as shown for example in CT26 animal models. When dgkα and/or dgkζ inhibitors enhance tumor killing, this activity may depend on cd8+ T cells and NK cells, as shown for example in the CT26 animal model. When dgkα and/or dgkζ inhibitors enhance tumor killing, this activity may be enhanced by CD4 cell clearance, for example, in a CT-26 animal model. In certain embodiments, the DGK alpha and/or DGK zeta inhibitors enhance AH1+ tetrameric antigen presentation in a CT-26 animal model. The dgkα and/or dgkζ inhibitors more preferably include one or more of the above-described properties, and may include all of these properties.

1. Exemplary inhibitors of DGK alpha and/or DGK zeta enzyme Activity

Exemplary compounds described herein (such as compounds of formula I and pharmaceutically acceptable salts thereof) are described in WO 2020/006018 and WO 2020/006016, the contents of both of which are specifically incorporated herein by reference. Exemplary compounds (such as the compounds of formula II and pharmaceutically acceptable salts thereof described herein) are as described in PCT/US2020/048070, the contents of which are specifically incorporated herein by reference.

In some embodiments, a combination therapy of T cell therapy and DGK inhibitor is provided that involves the administration or use of a dgkα and/or dgkζ inhibitor, which is a compound of formula (I):

Or a pharmaceutically acceptable salt thereof, wherein:

R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, C_3–4 cycloalkyl substituted with 0 to 4R_1a, C_1–3 alkoxy substituted with 0 to 4R_1a, -NR_aR_a、–S(O)_nR_e or-P (O) R_eR_e;

Each R_1a is independently F, cl, -CN, -OH, -OCH₃, or-NR_aR_a;

Each R_a is independently H or C_1–3 alkyl;

each R_e is independently C_3–4 cycloalkyl or C_1–3 alkyl optionally substituted with 0 to 4R_1a;

R₂ is H, C_1–3 alkyl optionally substituted with 0 to 4R_2a, or C_3–4 cycloalkyl substituted with 0 to 4R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), C_3–4 cycloalkyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₃ is H, F, cl, br, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_3–4 cycloalkyl, C_3–4 fluorocycloalkyl or-NO₂;

R₄ is –CH₂R_4a、–CH₂CH₂R_4a、–CH₂CHR_4aR_4d、–CHR_4aR_4b or-CR_4aR_4bR_4c;

R_4a and R_4b are independently:

(i) C_1–6 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy, -NR_aR_a、–S(O)₂R_e, or-NR_aS(O)₂R_e;

(ii) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH)_1–3O(C_1–3 alkyl), C_1–3 Fluoroalkoxy 、–O(CH)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl, -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–6 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or

(Iii) C_1–4 alkyl substituted with one cyclic group selected from the group consisting of C_3–6 cycloalkyl, heterocyclyl, aryl and heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from the group consisting of F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–6 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f;

each R_f is independently F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–6 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

R_4d is-OCH₃;

Each R_c is independently H or C_1–2 alkyl;

R_d is phenyl substituted with 0 to 1 substituents selected from F, cl, -CN, -CH₃, and-OCH₃;

Each R₅ is independently-CN, C_1–6 alkyl substituted with 0 to 4R_g, C_2–4 alkenyl substituted with 0 to 4R_g, C_2–4 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 4R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 4R_g, - (CH₂)_1–2 (heterocyclyl substituted with 0 to 4R_g)), a compound of formula (i), - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

Each R_g is independently F, cl, -CN, -OH, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -O (CH₂)_1–2O(C_1–2 alkyl), or-NR_cR_c;

m is 0, 1,2 or 3, and

N is 0, 1 or 2.

In some embodiments, a combination therapy of T cell therapy and DGK inhibitor is provided that involves the administration or use of a dgkα and/or dgkζ inhibitor, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, cyclopropyl substituted with 0 to 3R_1a, C_1–3 alkoxy substituted with 0 to 3R_1a, -NR_aR_a、–S(O)_nCH₃ or-P (O) (CH₃)₂;

Each R_1a is independently F, cl or-CN;

Each R_a is independently H or C_1–3 alkyl;

R₂ is H or C_1–2 alkyl substituted with 0 to 2R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), cyclopropyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₃ is H, F, cl, br, -CN, C_1–2 alkyl, -CF₃, cyclopropyl or-NO₂;

R_4a and R_4b are independently:

(i) C_1–4 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy and-NR_aR_a;

(ii) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, -CH₂OH、–(CH₂)_1–2O(C_1–2 alkyl), 0 to 4, C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy 、–O(CH)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–4 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), and, cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or

(Iii) C_1–3 alkyl substituted with one cyclic group selected from the group consisting of C_3–6 cycloalkyl, heterocyclyl, phenyl and heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from the group consisting of F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–4 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f;

each R_f is independently F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–4 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

And each R₅ is independently selected from-CN, C_1–5 alkyl substituted with 0 to 4R_g, C_2–3 alkenyl substituted with 0 to 4R_g, c_2–3 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 3R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 3R_g, - (CH₂)_1–2 (heterocyclyl substituted with 0 to 4R_g)), a compound of formula (i), - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl).

In some embodiments, a combination therapy of T cell therapy and DGK inhibitor is provided that involves the administration or use of a dgkα and/or dgkζ inhibitor, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof, having the structure:

Wherein:

R₁ is-CN;

R₂ is-CH₃;

R₃ is H, F or-CN;

r₄ is:

In some embodiments, a combination therapy of T cell therapy and DGK inhibitor is provided that involves the administration or use of a dgkα and/or dgkζ inhibitor, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof, having the following structure or formula (or isomers thereof):

1- (bis (4-fluorophenyl) methyl) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl) piperazine-2-carboxylic acid methyl ester

4- ((2 R,5 s) -4- (bis (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -6-bromo-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridine-3-carbonitrile

(R) -8- (4- (bis (4-fluorophenyl) methyl) -3-methylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2, 7-dicarboxylic acid carbonitrile

8- [ (2S, 5R) -4- [ (4-chlorophenyl) (5-methylpyridin-2-yl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

4- [ (2 S,5 r) -4- [ (4-chlorophenyl) (4-fluorophenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -6-methoxy-1-methyl-1, 2-dihydro-1, 5-naphthyridin-2-one

8- [ (2S, 5R) -4- { [2- (difluoromethyl) -4-fluorophenyl ] methyl } -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

8- [ (2S, 5R) -4- [ (4-fluorophenyl) (4-methylphenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

8- [ (2S, 5R) -4- [1- (2, 6-difluorophenyl) ethyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

8- ((3R) -4- ((4-chlorophenyl) (5-fluoropyridin-2-yl) methyl) -3-methylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2, 7-carbonitrile

8- (4- (Bis (4-fluorophenyl) methyl) piperazin-1-yl) -5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

And

8- [ (2S, 5R) -4- [ bis (4-methylphenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

In some embodiments, a combination therapy of T cell therapy and DGK inhibitor is provided that involves the administration or use of a dgkα and/or dgkζ inhibitor, which is of formula (II):

Or a salt thereof, wherein:

R₁ is H, F, cl, br, -CN, -OH, C_1–3 alkyl substituted with 0 to 4R_1a, C_3–4 cycloalkyl substituted with 0 to 4R_1a, C_1–3 alkoxy substituted with 0 to 4R_1a, -NR_aR_a、–S(O)_nR_e, or-P (O) R_eR_e;

Each R_1a is independently F, cl, -CN, -OH, -OCH₃, or-NR_aR_a;

Each R_a is independently H or C_1–3 alkyl;

Each R_e is independently C_3–4 cycloalkyl or C_1–3 alkyl substituted with 0 to 4R_1a;

R₂ is H, C_1–3 alkyl substituted with 0 to 4R_2a, or C_3–4 cycloalkyl substituted with 0 to 4R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), C_3–4 cycloalkyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₄ is –CH₂R_4a、–CH₂CH₂R_4a、–CH₂CHR_4aR_4d、–CHR_4aR_4b or-CR_4aR_4bR_4c;

R_4a and R_4b are independently:

(i) -CN or C_1–6 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy, -NR_aR_a、–S(O)₂R_e, or-NR_aS(O)₂R_e;

(ii) C_3–8 carbocyclyl, 4-to 10-membered heterocyclyl, phenyl or 5-to 10-membered heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, through 0 to 4, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy, -O (C_1–4 hydroxyalkyl), a, -O (CR_xR_x)_1–3O(C_1–3 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–CH₂NR_aR_a、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -CR_xR_x)_0–2NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl, - (CR_xR_x)_1–2(C_3–4 cycloalkyl), - (CR_xR_x)_1–2 (morpholinyl), - (CR_xR_x)_1–2 (difluoromethyl), - (CR_xR_x)_1–2 (dimethylmorpholinyl)) and the like, - (CR_xR_x)_1–2 (oxaazabicyclo [2.2.1] heptane), (CR_xR_x)_1–2 (oxaazaspiro [3.3] heptane), - (CR_xR_x)_1–2 (methylpiperazinonyl), - (CR_xR_x)_1–2 (acetylpiperazinyl)) and the like, - (CR_xR_x)_1–2 (piperidinyl), - (CR_xR_x)_1–2 (difluoropiperidinyl), - (CR_xR_x)_1–2 (methoxypiperidinyl), - (CR_xR_x)_1–2 (hydroxypiperidinyl)), and the like, -O (CR_xR_x)_0–2(C_3–6 cycloalkyl), -O (CR_xR_x)_0–2 (methylcyclopropyl), -O (CR_xR_x)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CR_xR_x)_0–2 (oxetanyl)), -O (CR_xR_x)_0–2 (methylazetidinyl)), -O (CR_xR_x)_0–2 (tetrahydropyranyl) -O (CR_xR_x)_1–2 (morpholinyl)), -O (CR_xR_x)_0–2 (thiazolyl), Cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, dioxolanyl, pyrrolidone group and R_d, or

(Iii) C_1–4 alkyl substituted with one cyclic group selected from C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, mono-or bicyclic aryl or 5-to 10-membered heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–6 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f;

each R_f is independently F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–6 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

R_4d is-OCH₃;

Each R_c is independently H or C_1–2 alkyl;

R_d is phenyl substituted with 0 to 1 substituents selected from F, cl, -CN, -CH₃, and-OCH₃;

Each R₅ is independently-CN, C_1–6 alkyl substituted with 0 to 4R_g, C_2–4 alkenyl substituted with 0 to 4R_g, C_2–4 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 4R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 4R_g, - (CH₂)_1–2 (4-to 10-membered heterocyclyl substituted with 0 to 4R_g), and, - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

Each R_g is independently F, cl, -CN, -OH, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -O (CH₂)_1–2O(C_1–2 alkyl), or-NR_cR_c;

m is 0, 1,2 or 3, and

N is 0, 1 or 2.

In some embodiments, a combination therapy of T cell therapy and DGK inhibitor is provided that involves the administration or use of a dgkα and/or dgkζ inhibitor, which is a compound of formula (II):

Or a salt thereof, wherein:

R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, C_3–4 cycloalkyl substituted with 0 to 4R_1a, C_1–3 alkoxy substituted with 0 to 4R_1a, -NR_aR_a、–S(O)_nR_e or-P (O) R_eR_e;

Each R_1a is independently F, cl, -CN, -OH, -OCH₃, or-NR_aR_a;

Each R_a is independently H or C_1–3 alkyl;

Each R_e is independently C_3–4 cycloalkyl or C_1–3 alkyl substituted with 0 to 4R_1a;

R₂ is H, C_1–3 alkyl substituted with 0 to 4R_2a, or C_3–4 cycloalkyl substituted with 0 to 4R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), C_3–4 cycloalkyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₄ is –CH₂R_4a、–CH₂CH₂R_4a、–CH₂CHR_4aR_4d、–CHR_4aR_4b or-CR_4aR_4bR_4c;

R_4a and R_4b are independently:

(i) C_1–6 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy, -NR_aR_a、–S(O)₂R_e, or-NR_aS(O)₂R_e;

(ii) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH₂)_1–3O(C_1–3 alkyl), C_1–3 Fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl, -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–6 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or

(Iii) C_1–4 alkyl substituted with one cyclic group selected from the group consisting of C_3–6 cycloalkyl, heterocyclyl, aryl and heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from the group consisting of F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–6 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f;

each R_f is independently F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–6 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

R_4d is-OCH₃;

Each R_c is independently H or C_1–2 alkyl;

r_d is phenyl substituted with 0 or 1 substituent selected from F, cl, -CN, -CH₃, and-OCH₃;

Each R₅ is independently-CN, C_1–6 alkyl substituted with 0 to 4R_g, C_2–4 alkenyl substituted with 0 to 4R_g, C_2–4 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 4R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 4R_g, - (CH₂)_1–2 (4-to 10-membered heterocyclyl substituted with 0 to 4R_g), and, - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

Each R_g is independently F, cl, -CN, -OH, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -O (CH₂)_1–2O(C_1–2 alkyl), or-NR_cR_c;

m is 0, 1,2 or 3, and

N is 0, 1 or 2.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₁ is H, F, cl, br, -CN, -OH, C_1–3 alkyl substituted with 0 to 4R_1a, cyclopropyl substituted with 0 to 3R_1a, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, C_1–3 alkoxy substituted with 0 to 3R_1a, -NR_aR_a、–S(O)_nCH₃ or-P (O) (CH₃)₂;R₂ is H or C_1–2 alkyl optionally substituted with 0 to 2R_2a; each R_2a is independently F, Cl, -CN, -OH, -O (C_1–2 alkyl), cyclopropyl, c_3–4 alkenyl or C_3–4 alkynyl, R_4a and R_4b are independently (i) -CN or from 0 to 4 are independently selected from F, Cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy and-NR_aR_a substituted C_1–4 alkyl, (ii) C_3–6 carbocyclyl, 4-to 10-membered heterocyclyl, phenyl or 5-to 10-membered heteroaryl, each of 0 to 4 independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1-2 hydroxyalkyl, -CH₂NR_aR_a、–(CH₂)_1–2O(C_1–2 alkyl), - (CH₂)_1–2NR_xC(O)O(C_1-2 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CR_xR_x)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy 、–O(CH₂)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl), -CH₂)_1–2(C_3–4 cycloalkyl, -CR_xR_x (morpholinyl), -CR_xR_x (difluoromethyl morpholinyl), -CR_xR_x (dimethylmorpholinyl), -CR_xR_x (oxaazabicyclo [2.2.1] heptanyl), -CR_xR_x (oxaazaspiro [3.3] heptanyl), and-, -CR_xR_x (methylpiperazinonyl), -CR_xR_x (acetylpiperazinyl), -CR_xR_x (piperidinyl), -CR_xR_x (difluoropiperidinyl), a compound of formula (I), -CR_xR_x (methoxypiperidinyl), -CR_xR_x (hydroxypiperidinyl) -O (CH₂)_0–2(C_3–4 cycloalkyl), -O (CH₂)_0–2 (methylcyclopropyl), -a catalyst, and a process for preparing the same, -O (CH₂)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CH₂)_0–2 (oxetanyl), -O (CH₂)_0–2 (methylazetidinyl), -O (CH₂)_1–2 (morpholinyl)) -O (CH₂)_0–2 (tetrahydropyranyl), -O (CH₂)_0–2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, dioxolanyl, pyrrolidonyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or (iii) substituted with one substituent selected from C_3–6 cycloalkyl, C_1–3 alkyl substituted with 4-to 10-membered heterocyclyl, phenyl and heteroaryl cyclic groups, said cyclic groups being independently selected from F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, 0 to 3, C_1–3 alkoxy, C_1–2 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–4 cycloalkyl, or R_4a and R_4b together with the carbon atom to which they are attached form C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f, each R_f is independently F, Cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl and bicyclic heteroaryl, each cyclic group being independently selected from F, cl, br, -OH, -CN, C_1–4 alkyl, 0 to 3 per ring group, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy and-NR_cR_c, R_4c is C_1–4 alkyl or C_3–6 cycloalkyl, each independently selected from F, 0 to 4, and a pharmaceutically acceptable salt thereof, Cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN, each R₅ is independently-CN, C_1–5 alkyl substituted with 0 to 4R_g, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, C_2–3 alkenyl substituted with 0to 4R_g, C_2–3 alkynyl substituted with 0to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, phenyl substituted with 0 to 3R_g, oxadiazolyl substituted with 0 to 3R_g, Pyridinyl substituted with 0 to 3R_g, - (CH₂)_1–2 (4-to 10-membered heterocyclyl substituted with 0 to 4R_g), - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), alkyl, - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

each R_x is independently H or-CH₃, and m is 1,2, or 3.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, cyclopropyl substituted with 0 to 3R_1a, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, C_1–3 alkoxy substituted with 0 to 3R_1a, -NR_aR_a、–S(O)_nCH₃, or-P (O) (CH₃)₂; each R_1a is independently F, Cl or-CN, each R_a is independently H or C_1–3 alkyl, R₂ is H or C_1–2 alkyl substituted with 0 to 2R_2a, each R_2a is independently F, Cl, -CN, -OH, -O (C_1–2 alkyl), cyclopropyl, c_3–4 alkenyl or C_3–4 alkynyl, R_4a and R_4b are independently (i) from 0 to 4 are independently selected from F, Cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy and-NR_aR_a (ii) C_1–4 alkyl substituted by C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, phenyl or 5-to 10-membered heteroaryl, each of 0 to 4 independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, -CH₂OH、–(CH₂)_1–2O(C_1–2 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH₂)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–4 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), and, Cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, and R_d, or (iii) C_1–3 alkyl substituted with one cyclic group selected from C_3–6 cycloalkyl, heterocyclyl, phenyl, and heteroaryl, said cyclic group being substituted with 0 to 3 cyclic groups independently selected from F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–4 cycloalkyl, or R_4a and R_4b together with the carbon atom to which they are attached form C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f, each R_f is independently F, Cl, br, -OH, -CN, = O, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl and bicyclic heteroaryl, each cyclic group being independently selected from F, cl, br, -OH, -CN, C_1–4 alkyl, 0 to 3 per ring group, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy and-NR_cR_c, R_4c is C_1–4 alkyl or C_3–6 cycloalkyl, each independently selected from F, 0 to 4, and a pharmaceutically acceptable salt thereof, Cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN, each R₅ is independently-CN, C_1–5 alkyl substituted with 0 to 4R_g, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, C_2–3 alkenyl substituted with 0to 4R_g, C_2–3 alkynyl substituted with 0to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, phenyl substituted with 0 to 3R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 3R_g, - (CH₂)_1–2 (heterocyclyl substituted with 0 to 4R_g), - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), alkyl, - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl), and m is 1,2 or 3.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₁ is Cl or-CN; R₂ is-CH₃;R₄ is-CH₂R_4a or-CHR_4aR_4b;R_4a is cyclopropyl, cyclobutyl, cyclohexyl, bicyclo [1.1.1] pentyl, phenyl, pyridinyl, pyrimidinyl, oxadiazolyl, benzo [ d ] [1,3] dioxolyl or oxo-dihydrobenzo [ d ] oxazolyl, each substituted with 0 to 3 substituents independently selected from F、Cl、–CN、–CH₃、–CH(CH₃)₂、–CF₃、–OCH₃、–OCH(CH₃)₂、–OCHF₂、–OCF₃、–OCH₂( cyclopropyl) and cyclopropyl; R_4b is (i) -CH₃ and-CH₂CH₃, or (II) phenyl, isoxazolyl, oxadiazolyl, or thiazolyl, each substituted with 0 to 3 substituents independently selected from F, cl, -CH₃、–C(CH₃)₃、–CF₃、–OCF₃, and cyclopropyl, each R₅ is independently-CH₃、–CH₂CH₃、–CH₂ OH or-CH₂OCH₃, and m is 2.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₁ is Cl、–CN、–OH、–CHF₂、–CH₂OH、–CH₂OCH₃、–OCH₃、–OCH₂CH₃、–OCHF₂、–OCH₂CH₂OCH₃ or-OCH₂CH₂N(CH₃)₂;R₂ is H, -CH₃ or-CD₃;R₄ is-CH₂R_4a or-CHR_4aR_4b;R_4a is cyclohexyl, Phenyl, pyridyl, pyrimidinyl, oxadiazolyl, benzo [ d ] [1,3] dioxolyl or oxo-dihydrobenzo [ d ] oxazolyl, from 0 to 3 each independently selected from F、Cl、Br、–CN、–CH₃、–CH(CH₃)₂、–C(CH₃)₃、–CH₂OH、–CHF₂、–CF₃、–CH₂Br、–CH₂NH₂、–CH₂NHC(O)OCH₃、–C(CH₃)₂CN、–OCH₃、–OCD₃、–OCH₂CH₃、–OCH(CH₃)₂、–OCHF₂、–OCF₃、–OCH₂CH₂CF₃、–OC(CH₃)₂CN、–OC(CH₃)₂CH₂OH、–OC(CH₃)₂CH₂OCH₃、–N(CH₃)₂、–C(O)OCH₃、 cyclopropyl, cyanocyclopropyl, methylcyclopropyl, -O (cyclopropyl), -O ((ethoxycarbonyl) cyclopropyl), morpholinyl, pyrrolidinonyl, tetrahydropyranyl, dioxolanyl, -CH₂ (morpholinyl), morpholinyl, -CH₂ (difluoromethyl morpholinyl), -CH₂ (dimethylmorpholinyl), -CH₂ (oxaazabicyclo [2.2.1] heptanyl), -CH₂ (oxaazaspiro [3.3] heptanyl), -a process for preparing the same, and use of the same for preparing a pharmaceutical composition, -CH₂ (methylpiperazinonyl), -CH₂ (acetylpiperazinyl), -CH₂ (piperidinyl), -CH₂ (difluoropiperidinyl), -CH₂ (methoxypiperidinyl), -CH₂ (hydroxypiperidinyl), -C (CH₃)₂ (morpholinyl), -OCH₂ (cyclopropyl), -C (N-hydroxy-piperidinyl)), -OCH₂ (methylcyclopropyl), -OCH₂ (methylazetidinyl), -OCH₂ (oxetanyl), -OCH₂ (tetrahydropyranyl), and, -OCH₂ (thiazolyl) or-OCH₂CH₂ (cyclopropyl), R_4b is (i) -CN, -CH₃、–CH₂CH₃、–CH₂CH₂CH₃ or-CH (CH₃)₂; or (ii) phenyl, isoxazolyl, oxadiazolyl, thiazolyl or triazolyl, each independently selected from F, cl, br, -CH₃、–C(CH₃)₃、–CF₃、–OCF₃ and cyclopropyl, each R₅ is independently –CH₃、–CH₂CH₃、–CH₂CH₂CH₃、–CH₂OH、–CH₂OCH₃、–CH₂OCH₂CH₃、–CH₂NH₂、–CH₂N₃ or-CH₂NHC(O)OCH₃, and m is 2.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₁ is H, F, cl, br, -CN, -OH, C_1–3 alkyl substituted with 0 to 4R_1a, cyclopropyl substituted with 0 to 3R_1a, C_1–3 alkoxy substituted with 0 to 3R_1a, -NR_aR_a、–S(O)_nCH₃, or-P (O) (CH₃)₂. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R₁ is Cl、–CN、–OH、–CHF₂、–CH₂OH、–CH₂OCH₃、–OCH₃、–OCH₂CH₃、–OCHF₂、–OCH₂CH₂OCH₃ or-OCH₂CH₂N(CH₃)₂.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, cyclopropyl substituted with 0 to 3R_1a, C_1–3 alkoxy substituted with 0 to 3R_1a, -NR_aR_a、–S(O)_nCH₃, or-P (O) (CH₃)₂. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R₁ is H, F, cl, br, -CN, -CH₃, cyclopropyl, -OCH₃, or-NH₂, in some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R₁ is Cl or-CN., in some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R₁ is-CN.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₂ is H, C_1–2 alkyl substituted with 0 to 4R_2a, or C_3–4 cycloalkyl substituted with 0 to 2R_2a. In some embodiments, a compound of formula (II) or a salt thereof, wherein R₂ is H or C_1–2 alkyl substituted with 0 to 2R_2a, is administered. In some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein R₂ is H or-CH₃. In some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein R₁ is-CH₃. In some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein R₂ is H, -CH₃, or-CD₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₂ is H.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₂ is-CD₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is-CH₂R_4a or-CH₂CH₂R_4a. In some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein R₄ is-CH₂R_4a or-CD₂R_4a. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is phenyl, pyridinyl, tetrahydropyranyl, benzoxazinyl, benzo [ d ] [1,3] dioxolyl, benzoxazinonyl, indazolyl, indolyl, or quinolinyl, each substituted with 0 to 3 substituents independently selected from F、Cl、Br、–CN、–OH、–CH₃、–CH₂CH₃、–CH(CH₃)₂、–C(CH₃)₃、–CHF₂、–CF₃、–OCH₃、–OCH₂CH₃、–OCH(CH₃)₂、–OCHF₂、–OCF₃、–C(O)CH₃、–C(O)OC(CH₃)₃、–N(CH₃)₂、 cyanocyclopropyl and phenyl. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is phenyl, pyridinyl, or benzo [ d ] [1,3] dioxolyl, each substituted with 0 to 3 substituents independently selected from F、Cl、–CN、–CH₃、–CH(CH₃)₂、–CF₃、–OCH₃、–OCH(CH₃)₂、–OCHF₂、–OCF₃、–OCH₂( cyclopropyl) and cyclopropyl.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is-CH₂R_4a. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is phenyl, pyridinyl, tetrahydropyranyl, benzoxazinyl, benzo [ d ] [1,3] dioxolyl, benzoxazinonyl, indazolyl, indolyl, or quinolinyl, each substituted with 0 to 3 substituents independently selected from F、Cl、Br、–CN、–OH、–CH₃、–CH₂CH₃、–CH(CH₃)₂、–C(CH₃)₃、–CHF₂、–CF₃、–OCH₃、–OCH₂CH₃、–OCH(CH₃)₂、–OCHF₂、–OCF₃、–C(O)CH₃、–C(O)OC(CH₃)₃、–N(CH₃)₂、 cyanocyclopropyl and phenyl. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is phenyl, pyridinyl, or benzo [ d ] [1,3] dioxolyl, each substituted with 0 to 3 substituents independently selected from F、Cl、–CN、–CH₃、–CH(CH₃)₂、–CF₃、–OCH₃、–OCH(CH₃)₂、–OCHF₂、–OCF₃、–OCH₂( cyclopropyl) and cyclopropyl.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CH₂R_4a and R_4a is C_3–8 carbocyclyl, 4-to 10-membered heterocyclyl, phenyl or 5-to 10-membered heteroaryl, each of 0 to 4 independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy, -O (C_1–4 hydroxyalkyl), -O (CR_xR_x)_1–3O(C_1–3 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–CH₂NR_aR_a、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl, -CR_xR_x)_0–2NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -CR_xR_x)_1–2(C_3–4 cycloalkyl, -CR_xR_x)_1–2 (morpholinyl), - (CR_xR_x)_1–2 (difluoromethyl), - (CR_xR_x)_1–2 (dimethylmorpholinyl), - (CR_xR_x)_1–2 (oxaazabicyclo [2.2.1] heptanyl) and (CR_xR_x)_1–2 (oxaazaspiro [3.3] heptanyl)) and a pharmaceutically acceptable salt thereof, - (CR_xR_x)_1–2 (methylpiperazinonyl), - (CR_xR_x)_1–2 (acetylpiperazinyl), - (CR_xR_x)_1–2 (piperidinyl), - (CR_xR_x)_1–2 (difluoropiperidinyl)), a solvent, - (CR_xR_x)_1–2 (methoxypiperidinyl), - (CR_xR_x)_1–2 (hydroxypiperidinyl), -O (CR_xR_x)_0–2(C_3–6 cycloalkyl), -O (CR_xR_x)_0–2 (methylcyclopropyl)), a solvent, -O (CR_xR_x)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CR_xR_x)_0–2 (oxetanyl), -O (CR_xR_x)_0–2 (methylazetidinyl), -O (CR_xR_x)_0–2 (tetrahydropyranyl)), -O (CR_xR_x)_0–2), -O (CR_xR_x)_1–2 (morpholinyl)), O (CR_xR_x)_0–2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, dioxolanyl, pyrrolidone group and substituents of R_d.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CH₂R_4a and R_4a is C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, phenyl or 5-to 10-membered heteroaryl, each of 0 to 4 independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1-2 hydroxyalkyl, -CH₂NR_aR_a、–(CH₂)_1–2O(C_1–2 alkyl), - (CH₂)_1–2NR_xC(O)O(C_1-2 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CR_xR_x)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy 、–O(CH₂)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl), -CH₂)_1–2(C_3–4 cycloalkyl, -CR_xR_x (morpholinyl), -CR_xR_x (difluoromethyl morpholinyl), -CR_xR_x (dimethylmorpholinyl), -CR_xR_x (oxaazabicyclo [2.2.1] heptanyl), -CR_xR_x (oxaazaspiro [3.3] heptanyl), and-, -CR_xR_x (methylpiperazinonyl), -CR_xR_x (acetylpiperazinyl), -CR_xR_x (piperidinyl), -CR_xR_x (difluoropiperidinyl), a compound of formula (I), -CR_xR_x (methoxypiperidinyl), -CR_xR_x (hydroxypiperidinyl) -O (CH₂)_0–2(C_3–4 cycloalkyl), -O (CH₂)_0–2 (methylcyclopropyl), -a catalyst, and a process for preparing the same, -O (CH₂)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CH₂)_0–2 (oxetanyl), -O (CH₂)_0–2 (methylazetidinyl), -O (CH₂)_1–2 (morpholinyl)) -substituents for O (CH₂)_0–2 (tetrahydropyranyl), -O (CH₂)_0–2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, dioxolanyl, pyrrolidonyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is cyclohexyl, phenyl, or benzo [ d ] [1,3] dioxolyl, each substituted with 1 to 3 substituents independently selected from F, cl, -CH (CH₃)₂、–CF₃、–OCH₂CH₃、–OCF₃, cyclopropyl, and-OCH₂ (cyclopropyl).

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is (i) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, each of 0 to 4, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH₂)_1–3O(C_1–3 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–6 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), and, Cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, and R_d, or (ii) C_1–4 alkyl substituted with one cyclic group selected from C_3–6 cycloalkyl, heterocyclyl, aryl, and heteroaryl, said cyclic group being substituted with 0 to 3 cyclic groups independently selected from F, Cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–6 cycloalkyl, and R_4b is phenyl or heteroaryl, each independently selected from 0 to 4 of F, Cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH₂)_1–3O(C_1–3 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–6 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), and, cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl and methylpiperidinyl. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is (i) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, 0 to 4, C_1–3 fluoroalkyl, -CH₂OH、–(CH₂)_1–2O(C_1–2 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH₂)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl, -O (CH₂)_1–2(C_3–4 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or (ii) substituted with one substituent selected from C_3–6 cycloalkyl, C_1–3 alkyl substituted with 0 to 3 cyclic groups independently selected from F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 Fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, and C_3–4 cycloalkyl, and R_4b is phenyl, isoxazolyl, oxadiazolyl, or thiazolyl, each substituted with 0 to 3 substituents independently selected from F, cl, -CH₃、–C(CH₃)₃、–CF₃、–OCF₃, and cyclopropyl. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is phenyl, pyridinyl or benzo [ d ] [1,3] dioxolyl, each substituted with 0 to 3 substituents independently selected from F、Cl、–CN、–CH₃、–CH(CH₃)₂、–CF₃、–OCH₃、–OCH(CH₃)₂、–OCHF₂、–OCF₃、–OCH₂( cyclopropyl) and cyclopropyl, and R_4b is phenyl, hydroxy, or hydroxy, is optionally substituted with one or more substituents independently selected from F、Cl、–CN、–CH₃、–CH(CH₃)₂、–CF₃、–OCH₃、–OCH(CH₃)₂、–OCHF₂、–OCF₃、–OCH₂( cyclopropyl isoxazolyl, oxadiazolyl, or thiazolyl, each substituted with 0 to 3 substituents independently selected from F, cl, -CH₃、–C(CH₃)₃、–CF₃、–OCF₃, and cyclopropyl.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is (i) C_3–6 carbocyclyl, 4-to 10-membered heterocyclyl, phenyl, or 5-to 10-membered heteroaryl, each from 0 to 4 independently selected from F, Cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy, -O (C_1–4 hydroxyalkyl), -O (CR_xR_x)_1–3O(C_1–3 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–CH₂NR_aR_a、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl, -CR_xR_x)_0–2NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -CR_xR_x)_1–2(C_3–4 cycloalkyl, -CR_xR_x)_1–2 (morpholinyl), - (CR_xR_x)_1–2 (difluoromethyl), - (CR_xR_x)_1–2 (dimethylmorpholinyl), - (CR_xR_x)_1–2 (oxaazabicyclo [2.2.1] heptanyl) and (CR_xR_x)_1–2 (oxaazaspiro [3.3] heptanyl)) and a pharmaceutically acceptable salt thereof, - (CR_xR_x)_1–2 (methylpiperazinonyl), - (CR_xR_x)_1–2 (acetylpiperazinyl), - (CR_xR_x)_1–2 (piperidinyl), - (CR_xR_x)_1–2 (difluoropiperidinyl)), a solvent, - (CR_xR_x)_1–2 (methoxypiperidinyl), - (CR_xR_x)_1–2 (hydroxypiperidinyl), -O (CR_xR_x)_0–2(C_3–6 cycloalkyl), -O (CR_xR_x)_0–2 (methylcyclopropyl)), a solvent, -O (CR_xR_x)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CR_xR_x)_0–2 (oxetanyl), -O (CR_xR_x)_0–2 (methylazetidinyl), -O (CR_xR_x)_0–2 (tetrahydropyranyl)), -O (CR_xR_x)_0–2), -O (CR_xR_x)_1–2 (morpholinyl), -O (CR_xR_x)_0–2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, dioxolanyl, pyrrolidone group, and substituents of R_d; or (ii) through a member selected from the group consisting of C_3–6 carbocyclyl, c_1–4 alkyl substituted with a 4-to 10-membered heterocyclyl, 6-to 10-membered aryl or 5-to 10-membered heteroaryl cyclic group containing 0 to 3 groups independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl, -NR_aC(O)O(C_1–4 alkyl) and C_3–6 cycloalkyl, and R_4b is phenyl, Isoxazolyl, oxadiazolyl, thiazolyl, or triazolyl, each substituted with 0 to 3 substituents independently selected from F, cl, br, -CH₃、–C(CH₃)₃、–CF₃、–OCF₃, and cyclopropyl.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is (i) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, 0 to 4, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH₂)_1–3O(C_1–3 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–6 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), and, cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, and R_d, or (ii) C_1–4 alkyl substituted with one cyclic group selected from C_3–6 cycloalkyl, heterocyclyl, mono-or bicyclic aryl, and heteroaryl, said cyclic group being substituted with 0 to 3 cyclic groups independently selected from F, Cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–6 cycloalkyl, and R_4b is substituted with 0 to 4 substituents independently selected from F, C_1–6 alkyl substituted with Cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy, -NR_aR_a、–S(O)₂R_e, or-NR_aS(O)₂R_e substituents. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, each over 0 to 4, C_1–3 fluoroalkyl, -CH₂OH、–(CH₂)_1–2O(C_1–2 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH₂)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl, -O (CH₂)_1–2(C_3–4 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or (ii) substituted with one substituent selected from C_3–6 cycloalkyl, C_1–3 alkyl substituted with 0 to 3 cyclic groups independently selected from F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, and C_3–4 cycloalkyl, and R_4b is substituted with 0 to 4 substituents independently selected from F, Cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy and-NR_aR_a C_1–4 alkyl. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is phenyl, Pyridyl or benzo [ d ] [1,3] dioxolyl, each substituted with 0 to 3 substituents independently selected from F、Cl、–CN、–CH₃、–CH(CH₃)₂、–CF₃、–OCH₃、–OCH(CH₃)₂、–OCHF₂、–OCF₃、–OCH₂( cyclopropyl) and cyclopropyl, and R_4b is-CH₃ and-CH₂CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is (i) C_3–8 carbocyclyl, 4-to 10-membered heterocyclyl, phenyl, or 5-to 10-membered heteroaryl, each from 0 to 4 independently selected from F, Cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy, -O (C_1–4 hydroxyalkyl), -O (CR_xR_x)_1–3O(C_1–3 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–CH₂NR_aR_a、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl, -CR_xR_x)_0–2NR_aC(O)O(C_1–4 alkyl, -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -CR_xR_x)_1–2(C_3–4 cycloalkyl, -CR_xR_x)_1–2 (morpholinyl), - (CR_xR_x)_1–2 (difluoromethyl), - (CR_xR_x)_1–2 (dimethylmorpholinyl), - (CR_xR_x)_1–2 (oxaazabicyclo [2.2.1] heptanyl) and (CR_xR_x)_1–2 (oxaazaspiro [3.3] heptanyl)) and a pharmaceutically acceptable salt thereof, - (CR_xR_x)_1–2 (methylpiperazinonyl), - (CR_xR_x)_1–2 (acetylpiperazinyl), - (CR_xR_x)_1–2 (piperidinyl), - (CR_xR_x)_1–2 (difluoropiperidinyl)), a solvent, - (CR_xR_x)_1–2 (methoxypiperidinyl), - (CR_xR_x)_1–2 (hydroxypiperidinyl), -O (CR_xR_x)_0–2(C_3–6 cycloalkyl), -O (CR_xR_x)_0–2 (methylcyclopropyl)), a solvent, -O (CR_xR_x)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CR_xR_x)_0–2 (oxetanyl), -O (CR_xR_x)_0–2 (methylazetidinyl), -O (CR_xR_x)_0–2 (tetrahydropyranyl)), -O (CR_xR_x)_0–2), -O (CR_xR_x)_1–2 (morpholinyl)), -O (CR_xR_x)_0–2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, dioxolanyl, pyrrolidone group and R_d, or (ii) substituted with one substituent selected from C_3–6 cycloalkyl, C_1–4 alkyl substituted with a 4-to 10-membered heterocyclyl, mono-or bicyclic aryl, or 5-to 10-membered heteroaryl cyclic group containing 0 to 3 groups independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, and C_3–6 cycloalkyl, and R_4b is-CN or is substituted with 0 to 4 substituents independently selected from F, C_1–6 alkyl substituted with Cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy, -NR_aR_a、–S(O)₂R_e, or-NR_aS(O)₂R_e substituents. In some embodiments, a compound of formula (II) or a salt thereof is administered, wherein R_4a is (i) C_3–6 carbocyclyl, 4-to 10-membered heterocyclyl, phenyl, or 5-to 10-membered heteroaryl, each from 0 to 4 independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1-2 hydroxyalkyl, -CH₂NR_aR_a、–(CH₂)_1–2O(C_1–2 alkyl), - (CH₂)_1–2NR_xC(O)O(C_1-2 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CR_xR_x)_1–2O(C_1–2 alkyl), C_1–3 Fluoroalkoxy, C_1–3 cyanoalkoxy 、–O(CH₂)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl, - (CH₂)_1–2(C_3–4 cycloalkyl), -CR_xR_x (morpholinyl), -CR_xR_x (difluoromethyl), -CR_xR_x (dimethylmorpholinyl), -CR_xR_x (oxaazabicyclo [2.2.1] heptanyl), -CR_xR_x (oxaazaspiro [3.3] heptanyl), -CR_xR_x (methylpiperazinonyl), -CR_xR_x (acetylpiperazinyl), and-, -CR_xR_x (piperidinyl), -CR_xR_x (difluoropiperidinyl), -CR_xR_x (methoxypiperidinyl), -CR_xR_x (hydroxypiperidinyl), -O (CH₂)_0–2(C_3–4 cycloalkyl), -O (CH₂)_0–2 (methylcyclopropyl), -O (CH₂)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CH₂)_0–2 (oxetanyl)), -O (CH₂)_0–2 (methylazetidinyl), -O (CH₂)_1–2 (morpholinyl), -O (CH₂)_0–2 (tetrahydropyranyl), -O (CH₂)_0–2 (thiazolyl)), Cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, dioxolanyl, pyrrolidonyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or (ii) substituted with one substituent selected from C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, A C_1–3 alkyl substituted with a monocyclic or bicyclic aryl or 5-to 10-membered heteroaryl cyclic group, said cyclic group being independently selected from F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, through 0 to 3, c_1–3 alkoxy, C_1–2 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl, and C_3–4 cycloalkyl, and R_4b is-CN or is substituted with 0 to 4 substituents independently selected from F, Cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy and-NR_aR_a C_1–4 alkyl.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is-CHR_4aR_4b, and R_4b is-CN, -CH₃、–CH₂CH₃、–CH₂CH₂CH₃ or-CH (CH₃)₂).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is-CHR_4aR_4b, and R_4b is-CN, -CH₃, or-CH₂CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is-CHR_4aR_4b, and R_4b is-CN.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is-CHR_4aR_4b, and R_4b is-CH₃ or-CH₂CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is-CHR_4aR_4b, and R_4b is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is-CHR_4aR_4b, and R_4b is-CH₂CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein m is 1,2 or 3, and each R₅ is independently-CN, C_1–5 alkyl optionally substituted with 0 to 4R_g, C_2–3 alkenyl substituted with 0 to 4R_g, C, C_2–3 alkynyl optionally substituted with 0 to 4R_g, C_3–4 cycloalkyl optionally substituted with 0 to 4R_g, phenyl substituted with 0 to 3R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 3R_g, - (CH₂)_1–2 (4-to 10-membered heterocyclyl substituted with 0 to 4R_g), and, - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl). in some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein each R₅ is independently-CH₃、–CH₂CH₃、–CH₂ OH or-CH₂OCH₃.

In one embodiment, a compound of formula (II) or a salt thereof, wherein m is 0, is administered.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein m is 1,2 or 3. In some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein m is 1 or 2. In some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein m is 1.

In one embodiment, a compound of formula (II) or a salt thereof, wherein m is 2 or 3, is administered. In some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein m is 2.

In one embodiment, a compound of formula (II) or a salt thereof, wherein m is 3, is administered.

In one embodiment, a compound of formula (II), or a salt thereof, is administered having the structure of formula (III):

Wherein one, two or three of R_5a、R_5b、R_5c and R_5d are each R₅ and the remainder of R_5a、R_5b、R_5c and R_5d are each hydrogen. In some embodiments, a compound of formula (III), or a salt thereof, is administered, wherein each R₅ is independently –CN、–CH₃、–CH₂CH₃、–CH(CH₃)₂、–CHC(CH₃)₂、–CH₂F、–C(CH₃)₂F、–CF(CH₃)CH(CH₃)₂、–CH₂OH、–C(CH₃)₂OH、–C(CH₃)(OH)CH(CH₃)₂、–CH₂OCH₃、–C(O)C(CH₃)₂、–C(O)OH、–C(O)OCH₃、–C(O)OC(CH₃)₂、–C(O)NH₂、–C(O)NH( cyclopropyl), -C (O) O (cyclopropyl), cyclopropyl, phenyl, methyl oxadiazolyl, or methyl pyridinyl. In some embodiments, a compound of formula (III), or a salt thereof, is administered, wherein each R₅ is independently –CH₃、–CH₂CH₃、–CH₂CH₂CH₃、–CH₂OH、–CH₂OCH₃、–CH₂OCH₂CH₃、–CH₂NH₂、–CH₂N₃ or-CH₂NHC(O)OCH₃.

In one embodiment, a compound of formula (II), or a salt thereof, is administered having the structure of formula (IV):

Wherein R_5a and R_5c are each R₅, and R_5b and R_5d are each hydrogen. In some embodiments, a compound of formula (IV) or a salt thereof is administered, wherein (i) R_5a is-CH₃ or-CH₂CH₃ and R_5c is-CH₃ or-CH₂CH₃, or (ii) R_5a is-CH₃ and R_5c is-CH₂ OH or-CH₂OCH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₃ and R_5c is-CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₃ and R_5c is-CH₂CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₂CH₃ and R_5c is-CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₂CH₃ and R_5c is-CH₂CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₃ and R_5c is-CH₂ OH.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₃ and R_5c is-CH₂OCH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₃ and R_5c is-CH₂OCH₂CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₃ and R_5c is-CH₂CH₂CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₃ and R_5c is-CH₂N₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₃ and R_5c is-CH₂NH₂.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₃ and R_5c is-CH₂NHC(O)OCH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₂ OH and R_5c is-CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R_5a is-CH₂OCH₃ and R_5c is-CH₃.

In one embodiment, a compound of formula (II), or a salt thereof, is administered having the structure:

In one embodiment, a compound of formula (II), or a salt thereof, is administered having the structure:

In one embodiment, a compound of formula (II), or a salt thereof, is administered having the structure:

In one embodiment, a compound of formula (II), or a salt thereof, is administered having the structure:

in one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is:

(i)

(ii)

Or (b)

(iii)

In some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein R₁ is H, br, -CN, or-OCH₃, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is:

In some embodiments, a compound of formula (II), or a salt thereof, is administered, wherein R₁ is H, br, -CN, or-OCH₃, and R₂ is-CH₃. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof, wherein R₁ is-CN and R₂ is-CH₃, is administered.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R₁ is-CN, R₂ is-CH₃;R_5a is-CH₃, and R_5c is-CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R₁ is-CN, R₂ is-CH₃;R_5a is-CH₃, and R_5c is-CH₂CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R₁ is-CN, R₂ is-CH₃;R_5a is-CH₃, and R_5c is-CH₂CH₂CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R₁ is-CN, R₂ is-CH₃;R_5a is-CH₂CH₃, and R_5c is-CH₂CH₃.

In one embodiment, a compound of formula (IV) or a salt thereof is administered, wherein R₁ is-CN, R₂ is-CH₃;R_5a is-CH₃, and R_5c is-CH₃、–CH₂CH₃ or-CH₂CH₂CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₁ is-CN, R₂ is-CH₃;R₄ is-CHR_4aR_4b, and R_4b is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₁ is-CN, R₂ is-CH₃;R₄ is-CHR_4aR_4b, and R_4b is-CH₂CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₁ is-CN, R₂ is-CH₃;R₄ is-CHR_4aR_4b, and R_4b is-CH₂CH₂CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₁ is-CN, R₂ is-CH₃;R₄ is-CHR_4aR_4b, and R_4b is-CH₃、–CH₂CH₃ or-CH₂CH₂CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b, and R_4a is phenyl substituted with 1 to 2 substituents independently selected from F, cl, -CF₃、–OCF₃, or-OCH₂ (cyclopropyl). In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is phenyl substituted with-CF₃ or-OCF₃, and R_4b is-CH₃、–CH₂CH₃ or-CH₂CH₂CH₃. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is phenyl substituted with-CF₃ or-OCF₃, and R_4b is-CH₃. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is phenyl substituted with-CF₃ or-OCF₃, and R_4b is-CH₂CH₃. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is phenyl substituted with-CF₃ or-OCF₃, and R_4b is-CH₂CH₂CH₃. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is phenyl substituted with 1 to 2 substituents independently selected from F, cl, -CF₃、–OCF₃, or-OCH₂ (cyclopropyl), and R_4b is-CH₃、–CH₂CH₃ or-CH₂CH₂CH₃. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein R₄ is-CHR_4aR_4b, and R_4a is pyridinyl. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b and R_4a is pyridinyl substituted with-CF₃. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is pyridinyl, and R_4a is phenyl substituted with Cl. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a is pyridinyl substituted with-CF₃, and R_4b is phenyl substituted with F. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein R₄ is-CHR_4aR_4b;R_4a and one of R_4b is F-substituted phenyl and the other of R_4a and R_4b is cyclopropyl-substituted oxadiazolyl. In some embodiments, a compound of formula (II), or a salt thereof, is administered wherein R₁ is-CN, and R₂ is-CH₃.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein the compound is 4- ((2S, 5R) -4- ((2, 2-difluorobenzo [ d ] [1,3] dioxolan-5-yl) methyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (2-fluoro-4- (trifluoromethoxy) benzyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (4- (trifluoromethoxy) benzyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5-diethyl-4- (2-fluoro-4- (trifluoromethoxy) benzyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido ] pyrimidine-6-carbonitrile, 4- ((2S, 2-difluoro-2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-methyl-4- (4- (trifluoromethoxy) piperazin-1-yl) methyl-2-oxo-6-carbonitrile Carbonitriles; 4- ((2 s,5 r) -5-ethyl-4- (2-fluoro-4- (trifluoromethoxy) benzyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -2, 5-dimethyl-4- (3, 4, 5-trifluorobenzyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (3, 4-difluorobenzyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (2-chloro-4, 5-difluorobenzyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((2, 2-difluorobenzo [ d ] [1,3] dioxol-5-yl) methyl) -2, 5-dimethyl-1-piperazin-1-yl) Methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (2-chloro-4-fluorobenz-yl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (4-isopropylbenzyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (4- (cyclopropylmethoxy) benzyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (2-fluoro-4- (trifluoromethyl) benzyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido-pyrido [3, 5-diethylpiperazin-1-yl ] -4- ((2S, 5R) -4- (cyclopropylmethoxy) benzyl) -1-methyl-2-diethyl-6-carbonitrile ) Piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (4-cyclopropyl-2-fluorobenzyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((4, 4-difluorocyclohexyl) methyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, or 4- ((2S, 5R) -4- (4-ethoxybenzyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein the compound is 4- ((2S, 5R) -2, 5-diethyl-4- ((4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- ((4-fluorophenyl) (isoxazol-3-yl) methyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((5-cyclopropylisoxazol-3-yl) (4- (trifluoromethoxy) phenyl) methyl) -2, 5-diethyl piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3, 2-dihydropyrido ] pyrimidine-1-yl) -1-methyl-2- ((2, 2-fluoro) methyl-2-fluoro-4- ((2-methyl-2-d) pyrimidine-6-carbonitrile, 4- ((2S, 5-d) methyl) pyrimidine-6-carbonitrile -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (21-22); 4- ((2S, 5R) -4- ((4-cyclopropylthiazol-2-yl) (4-fluorophenyl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -5-ethyl-4- ((4-fluorophenyl) (6- (trifluoromethyl) pyridin-2-yl) methyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -5-ethyl-4- ((4-fluorophenyl) (isoxazol-3-yl) methyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido-pyrido [3, 2-dihydropyridin-2-yl ] pyrimidine-6-carbonitrile, 4- ((2S, 2-dihydropyridin-2-yl) methyl-2-methyl-4- ((4-fluorophenyl) 2-methyl-2-dihydropyridin-2-yl) methyl-4- ((4-fluoro) 2-dihydropyridin-yl) methyl-2-methyl-2-yl-methyl-2-yl-methyl-2-carboxylate -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-4- ((4-fluorophenyl) (pyridin-2-yl) methyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (pyridin-2-yl (4- (trifluoromethoxy) phenyl) methyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((4-chlorophenyl) (pyridin-2-yl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido-2-dihydro-1-methyl-4- (pyridin-2-yl) methyl-1-yl) -1-methyl-2-methyl-piperazin-6-carbonitrile, 4- ((2S, 2-dihydro-2-methyl) -1-methyl-2-yl) -2-dihydropyridin-6-carbonitrile, 4- ((4-2-dihydropyrido) 2-dihydropyrido-2-yl-2-methyl-2-yl) -4- ((4-2-methyl-2-carbonyl-2-methyl-carbonyl - (trifluoromethyl) pyridin-3-yl) methyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((5-cyclopropylisoxazol-3-yl) (4- (trifluoromethoxy) phenyl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((5-cyclopropylisoxazol-3-yl) (4-fluorophenyl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((2-cyclopropylthiazol-5-yl) (4-fluorophenyl) methyl) -5-methyl-2-oxo-1, 2-dihydropyrazin-6-carbonitrile, 4- ((2S, 5R) -4- ((5-cyclopropyli-methyl-2-methyl-1-d) pyrimidine-6-carbonitrile, 1-chloro-4- ((2-methyl-2-d) pyrimidine-1-methyl-2-d-carbonitrile 4- ((2S, 5R) -4- ((3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methylpyridin-2 (1H) -one, 4- ((2S, 5R) -4- ((3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (25-26), 4- ((2S, 5R) -4- ((3- (tert-butyl) -1,2, 4-oxadiazol-5-yl) (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4R) -4, 5-dimethylpiperazin-1-yl) -1-oxo-6-carbonitrile, 4- ((3, 2-dihydropyridin-1-yl) pyrimidine-6-carbonitrile (25-26), 4- ((2S, 5R) -4- ((3- (tert-butyl) -1,2, 4-oxadiazol-5-yl) methyl) -1-dihydropyrazin-5-yl-2 (4-fluorophenyl) methyl) -2, 5-dihydropyrazin-yl-one Pyrido [3,2-d ] pyrimidine-6-carbonitrile (25-26), 4- ((2S, 5R) -4- ((3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) (4-fluorophenyl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2-ethyl-4- ((4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -5-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2-ethyl-4- ((4-fluorophenyl) (6- (trifluoromethyl) pyridin-2-yl) methyl) -5-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (bis (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (27-28), 4- ((2S, 5R) -4- ((4-cyclopropylthiazol-2-yl) (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((4-fluorophenyl) (isoxazol-3-yl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-oxo-2, 2-dihydropyrazin-6-carbonitrile, 4- ((2S, 5R) -4- ((4-cyclopropyl) thiazol-1-yl) methyl-2-oxo-1, 5-dihydropyrazin-6-carbonitrile 5-Cyclopropylisoxazol-3-yl) (4- (trifluoromethoxy) phenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridazo [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((4-fluorophenyl) (2- (trifluoromethyl) thiazol-4-yl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridazo [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5S) -4- ((4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -5- (methoxymethyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridazo [3,2-d ] pyrimidine-6-carbonitrile, 6-chloro-4- ((2S, 5S) -4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-methyl) -2-hydroxy-pyrido [ 1-methyl) -1-d ] piperazin-1-carbonitrile Pyrimidin-2 (1H) -one, 4- ((2S, 5S) -4- ((4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -5- (hydroxymethyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- ((4-fluorophenyl) (2- (trifluoromethyl) thiazol-4-yl) methyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((6- (difluoromethyl) pyridin-2-yl) (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2, 5R) -4-bromo-1, 2-dihydropyridin-1-yl) 4- ((2, 5R) -4-bromo-1-2-triazol-2-yl) pyrimidine-6-carbonitrile 1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((3-cyclopropyl-1-methyl-1H-1, 2, 4-triazol-5-yl) (4-fluorophenyl) methyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-4- ((4-fluorophenyl) (2- (trifluoromethyl) thiazol-4-yl) methyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((6- (difluoromethyl) pyridin-2-yl) (4-fluorophenyl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-oxo-2-chloro-1-chloro-2-methyl-2-methylpyridin-1-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((2-fluoro) methyl-2-dihydropyrazin-1-d) pyrimidine-6-carbonitrile 1,2, 4-oxadiazol-3-yl) (4-fluorophenyl) methyl) -2, 5-diethylpiperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one; 4- ((2S, 5R) -4- ((5-cyclopropyl-1, 2, 4-oxadiazol-3-yl) (4-fluorophenyl) methyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (bis (4-chlorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((4-cyanophenyl) (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, or 4- ((2S, 5R) -4- ((4-fluorophenyl) (3- (trifluoromethyl) bicyclo [ 1.1.1-yl) methyl ] pentane -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered wherein the compound is 4- ((2S, 5R) -4- (1- (4-cyclopropylphenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (17-18), 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (19-20), 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (19-18), 4R) -5-diethyl-1- (4-fluoro) phenyl) propyl ] pyrimidine-1-2-methyl-carbonitrile -6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (2-fluoro-4-methoxyphenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (2, 2-difluorobenzo [ d ] [1,3] dioxolan-5-yl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4-methoxyphenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1-methoxyphenyl) pyrimidin-6-carbonitrile, and 4- ((2S, 5R) -2, 5-diethyl-4- (1-methoxyphenyl) piperazin-1-yl) 2-dihydro-1-methyl-2-oxo-1-d ] pyrimidine-carbonitrile - (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -2, 5-diethyl-4- (4- (trifluoromethoxy) benzyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (cyclopropylmethoxy) phenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (2-fluoro-4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-cyclopropylphenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-oxo-2, 2-oxo-1-methyl-2-carbonitrile Dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (3-fluoro-4- (trifluoromethoxy) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (cyclopropylmethoxy) -2-fluorophenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-1- (2- (trifluoromethoxy) phenyl) pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (2-trifluoromethoxy) phenyl) pyrimidine-6-carbonitrile Carbonitriles, 4- ((2S, 5R) -4- (1- (4-cyclopropyl-2-fluorophenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (6- (trifluoromethyl) pyridin-3-yl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethoxy) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-4- (1- (4-isopropoxyphenyl) ethyl) -2-methyl-piperazin-1-yl) -2-oxo-1, 2-dihydropyrido-methyl-4- ((2-methyl-4- (4-isopropoxyphenyl) ethyl) -2-methyl-1-d ] pyrimidine-6-carbonitrile - (1- (4-methoxyphenyl) ethyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2 s,5 r) -5-ethyl-4- (1- (2-fluoro-4- (trifluoromethyl) phenyl) ethyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2 s,5 r) -4- (1- (4-cyanophenyl) ethyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (4-cyclopropylphenyl) ethyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (cyclopropylmethoxy) phenyl) ethyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (cyclopropylmethoxy) -2-fluorophenyl) ethyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (2-difluorobenzo [ d ] [1,3] dioxolan-5-ethyl) -5-methyl-2-oxo-1, 2-dihydropyrido-pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-dihydropyran-6-carbonitrile (2 s,5 r) -4- (1- (4- (difluoromethoxy) phenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (2, 2-difluorobenzo [ d ] [1,3] dioxolan-5-yl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-4- (1- (2-fluoro-4- (trifluoromethoxy) phenyl) ethyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4-17-yl) ((2S, 5R) -5-ethyl-4- (1- (2-fluoro-4- (trifluoromethoxy) phenyl) propyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (4-cyclopropyl-2-fluorophenyl) ethyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -2-ethyl-5-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) -1-oxo-1, 2-dihydropyrido ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -1-methyl-2-dihydropyrido-6-carbonitrile) -2-ethyl-4- (1- (2-fluoro-4- (trifluoromethyl) phenyl) ethyl) -5-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -2-ethyl-5-methyl-4- (1- (4- (trifluoromethoxy) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-fluorophenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-dimethyl-4- (1- (4- (trifluoromethoxy) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-dimethyl-4- (1- (3, 4, 5-trifluorophenyl) pyrimidine-6-carbonitrile ) Ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -2, 5-dimethyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (2-fluoro-4- (trifluoromethoxy) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (2, 4-difluorophenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-chloro-2-fluorophenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-oxo-2-dihydropyrido-1-carbonitrile [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (3, 4-difluorophenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (2-fluoro-4- (trifluoromethyl) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (2-fluoro-4- (trifluoromethoxy) phenyl) propyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -2, 5-dimethyl-4- (1- (4- (trifluoromethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (difluoromethoxy) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (3-fluoro-4- (trifluoromethoxy) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (2, 2-difluorobenzo [ d ] [1,3] dioxolan-5-ethyl) -2, 2-dihydropyrido ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (3-fluoro-4-dimethylpiperazin-1-yl) ethyl-2-oxo-2-dihydropyrazin-6-carbonitrile, 4- ((2S, 5R) -4- (3-fluoro-4-fluoro-phenyl) ethyl-1-oxo-2-dihydropyran-2-carbonitrile Cyclopropyl-2-fluorophenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 6-chloro-4- ((2S, 5S) -5- (hydroxymethyl) -2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 6-chloro-4- ((2S, 5S) -5- (methoxymethyl) -2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2S, 5S) -5- (methoxymethyl) -2-methyl-4- (1- (4- (trifluoromethyl) phenyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3, 2-methyl-4- (trifluoromethyl) phenyl) piperazin-1-yl) -1-methyl-2-dihydropyrido [3,2-d ] pyrimidin-1-yl ] -5-methyl-4- ((2, 5-methyl) -4-d ] pyrimidine-2- (4-methyl-2-yl) -4- ((2-methyl) -4-d) pyrimidine-2-carbonitrile 1- (4- (trifluoromethoxy) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5S) -5- (hydroxymethyl) -2-methyl-4- (1- (4- (trifluoromethoxy) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 6-chloro-4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- ((1-hydroxy-2-methylpropan-2-yl) oxy) phenyl) ethyl) piperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 6-chloro-4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- ((1-methoxy-2-methylpropan-2-yl) oxy) phenyl) ethyl) piperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidine-2 (1H) -one, 6-chloro-4- (. Sub. 2S, 5R) -2, 5-diethyl-4- (1- (4- ((1-methoxy-2-methylpropan-2-yl) oxy) phenyl) piperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (methoxy-d₃) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (6-cyclopropylpyridin-3-yl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (2-morpholinyl-4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-oxo-2, 2-dihydropyrido [3, 5-diethyl-6-carbonitrile, 4- ((2S, 5R) -4-dimethylpiperazin-1-yl-2-methyl-2-d ] carbonitrile (4- (2-cyanopropan-2-yl) phenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (4- (cyclopropylmethoxy) -2-fluorophenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (29-30), 4- ((2S, 5R) -4- (1- (4-cyclopropylphenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (3-methyl-2-oxo-2, 3-dihydrobenzo [ d ] oxazol-5-yl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4 - (1- (2-morpholinylpyrimidin-5-yl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2 s,5 r) -2, 5-diethyl-4- (1- (4- (methoxy-d₃) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2 s,5 r) -2, 5-diethyl-4- (1- (4-methoxyphenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -2, 5-diethyl-4- (1- (6-methoxypyridin-2-yl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (cyclopropylmethoxy) phenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (1-methylcyclopropyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-cyanophenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -1-dimethyl-2-oxo-2-fluoro-1, 2-dihydropyrido ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -1-diethyl-1-methyl-2-dihydropyridin-2-yl) ethyl-1-dihydropyran-2-yl-carbonitrile -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -2, 5-diethyl-4- (1- (6- (trifluoromethyl) pyridin-3-yl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (2- (trifluoromethyl) pyrimidin-5-yl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (6-methylpyridin-3-yl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (2-oxo-pyrrolidin-1-yl) phenyl) piperazin-1-yl) Phenyl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (difluoromethoxy) -2-fluorophenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4-isopropoxyphenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (p-tolyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2- (4-isopropoxyphenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -1-fluoro-2-fluoro-1-fluoro-phenyl) pyrimidine-6-carbonitrile, 4- ((2, 5-R) -1-diethyl-piperazin-1-yl) -1-methyl-2-diethyl-2-dihydropyrido-yl 2s,5 r) -4- (1- (6- (difluoromethoxy) pyridin-2-yl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) butyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (31-32), 4- ((2S, 5R) -2, 5-diethyl-4- (1- (6- (trifluoromethoxy) pyridin-2-yl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (2-morpholinopropane-2-yl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2,5 -diethyl-4- (1- (4-methoxyphenyl) -2-methylpropyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (4- (2-cyanopropan-2-yl) phenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridazo [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (2-cyclopropylbenzo [ d ] oxazol-5-yl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridazo [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-cyclopropylphenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridazo [3,2-d ] pyrimidine-6-carbonitrile, (1S, 2S) -2- (4- (1- ((2R, 5S) -4- (6-cyano-1-cyano-yl) pyrimidine-6-carbonitrile -methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidin-4-yl) -2, 5-diethylpiperazin-1-yl) -ethyl) phenoxy) cyclopropane-1-carboxylic acid ethyl ester, 4- ((2 s,5 r) -2, 5-diethyl-4- (1- (4-isopropoxyphenyl) -2-methylpropyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2 s,5 r) -4- (1- (4- (1-cyanocyclopropyl) phenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; Methyl 4- (1- ((2R, 5S) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidin-4-yl) -2, 5-diethylpiperazin-1-yl) ethyl) benzoate, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (morpholinomethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (hydroxymethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (bromomethyl) phenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-oxo-2-oxo-1, 2-dihydropyrido ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (hydroxymethyl) phenyl) ethyl) pyrimidin-6-carbonitrile, 4- ((2S, 5-diethyl-1-oxo-2-oxo-pyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5-diethyl-methyl) 1-2-yl) pyrimidine-carbonyl) carboxylate 1-yl) methyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- ((2, 2-dimethylmorpholinyl) methyl) phenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- ((4, 4-difluoropiperidin-1-yl) methyl) phenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (2-oxa-6-azaspiro [3.3] heptan-6-yl) methyl) ethyl) -2, 5-diethyl-pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- ((2-dimethylpiperidin-1-yl) phenyl) ethyl) -2, 5-dihydropyridin-6-carbonitrile, 2- ((2S, 5-diethyl-1-methyl) pyrimidine-6-carbonitrile 4- (1- (4- (piperidin-1-ylmethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- ((4-acetylpiperazin-1-yl) methyl) phenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- ((4-hydroxypiperidin-1-yl) methyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- ((4-methyl-3-oxo-piperazin-1-yl) methyl) phenyl) pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2-diethyl-4- (1-methyl-1-oxo-piperazin-1-yl) ethyl) piperazin-6-carbonitrile 4- ((2 s, 5R) -2, 5-diethyl-4- (1- (4- (((R) -3-hydroxypiperidin-1-yl) methyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (4- (((2S, 6R) -2, 6-dimethylmorpholinyl) methyl) phenyl) ethyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (3-methyl-2-oxo-2, 3-dihydrobenzo [ d ] oxazol-5-yl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (4-cyclopropylphenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo 1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-cyclopropyl-2-fluorophenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-4- (1- (4-methoxyphenyl) propyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-ethoxyphenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (cyclopropyl) methoxy) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-methoxyphenyl) propyl) -5-ethyl-2-methylpiperazin-1-2-yl) methyl-2-yl ] pyrimidine-2-carbonitrile Carbonitriles, 4- ((2S, 5R) -4- (1- (4-cyclopropoxyphenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-4- (1- (4-methoxyphenyl) -2-methylpropyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (2-cyanopropan-2-yl) phenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (3, 4-difluorophenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-bromophenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-4- (1- (4-isopropoxyphenyl) -2-methylpropyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1-cyanocyclopropyl) phenyl) ethyl) -5-ethyl-2-methylpiperazin-1-yl) -1-oxo-2-methylpyrazin-1-yl-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-4- (1- (4-isopropoxyphenyl) -2-methylpiperazin-1-yl-1-oxo-2-dihydropyran-6-carbonitrile, 4- ((2S, 5R) -1-dihydropyran-methyl-2-dihydropyran-2-carbonitrile - (cyclopropylmethoxy) -2, 6-difluorophenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (tetrahydro-2H-pyran-4-yl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (1, 3-dioxolan-2-yl) phenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-4- (1- (4-isopropoxyphenyl) propyl) -2-methyl-2-oxo-1-dihydropyrido-6-carbonitrile, 4- ((2S, 5R) -4- ((2-methyl-2-pyrido-2-d) pyrimidine-6-carbonitrile -ethyl-2-methyl-4- (1- (4- (3, 3-trifluoropropoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- ((tetrahydro-2H-pyran-4-yl) methoxy) phenyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (2-cyclopropylethoxy) phenyl) propyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4-oxo-3-phenyl) propyl) piperazin-1-yl) 2-oxo-1-dihydro-pyrido [2, 2-d ] methyl-1-yl ] piperazin-1-yl Pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- ((1-methylazetidin-3-yl) methoxy) phenyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- ((1-methylcyclopropyl) methoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (thiazol-2-ylmethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (3-bromo-phenyl) ethyl) -1-bromo-2-methyl-piperazin-6-carbonitrile 1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (3- (morpholinomethyl) -4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (3- ((dimethylamino) methyl) -4- (trifluoromethyl) phenyl) ethyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (3- (piperidin-ylmethyl) -4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1-dihydropyrido-2-pyrimidine-6-carbonitrile, 4- ((2S, 2-ethyl-2-methylpiperazin-1-yl) -4- ((3- (trifluoromethyl) phenyl) 5-ethyl-2-pyrimidine-6-carbonitrile, 4- ((2S, 2-dihydropyrido) 5-dihydropyrido-5-methyl-6-carbonitrile (trifluoromethyl) phenyl) ethyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (aminomethyl) phenyl) ethyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, (4- (1- ((2R, 5S) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidin-4-yl) -2-ethyl-5-methylpiperazin-1-yl) ethyl) -4- ((2S, 5R) -4- (1- (3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) ethyl) -2, 5-diethylpiperazin-1-methyl-2-oxo-1-2-dihydropyrido ] pyrimidine-6-carbonitrile; 4- ((2 s,5 r) -4- (1- (3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) -2-methylpropyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (5-cyclopropyl-1, 3, 4-oxadiazol-2-yl) -2-methylpropyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 2- ((2R, 5S) -4- (6-chloro-1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidin-4-yl) -2, 5-diethylpiperazin-1-yl) -2- (4-fluoro-pyrimidine-6-carbonitrile Phenyl) acetonitrile, 4- ((2S, 5R) -4- (1- (4- ((2-cyanopropan-2-yl) oxy) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-cyclopropylphenyl) propyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4-cyclopropyl-2-fluorophenyl) propyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 6-chloro-4- ((2S, 5R) -4- (1- (4- (hydroxymethyl) phenyl) ethyl) -2, 5-dimethyl-1-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (4-hydroxymethyl) phenyl) ethyl) -1-dimethyl-6-carbonitrile, 4- ((2, 4-R) -4- (4-cyclopropyl-2-fluorophenyl) propyl) pyrimidine-6-carbonitrile Phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- (bromomethyl) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- ((2, 2-dimethylmorpholinyl) methyl) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (((1S, 4S) -2-oxa-5-azabicyclo [2.2.1] heptan-5-yl) methyl) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-6-carbonitrile, 4- ((2S, 5-dimethylpiperazin-1-yl) ethyl) -1-methyl-2, 2-d-dihydropyran-6-carbonitrile, 4- ((2, 2-dimethyl-1-oxo-1-d-pyrimidine-6-carbonitrile 2s,6 r) -2, 6-dimethylmorpholinyl methyl) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1- (4- ((4, 4-difluoropiperidin-1-yl) methyl) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (4- ((2-oxa-6-azaspiro [3.3] heptan-6-yl) methyl) phenyl) ethyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 6-chloro-1-methyl-4- ((2S, 5R) -2-methyl-5-propyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) pyrido [3,2-d ] pyrimidin-2 (1H) -one, 1-methyl-4-oxo-1, 2-dihydropyridin-6-carbonitrile - ((2S, 5R) -2-methyl-5-propyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (33-34); 6-chloro-4- ((2S, 5S) -5- (methoxymethyl) -2-methyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one; 4- ((2S, 5S) -5- (methoxymethyl) -2-methyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5S) -5- (ethoxymethyl) -2-methyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methylpyridin-2 (1H) -one; 4- ((2S, 5S) -1-methyl-2-dihydropyrido-1-d-carbonitrile; -1-2-dihydropyran-2-yl-2-dihydropyran - ((2 s,5 s) -5- (azidomethyl) -2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6-chloro-1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2 s,5 r) -5- (aminomethyl) -2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6-chloro-1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2 s,5 s) -5- (methoxymethyl) -2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2R, 5R) -2- (hydroxymethyl) -5-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 6-chloro-4- ((2R, 5R) -2- (methoxymethyl) -5-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one; 4- ((2R, 5R) -2- (methoxymethyl) -5-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2R, 5R) -5-ethyl-2- (hydroxymethyl) -4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methylpyrido-2 (1H) -one; 4- ((2R, 5R) -5-methyl-1-2-dihydropyrido-1-d ] pyrimidine-6-carbonitrile Carbonitriles; 4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) piperazin-1-yl) -2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, 6-chloro-4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) piperazin-1-yl) -1- (methyl-d₃) pyrido [3,2-d ] pyrimidine-2 (1H) -one, 4S, 5R) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1- (methyl-d) -1- (methyl-₃) -2-oxo-1, 2-pyrido-2-methyl-4- (1, 2-d ] pyrimidine-6-carbonitrile, 6-chloro-4- ((2, 5R) -5-chloro-2-methyl-2-pyrido [2, 2-d ] pyrimidine-1H) -one Diethyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6- (hydroxymethyl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6- (methoxymethyl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 6-chloro-4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2, 5R) -2-diethyl-4- (trifluoromethyl) phenyl) piperazin-1-yl) -1-methylpyridin-2 (1H) -one, 4- ((2S, 5-diethyl-4- (4-methyl) phenyl) piperazin-1-yl) -piperazin-1-yl-methyl-pyrido-1 (1H) -one Methoxy-1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6-ethoxy-1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6- (2- (dimethylamino) ethoxy) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6- (2-methoxyethoxy) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 4- ((2S, 5R) -2, 5-diethyl-4- (trifluoromethyl) phenyl) propan-2 (1H) -one, 4- ((2S, 5R) -2, 5-dimethyl-4- (trifluoromethyl) phenyl) propan-2 (1H) -one 1H) -ketone, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -6-ethoxy-1-methylpyridoo [3,2-d ] pyrimidin-2 (1H) -ketone, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6- (difluoromethyl) -1-methylpyridoo [3,2-d ] pyrimidin-2 (1H) -ketone, 6- (difluoromethyl) -4- ((2S, 5R) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methylpyridoo [3,2-d ] pyrimidin-2 (1H) -ketone, 4- ((2S, 5R) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6-hydroxy-1-methylpyridoo [3,2-d ] pyrimidin-1 (1H) -ketone, 6- (difluoromethyl) -4- ((2S, 5R) -5-ethyl-1-methylpyridoo [3,2-d ] pyrimidin-1-H) -ketone 4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -6- (difluoromethoxy) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one, 6-chloro-4- ((2S, 5R) -2, 5-dimethyl-4- (1- (3- (trifluoromethyl) bicyclo [1.1.1] pentan-1-yl) propyl) piperazin-1-yl) -1-methylpyrido [3,2-d ] pyrimidin-2 (1H) -one (diastereomeric mixture), 4- ((2S, 5R) -2, 5-dimethyl-4- (1- (3- (trifluoromethyl) bicyclo [1.1.1] pentan-1-yl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile; 4- ((2S, 5R) -4- (1-cyclopropylpropyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridazo [3,2-d ] pyrimidine-6-carbonitrile, 4- ((2S, 5R) -4- (1- (3, 3-difluorocyclobutyl) propyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridazo [3,2-d ] pyrimidine-6-carbonitrile, or 4- ((2S, 5R) -4- (1- (4, 4-difluorocyclohexyl) propyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyridazo [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is:

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (17-18).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -2, 5-diethyl-4- ((S) -1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -2, 5-diethyl-4- ((R) -1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is:

in one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (19-20).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -5-ethyl-2-methyl-4- ((S) -1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -5-ethyl-2-methyl-4- ((R) -1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is:

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -2, 5-diethyl-4- (1- (4- (trifluoromethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -2, 5-diethyl-4- ((S) -1- (4- (trifluoromethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -2, 5-diethyl-4- ((R) -1- (4- (trifluoromethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -5-ethyl-4- ((4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (21-22).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -5-ethyl-4- ((S) - (4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -5-ethyl-4- ((R) - (4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethoxy) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -5-ethyl-2-methyl-4- ((S) -1- (4- (trifluoromethoxy) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -5-ethyl-2-methyl-4- ((R) -1- (4- (trifluoromethoxy) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is:

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -5-ethyl-2-methyl-4- ((S) -1- (4- (trifluoromethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -5-ethyl-2-methyl-4- ((R) -1- (4- (trifluoromethoxy) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -5-ethyl-2-methyl-4- ((S) -1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -5-ethyl-2-methyl-4- ((R) -1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -4- ((4-chlorophenyl) (pyridin-2-yl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (23-24).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -4- ((R) - (4-chlorophenyl) (pyridin-2-yl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -4- ((S) - (4-chlorophenyl) (pyridin-2-yl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is:

in one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -4- ((3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (25-26).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -4- ((R) - (3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) (4-fluorophenyl) methyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -4- ((S) - (3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) (4-fluorophenyl) methyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is:

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -4- ((4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (27-28).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -4- ((S) - (4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -4- ((R) - (4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is:

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -4- (1- (4- (cyclopropylmethoxy) -2-fluorophenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (29-30).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -4- ((S) -1- (4- (cyclopropylmethoxy) -2-fluorophenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -4- ((R) -1- (4- (cyclopropylmethoxy) -2-fluorophenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is:

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s,5 r) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) butyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (31-32).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2S, 5 r) -2, 5-diethyl-4- ((S) -1- (4- (trifluoromethyl) phenyl) butyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 4- ((2 s, 5R) -2, 5-diethyl-4- ((R) -1- (4- (trifluoromethyl) phenyl) butyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is:

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 1-methyl-4- ((2 s,5 r) -2-methyl-5-propyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (33-34).

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 1-methyl-4- ((2S, 5 r) -2-methyl-5-propyl-4- ((S) -1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

In one embodiment, a compound of formula (II) or a salt thereof is administered, wherein the compound is 1-methyl-4- ((2 s, 5R) -2-methyl-5-propyl-4- ((R) -1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile.

2. Compositions and dosages

The compounds described herein (e.g., according to formula (I) or (II), such as a compound selected from compounds 1-34 and/or pharmaceutically acceptable salts thereof) may be administered by any means suitable for the disorder to be treated, which may depend on the need for treatment of the particular site or the amount of compound to be delivered.

Also included herein are pharmaceutical compositions comprising a compound, e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1 through 34 and/or pharmaceutically acceptable salts thereof, and one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants (collectively referred to herein as "carrier" materials) and, if desired, other active ingredients. The compound (e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34) may be administered by any suitable route, preferably in the form of a pharmaceutical composition suitable for such route, and in a dose effective for the intended treatment. The compounds and compositions described herein may be administered, for example, orally, transmucosally, or parenterally (including intravascularly, intravenously, intraperitoneally, subcutaneously, intramuscularly, and intraperitoneally) in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. For example, the pharmaceutical carrier may contain mannitol or lactose or a mixture of microcrystalline cellulose. The mixture may contain additional ingredients such as lubricants, for example, magnesium stearate and disintegrants such as crospovidone. The carrier mixture may be filled into gelatin capsules or compressed into tablets. The pharmaceutical composition may be administered as an oral dosage form or, for example, as an infusion.

The dosage regimen of the DGK inhibitor will vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration, the species, age, sex, health, physical condition and weight of the recipient, the nature and extent of the symptoms, the type of concurrent treatment, the frequency of treatment, the route of administration, and the renal and hepatic function of the patient.

In some embodiments, the inhibitor is administered in a therapeutically effective dose. In some embodiments, the therapeutically effective dose provides a dose of inhibitor that is therapeutically effective for a period of time.

As a general guidance, the daily oral dosage of each active ingredient, when used for the indicated effect, will range from about 0.001 to about 5000mg per day, preferably between about 0.01 to about 1000mg per day, most preferably between about 0.1 to about 250mg per day. For intravenous administration, the most preferred dosage range is from about 0.01 to about 10 mg/kg/minute during continuous rate infusion. DGK inhibitors as described herein may be administered in a single daily dose or the total daily dose may be administered in two, three or four times.

These compounds are typically administered in admixture with suitable pharmaceutically acceptable diluents, excipients or carriers (collectively referred to herein as pharmaceutically acceptable carriers) that are suitably selected with respect to the intended form of administration (e.g., oral tablets, capsules, elixirs and syrups) and are consistent with conventional pharmaceutical practices.

Dosage forms suitable for administration (pharmaceutical compositions) may contain from about 1mg to about 2000 mg of active ingredient per dosage unit. In these pharmaceutical compositions, the active ingredient is typically present in an amount of about 0.1 to 95% by weight, based on the total weight of the composition.

A typical capsule for oral administration contains at least one DGK inhibitor (250 mg), lactose (75 mg) and magnesium stearate (15 mg). The mixture was passed through a 60 mesh screen and filled into size L gelatin capsules.

A typical injectable formulation is prepared by aseptically placing at least one DGK inhibitor (250 mg) in a vial, aseptically lyophilizing and sealing. In use, the contents of the vial are mixed with 2mL of physiological saline to produce an injectable formulation.

For oral administration, the pharmaceutical compositions described herein may be in the form of, for example, tablets, capsules, liquid capsules, suspensions, or liquids. The pharmaceutical compositions are preferably formulated in dosage unit form containing a specified amount of the active ingredient. For example, the pharmaceutical composition may be provided as a tablet or capsule containing the active ingredient in an amount of about 0.1 to 1000mg, preferably about 0.25 to 250mg, and more preferably about 0.5 to 100mg. The appropriate daily dosage for a human or other mammal may vary widely depending on the condition of the patient and other factors, but may be determined using conventional methods.

Regardless of the route of administration selected, the DGK inhibitors (which may be used in the form of suitable hydrates and/or pharmaceutical compositions containing the same) for administration as described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The actual dosage level of the active ingredient in the pharmaceutical composition containing the DGK inhibitor may be varied to obtain an amount of the active ingredient that is effective to achieve a therapeutic response to the particular patient, composition and mode of administration and that is non-toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound employed or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician of ordinary skill in the art can readily determine and prescribe an effective amount of the pharmaceutical composition required. For example, the physician may start the dosage of DGK inhibitor used in the pharmaceutical composition from a dosage lower than that required to achieve a therapeutic effect and gradually increase the dosage until the effect is achieved.

Generally, a suitable daily dose of the DGK inhibitor will be the least effective dose to produce a therapeutic effect. Such effective dosages will generally depend on the factors described above. Generally, the dosage of DGK inhibitor for use in patients orally, intravenously, intraventriculially and subcutaneously ranges from about 0.01 to about 50 milligrams per kilogram of body weight per day. If desired, an effective daily dose of the active compound may be administered separately as two, three, four, five, six or more divided doses at appropriate intervals throughout the day, optionally in unit dosage forms. In certain aspects, the dose is administered once daily.

Any pharmaceutical composition contemplated herein may be delivered orally, e.g., via any acceptable and suitable oral formulation. Exemplary oral formulations include, but are not limited to, for example, tablets, troches, lozenges, aqueous and oily suspensions, dispersible powders or granules, emulsions, hard and soft capsules, liquid capsules, syrups and elixirs. Pharmaceutical compositions for oral administration may be prepared according to any method known in the art for preparing pharmaceutical compositions for oral administration. To provide a pharmaceutically palatable preparation, the pharmaceutical composition may contain at least one ingredient selected from the group consisting of sweetening agents, flavouring agents, colouring agents, demulcents, antioxidants and preserving agents.

Tablets may be prepared, for example, by mixing at least one compound (e.g. a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34) and/or at least one pharmaceutically acceptable salt thereof with at least one non-toxic pharmaceutically acceptable excipient suitable for use in the preparation of tablets. Exemplary excipients include, but are not limited to, inert diluents such as, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate and sodium phosphate, granulating and disintegrating agents such as, for example, microcrystalline cellulose, croscarmellose sodium, corn starch and alginic acid, binders such as, for example, starch, gelatin, polyvinylpyrrolidone and acacia, and lubricants such as, for example, magnesium stearate, stearic acid and talc. Furthermore, the tablets may be uncoated or coated by known techniques to mask the harsh taste of the off-taste drug or delay the disintegration and absorption of the active ingredient in the gastrointestinal tract, thereby allowing the effect of the active ingredient to be maintained for a longer period of time. Exemplary water-soluble taste masking materials include, but are not limited to, hydroxypropyl methylcellulose and hydroxypropyl cellulose. Exemplary time delay materials include, but are not limited to, ethylcellulose and cellulose acetate butyrate.

Hard capsules may be prepared, for example, by mixing at least one compound (e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34) and/or at least one pharmaceutically acceptable salt thereof, with at least one inert solid diluent such as, for example, calcium carbonate, calcium phosphate and kaolin.

Soft capsules may be prepared, for example, by mixing at least one compound (e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34) and/or at least one pharmaceutically acceptable salt thereof, with at least one water-soluble carrier such as, for example, polyethylene glycol, and at least one oil medium such as, for example, peanut oil, liquid paraffin, and olive oil.

The aqueous suspension may be prepared, for example, by mixing at least one compound (e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34) and/or at least one pharmaceutically acceptable salt thereof, with at least one excipient suitable for use in preparing an aqueous suspension. Exemplary excipients suitable for use in preparing the aqueous suspension include, but are not limited to, suspending agents (such as, for example, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, alginic acid, polyvinylpyrrolidone, tragacanth, and acacia), dispersing or wetting agents (such as, for example, naturally-occurring phospholipids, for example, lecithin, condensation products of alkoxides with fatty acids, such as, for example, polyoxyethylene stearate, condensation products of ethylene oxide with long chain fatty alcohols, such as, for example, heptadecalkoxyethanol, condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, such as, for example, polyoxyethylene sorbitol monooleate, and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, such as, for example, polyoxyethylene sorbitan monooleate, aqueous suspensions may also contain at least one preservative, such as, for example, ethyl p-hydroxybenzoate and n-propyl p-hydroxybenzoate, at least one coloring agent, at least one flavoring agent, and/or at least one sweetener, including, but not limited to, for example, sucrose, saccharin and aspartame.

Oily suspensions may be prepared, for example, by suspending at least one compound (e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34) and/or at least one pharmaceutically acceptable salt thereof in a vegetable oil (such as, for example, peanut oil, olive oil, sesame oil and coconut oil) or in a mineral oil (such as, for example, liquid paraffin). The oily suspensions may also contain at least one thickening agent, such as, for example, beeswax, hard paraffin or cetyl alcohol. To provide a palatable oily suspension, at least one of the above-mentioned sweeteners and/or at least one flavoring agent may be added to the oily suspension. The oily suspension may further contain at least one preservative including, but not limited to, antioxidants such as, for example, butylated hydroxyanisole and alpha-tocopherol.

Dispersible powders and granules can be prepared, for example, by mixing at least one compound (e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34) and/or at least one pharmaceutically acceptable salt thereof with at least one dispersing and/or wetting agent, at least one suspending agent, and/or at least one preservative. Suitable dispersing, wetting and suspending agents are described above. Exemplary preservatives include, but are not limited to, for example, antioxidants (e.g., ascorbic acid). In addition, the dispersible powders and granules may also contain at least one excipient including, but not limited to, for example, sweeteners, flavoring agents and coloring agents.

Emulsions of at least one compound (e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34) and/or at least one pharmaceutically acceptable salt thereof, may be prepared, for example, as oil-in-water emulsions. The oil phase of an emulsion comprising a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34, may be constituted by known ingredients in a known manner. The oily phase may be provided by, for example, but not limited to, vegetable oils (such as, for example, olive oil and peanut oil), mineral oils (such as, for example, liquid paraffin), and mixtures thereof. While the phase may contain only an emulsifier, it may contain a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil. Suitable emulsifiers include, but are not limited to, for example, naturally occurring phospholipids (e.g., soy lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (such as, for example, sorbitan monooleate) and condensation products of partial esters with ethylene oxide (such as, for example, polyoxyethylene sorbitan monooleate). Preferably, a hydrophilic emulsifier is used together with a lipophilic emulsifier as a stabilizer. It is also preferred to include both oil and fat. The emulsifying agent (whether or not containing a stabilizer) together form a so-called emulsifying wax, and the emulsifying wax together with the oil and fat form an emulsifying ointment base called the oily dispersed phase forming the cream formulation. The emulsion may also contain sweeteners, flavoring agents, preservatives and/or antioxidants. Emulsifying agents and emulsion stabilizers suitable for use in formulations of the methods of treatment include Tween (Tween) 60, span (Span) 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate, alone or with waxes or other materials well known in the art.

The compound (e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1-34) and/or at least one pharmaceutically acceptable salt thereof, may also be delivered intravenously, subcutaneously, and/or intramuscularly, e.g., via any pharmaceutically acceptable and suitable injection form. Exemplary injectable forms include, but are not limited to, sterile aqueous solutions, sterile oil-in-water microemulsions, and aqueous or oily suspensions, for example, containing acceptable carriers and solvents such as water, ringer's solution, and isotonic sodium chloride solution.

Formulations for parenteral administration may be in the form of aqueous or nonaqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules using one or more of the carriers or diluents mentioned in the formulations for oral administration, or by using other suitable dispersing or wetting agents and suspending agents. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth, and/or various buffers. Other adjuvants and modes of administration are widely known in the pharmaceutical arts. The active ingredient may also be administered by injection as a composition with a suitable carrier, including saline, dextrose, or water, or with cyclodextrin (i.e., captisol), cosolvent solubilisation (i.e., propylene glycol), or micelle solubilisation (i.e., tween 80).

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be used are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (such as oleic acid) find use in the preparation of injectables.

Sterile injection oil-in-water microemulsions may be prepared, for example, by 1) dissolving at least one compound (e.g., a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34) and/or at least one pharmaceutically acceptable salt thereof in an oil phase (such as, for example, a mixture of soybean oil and lecithin), 2) combining the oil phase containing the compound of formula (I) and/or a pharmaceutically acceptable salt thereof with a mixture of water and glycerin, and 3) treating the mixture to form a microemulsion.

Sterile aqueous or oily suspensions may be prepared according to methods known in the art. For example, sterile aqueous solutions or suspensions may be prepared using non-toxic parenterally acceptable diluents or solvents such as, for example, 1, 3-butanediol, and sterile oily suspensions may be prepared using sterile non-toxic acceptable solvents or suspension media such as, for example, sterile fixed oils, for example, synthetic mono-or diglycerides, and fatty acids such as, for example, oleic acid.

Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) (such as d-alpha-tocopheryl polyethylene glycol 1000 succinate), surfactants for pharmaceutical dosage forms (such as tween), polyethoxylated castor oil (such as CREMOPHOR surfactant, BASF) or other similar polymer delivery matrices, serum proteins (such as human serum albumin), buffer substances (such as phosphate, glycine, sorbic acid, potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protein-amine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. Cyclodextrins (such as α -, β -and γ -cyclodextrins) or chemically modified derivatives (such as hydroxyalkyl cyclodextrins, including 2-and 3-hydroxypropyl-cyclodextrins) or other solubilizing derivatives may also be advantageously used to enhance delivery of the compounds of the formulas described herein.

The pharmaceutically active compounds described herein can be processed according to pharmaceutically conventional methods to produce pharmaceutical formulations for administration to patients (including humans and other mammals). The pharmaceutical compositions may be subjected to conventional pharmaceutical procedures (such as sterilization) and/or may contain conventional excipients (such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.). Tablets and pills may also be prepared with enteric coatings. These compositions may also contain adjuvants such as wetting agents, sweeteners, flavoring agents and perfuming agents.

The amount of compound administered and the dosage regimen of the compounds and/or compositions described herein in treating a disease will depend upon a variety of factors including the age, weight, sex, medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the particular compound used. Thus, dosing regimens may vary widely, but may be routinely determined using standard methods. The daily dosage may be about 0.001 to 100mg/kg body weight, preferably about 0.0025 to about 50mg/kg body weight, and most preferably about 0.005 to 10mg/kg body weight. The daily dose may be administered in one to four doses per day. Other dosing regimens include once weekly doses and once every two day cycle.

For therapeutic purposes, the active compounds described herein are generally combined with one or more adjuvants appropriate to the indicated route of administration. If administered orally, the compounds may be mixed with lactose, sucrose, starch powder, cellulose alkanoates, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone and/or polyvinyl alcohol and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain the controlled release formulation provided by a dispersion of the active compound in hydroxypropyl methylcellulose.

The pharmaceutical compositions described herein comprise at least one compound (e.g., a compound of formula (I)) and/or at least one pharmaceutically acceptable salt thereof and optionally additives selected from any pharmaceutically acceptable carrier, adjuvant, and vehicle. Alternative compositions described herein comprise a compound, such as a compound of formula (I) or (II), such as a compound selected from compounds 1 to 34 described herein, or a prodrug thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

In some embodiments, administration of the dgkα and/or dgkζ inhibitors begins before, after, during, simultaneously with, substantially simultaneously with, sequentially with, and/or intermittently with administration of a cell therapy (such as a T cell therapy). In some embodiments, the methods involve the administration of dgkα and/or dgkζ inhibitors beginning prior to administration of T cell therapy. In other embodiments, the methods involve administration of dgkα and/or dgkζ inhibitors beginning after administration of T cell therapy. In some embodiments, in other embodiments, the methods involve the administration of dgkα and/or dgkζ inhibitors beginning on the same day as the administration of T cell therapy (e.g., day 1 of combination therapy). In some embodiments, the methods involve the administration of a dgkα and/or dgkζ inhibitor beginning simultaneously with the administration of T cell therapy. In some embodiments, administration of the inhibitor begins during administration of the T cell therapy, e.g., a first dose of the inhibitor is administered at the time of T cell therapy infusion.

In some embodiments, the dgkα and/or dgkζ inhibitors are administered on an intermittent dosing regimen. In some embodiments, the intermittent dosing regimen involves non-daily administration of a dgkα and/or dgkζ inhibitor. In some embodiments, the intermittent dosing regimen involves periodic administration of a dgkα and/or dgkζ inhibitor.

In some embodiments, the dgkα and/or dgkζ inhibitor is administered periodically. In some embodiments, the cycle comprises an administration period in which the dgkα and/or dgkζ inhibitor is administered followed by a rest period in which the dgkα and/or dgkζ inhibitor is not administered. In some embodiments, the total number of days of the cycle (e.g., from the beginning of administration of the dgkα and/or dgkζ inhibitor) is greater than or about 21 days, 28 days, 30 days, 40 days, 50 days, 60 days, or more.

In some embodiments, the initiation of administration of the dgkα and/or dgkζ inhibitor and the initiation of administration of the T cell therapy are performed on the same day during at least one cycle, optionally simultaneously. In some embodiments, the beginning of the administration of the dgkα and/or dgkζ inhibitor for at least one cycle occurs prior to the beginning of the administration of the T cell therapy. In some embodiments, the start of administration of the dgkα and/or dgkζ inhibitor for at least one cycle is performed simultaneously or on the same day as the start of administration of the T cell therapy. In some embodiments, the methods involve the initiation of administration of the dgkα and/or dgkζ inhibitor on the same day as the administration of the T cell therapy (e.g., on day 1 of the combination therapy). In some embodiments, the administration of the inhibitor begins on day 1 of the combination therapy prior to the administration of the T cell therapy.

In some embodiments, the dgkα and/or dgkζ inhibitor is administered from or about 0to 30 days, such as 0to 15 days, 0to 6 days, 0to 96 hours, 0to 24 hours, 0to 12 hours, 0to 6 hours or 0to 2 hours, 2 hours to 15 days, 2 hours to 6 days, 2 hours to 96 hours, 2 hours to 24 hours, 2 hours to 12 hours, 2 hours to 6 hours, 6 hours to 30 days, 6 hours to 15 days, 6 hours to 6 days, 6 hours to 96 hours, 6 hours to 24 hours, 6 hours to 12 hours, 12 hours to 30 days, 12 hours to 15 days, 12 hours to 6 days, 12 hours to 96 hours, 12 hours to 24 hours, 24 hours to 30 days, 24 hours to 15 days, 24 hours to 6 days, 24 hours to 96 hours, 96 hours to 96 days, 96 hours to 30 days, 96 hours to 96 days, 6 hours to 30 days, 6 hours to 96 days, 6 days to 30 days, 6 days, 15 days, or 15 days before initiation of T cell therapy. In some aspects, the dgkα and/or dgkζ inhibitor is administered no more than about 96 hours, 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 2 hours, or 1 hour prior to initiation of T cell therapy.

In some of any such embodiments, the dgkα and/or dgkζ inhibitor is administered prior to the cell therapy (e.g., T cell therapy), and administration of the dgkα and/or dgkζ inhibitor is continued at regular intervals until the start of the cell therapy (e.g., T cell therapy) and/or for a period of time after the start of the cell therapy (e.g., T cell therapy).

In some embodiments, the dgkα and/or dgkζ inhibitor is administered after administration of the cell therapy (e.g., T cell therapy), or further administered. In some embodiments, the dgkα and/or dgkζ inhibitor is administered within 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 96 hours, 4 days, 5 days, 6 days or 7 days, 14 days, 15 days, 21 days, 24 days, 28 days, 30 days, 36 days, 42 days, 60 days, 72 days or 90 days or within about 1 hour, about 2 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 96 hours, about 4 days, about 5 days, about 6 days or about 7 days, about 14 days, about 15 days, about 21 days, about 24 days, about 28 days, about 30 days, about 36 days, about 42 days, about 60 days, about 72 days or within about 90 days after initiation of administration of the cell therapy (e.g., T cell therapy). In some embodiments, the provided methods involve continuous (such as at regular intervals) administration of the dgkα and/or dgkζ inhibitors after the initiation of administration of the cell therapy.

In some embodiments, the dgkα and/or kζ inhibitor is administered at most or at most about 1 day, at most or at most about 2 days, at most or at most about 3 days, at most or at most about 4 days, at most or at most about 5 days, at most or at most about 6 days, at most or at most about 7 days, at most or at most about 12 days, at most or at most about 14 days, at most or at most about 21 days, at most or at most about 24 days, at most or at most about 28 days, at most or at most about 30 days, at most or at most about 35 days, at most or at most about 42 days, at most or at most about 60 days, at most or at most about 90 days, at most or at most about 120 days, at most or at most about 180 days, at most or at most about 240 days, at most or at most about 360 days or at most about 720 days or longer after the initiation of administration of the cell therapy (e.g., T cell therapy).

In some embodiments, the administration of the inhibitor begins on days 1-6 (including days 1 and 6) of the combination therapy. In some embodiments, the administration of the inhibitor begins on days 1-4 (including days 1 and 4) of the combination therapy. In some embodiments, the administration of the inhibitor begins on day 1 or day 2 of the combination therapy. In some embodiments, the administration of the inhibitor begins on day 1 of the combination therapy. In some embodiments, day 1 of the combination therapy is the day of administration of the T cell therapy.

In some embodiments, administration of the inhibitor begins within 12 hours or about 12 hours of administration of the T cell therapy. In some embodiments, administration of the inhibitor begins within 6 hours or about 6 hours of administration of the T cell therapy. In some embodiments, administration of the inhibitor begins within 4 hours or about 4 hours of administration of the T cell therapy. In some embodiments, administration of the inhibitor begins within 2 hours or about 2 hours of administration of the T cell therapy. In some embodiments, administration of the inhibitor begins within 1 hour or about 1 hour of administration of the T cell therapy.

In some of any of the above embodiments, the dgkα and/or dgkζ inhibitor is administered before and after the initiation of administration of the cell therapy (e.g., T cell therapy).

In some embodiments, the administration of the dgkα and/or dgkζ inhibitor is initiated at or after a detectable peak or maximum level of cells of the T cell therapy in the blood of the subject, optionally immediately thereafter or within 1 to 3 days thereafter. In some embodiments, the detectable number of cells of the T cell therapy in the blood is undetectable or reduced after being detectable in the blood, optionally at or after a reduction compared to the previous time point after T administration of the cell therapy, optionally immediately thereafter or within 1 to 3 days thereafter, starting administration of the dgkα and/or dgkζ inhibitor. In some embodiments, the administration of the dgkα and/or dgkζ inhibitor is initiated immediately or within 1 to 3 days after or at a time or after the decrease in the cell number of the detectable T cell therapy in the blood, at a peak or maximum level or more than 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold or more of the cell number of the detectable T cell therapy in the blood of the subject after the initiation of administration of the T cell therapy. In some embodiments, administration of the dgkα and/or dgkζ inhibitor is initiated immediately after or within 1 to 3 days after a peak or maximum level of detectable cell number of T cell therapy in the subject's blood, when the detectable cell number of T cells or the number of T cell-derived cells in the subject's blood is less than 10%, less than 5%, less than 1%, or less than 0.1% of total Peripheral Blood Mononuclear Cells (PBMCs) in the subject's blood. In some embodiments, the number of cells of the T cell therapy detectable in the blood that is depleted after the start of administration of the T cell therapy is increased, e.g., by a peak or maximum number of cells of the T cell therapy detectable in the blood of a non-depleted subject or more than 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, more fold after 10-fold, or after, optionally immediately after or within 1 to 3 days after, the start of administration of the dgkα and/or dgkζ inhibitor. In some embodiments, the administration of the dgkα and/or dgkζ inhibitor is initiated immediately thereafter or within 1 to 3 days thereafter, optionally when or after the subject exhibits disease progression and/or post-remission recurrence following treatment with T cell therapy. In some embodiments, the administration of the dgkα and/or dgkζ inhibitor is initiated immediately or within 1 to 3 days after or after the subject exhibits an increased tumor burden as compared to the tumor burden at a time before or after the administration of the T cells and before the initiation of the administration of the dgkα and/or dgkζ inhibitor.

In some embodiments, the administration of the dgkα and/or dgkζ inhibitor is initiated at least one cycle after the initiation of the administration of the T cell therapy. In some embodiments, the administration of the dgkα and/or dgkζ inhibitor is initiated at least or about at least 1 day, at least or about at least 2 days, at least or about at least 3 days, at least or about at least 4 days, at least or about at least 5 days, at least or about at least 6 days, at least or about at least 7 days, at least or about at least 8 days, at least or about at least 9 days, at least or about at least 10 days, at least or about at least 12 days, at least or about at least 14 days, at least or about 15 days, at least or about at least 21 days, at least or about 24 days, at least or about at least 28 days, at least or about at least 30 days, at least or about at least 35 days, or at least about at least 42 days, at least or about at least 60 days, or at least or about at least 90 days after the initiation of the administration of the T cell therapy. In some embodiments, the onset of administration of the dgkα and/or dgkζ inhibitor is at least 2 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks after the onset of administration of the T cell therapy. In some embodiments, the onset of administration of the dgkα and/or dgkζ inhibitor occurs 2 to 35 days after the onset of administration of the T cell therapy. In some embodiments, the onset of administration of the dgkα and/or dgkζ inhibitor occurs 2 to 28 days after the onset of administration of the T cell therapy. In some embodiments, the onset of administration of the dgkα and/or dgkζ inhibitor occurs 2 to 21 days after the onset of administration of the T cell therapy. In some embodiments, the onset of administration of the dgkα and/or dgkζ inhibitor occurs 2 to 14 days after the onset of administration of the T cell therapy. In some embodiments, the onset of administration of the dgkα and/or dgkζ inhibitor occurs 7 to 21 days after the onset of administration of the T cell therapy. In some embodiments, the onset of administration of the dgkα and/or dgkζ inhibitor occurs 7 to 14 days after the onset of administration of the T cell therapy. In some embodiments, the initiation of administration of the dgkα and/or dgkζ inhibitor is performed at a time greater than or greater than about 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 24 days, or 28 days after initiation of administration of the T cell therapy.

In some embodiments, the dgkα and/or dgkζ inhibitor is administered several times a day, two days a day, daily, every other day, three times a week, twice a week, or once a week after the initiation of cell therapy. In some embodiments, the dgkα and/or dgkζ inhibitor is administered every three days. In some embodiments, the dgkα and/or dgkζ inhibitor is administered every two days. In some embodiments, the dgkα and/or dgkζ inhibitor is administered daily, such as in a continuous dosing regimen. In some embodiments, the dgkα and/or dgkζ inhibitor is administered twice daily. In some embodiments, the dgkα and/or dgkζ inhibitor is administered three times daily. In other embodiments, the dgkα and/or dgkζ inhibitor is administered every other day. In some embodiments, the dgkα and/or dgkζ inhibitor is administered during administration for a plurality of consecutive days (such as up to about 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 consecutive days). In some embodiments, the dgkα and/or dgkζ inhibitor is administered for greater than or about 7 consecutive days, greater than or about 14 consecutive days, greater than or about 21 consecutive days, or greater than or about 28 consecutive days. In some embodiments, the dgkα and/or dgkζ inhibitor is administered during an administration period of up to 21 consecutive days. In some embodiments, the dgkα and/or dgkζ inhibitor is administered during an administration period of up to 21 consecutive days, wherein the period comprises more than 30 days, starting with the beginning of the administration of the dgkα and/or dgkζ inhibitor.

In some embodiments, the dgkα and/or dgkζ inhibitor is administered at least until the number of engineered T cells of the T cell therapy reaches a peak in the subject following administration of the T cell therapy. In some embodiments, the dgkα and/or dgkζ inhibitor is administered until the number of engineered T cells of the T cell therapy reaches a peak in the subject following administration of the T cell therapy.

In some embodiments, the dgkα and/or dgkζ inhibitor is optionally administered over a plurality of cycles, for a period of time between or between about 21 and 42 days (including 21 and 42 days). In some embodiments, the inhibitor is optionally administered over a plurality of cycles, over a period of time between or between about 21 and 35 days (including 21 and 35 days). In some embodiments, the inhibitor is optionally administered over a plurality of cycles, over a period of time between or between about 21 and 28 days (including 21 and 28 days). In some embodiments, the inhibitor is optionally administered over a plurality of cycles, over a period of time between or between about 28 and 42 days (including 28 and 42 days). In some embodiments, the inhibitor is optionally administered over a plurality of cycles, over a period of time between or between about 28 and 35 days (including 28 and 35 days). In some embodiments, the inhibitor is optionally administered over a plurality of cycles, over a period of time of at or about 28 days. In some embodiments, the inhibitor is optionally administered over a plurality of cycles, over a period of time of at or about 30 days.

In some embodiments, the dgkα and/or dgkζ inhibitor is administered during no more than about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or no more than 30 consecutive days of administration. In certain embodiments, the dgkα and/or dgkζ inhibitor is administered once daily for 14 days over a 21 day treatment period. In certain embodiments, the dgkα and/or dgkζ inhibitor is administered once daily for 21 days over a 28 day treatment period. In some embodiments, the dgkα and/or dgkζ inhibitor is administered during no more than 14 consecutive days of administration.

In some embodiments, the dgkα and/or dgkζ inhibitor is administered in a cycle, wherein the cycle comprises a rest period of days of administration of the dgkα and/or dgkζ inhibitor followed by no administration of the dgkα and/or dgkζ inhibitor. In some embodiments, the rest period is more than about 1 day, more than about 3 days, more than about 5 days, more than about 7 days, more than about 8 days, more than about 9 days, more than about 10 days, more than about 11 days, more than about 12 days, more than about 13 days, more than about 14 days, more than about 15 days, more than about 16 days, more than about 17 days, more than about 18 days, more than about 19 days, more than about 20 days, or more than about 21 days or more. In some embodiments, the rest period is more than 7 consecutive days, more than 14 consecutive days, more than 21 days, or more than 28 days. In some embodiments, the rest period is greater than about 14 consecutive days. In some embodiments, the administration cycle of the dgkα and/or dgkζ inhibitor does not contain a rest period.

In some embodiments, the dgkα and/or dgkζ inhibitor is administered in a cycle, wherein the cycle is repeated at least once. In some embodiments, the dgkα and/or dgkζ inhibitor is administered for at least 2 cycles, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, at least 7 cycles, at least 8 cycles, at least 9 cycles, at least 10 cycles, at least 11 cycles, or at least 12 cycles. In some embodiments, the dgkα and/or dgkζ inhibitor is administered for 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 cycles.

In some embodiments, the dgkα and/or dgkζ inhibitor is administered six times a day, five times a day, four times a day, three times a day, two times a day, once a day, every other day, every third day, twice a week, once a week, or only once a day, before or after starting to administer the T cell therapy. In some embodiments, the dgkα and/or dgkζ inhibitor is administered once daily of the inhibitor. In some embodiments, the dgkα and/or dgkζ inhibitor is administered at a plurality of doses at regular intervals before, during, and/or after the T cell therapy administration period. In some embodiments, the dgkα and/or dgkζ inhibitor is administered at one or more doses at regular intervals prior to administration of the T cell therapy. In some embodiments, the dgkα and/or dgkζ inhibitor is administered at one or more doses at regular intervals after administration of the T cell therapy. In some embodiments, one or more doses of the dgkα and/or dgkζ inhibitor may be concurrent with the dose administered for T cell therapy.

In some embodiments, the methods involve administering T cell therapy to a subject who has previously been administered a therapeutically effective amount of a dgkα and/or dgkζ inhibitor. In some embodiments, the dgkα and/or dgkζ inhibitor is administered to the subject prior to administering a dose of cells expressing the recombinant receptor to the subject. In some embodiments, treatment with a dgkα and/or dgkζ inhibitor occurs simultaneously with administration of the dose of cells. In some embodiments, the dgkα and/or dgkζ inhibitor is administered after the dose of cells is administered.

In some embodiments, the dgkα and/or dgkζ inhibitor is administered daily for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or more than 21 days. In some embodiments, the dgkα and/or dgkζ inhibitor is administered twice daily for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or more than 21 days. In some embodiments, the dgkα and/or dgkζ inhibitor is administered three times daily for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or more than 21 days. In some embodiments, the dgkα and/or dgkζ inhibitor is administered every other day for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or more than 21 days.

In some embodiments of the methods provided herein, the dgkα and/or dgkζ inhibitor and the T cell therapy are administered simultaneously or near simultaneously. In certain embodiments, the dgkα and/or dgkζ inhibitors (duration and amount therapeutically sufficient to at least partially reverse the T cell depleted state of T cell therapy) are administered to the patient at one time.

C. Administration of checkpoint inhibitor antagonists

In some of any of the preceding embodiments, the combination therapy (e.g., a combination therapy comprising administration of a dgkα and/or dgkζ inhibitor and administration of a T cell therapy) further comprises administration of a checkpoint antagonist (also referred to as a checkpoint inhibitor) to a subject suffering from a disease or disorder. In some embodiments, the checkpoint antagonist is administered prior to (prior to), concurrently with, or subsequent to (subsequent to or subsequent to) administration of the dgkα and/or dgkζ inhibitor. In some embodiments, the checkpoint antagonist is administered prior to (prior to), concurrently with, or subsequent to (subsequent to or subsequent to) administration of the T cell therapy.

In some embodiments, the combination therapy comprises administration of a dgkα and/or dgkζ inhibitor, administration of a T cell therapy, and administration of an antagonist of a checkpoint inhibitor. In some embodiments, the combination therapy comprises administration of a dgkα and/or dgkζ inhibitor, administration of a T cell therapy, and administration of two or more antagonists of one or more checkpoint inhibitors.

In some embodiments, the checkpoint antagonist is an antagonist or inhibitor of the PD1/PD-L1 axis. In some embodiments, the checkpoint antagonist is an antagonist or inhibitor of CTLA-4. In some embodiments, two or more antagonists may be administered, one of which is an antagonist or inhibitor of the PD1/PD-L1 axis and the other is an antagonist or inhibitor of CTLA-4.

In some embodiments, the combination therapy comprises administration of a dgkα and/or dgkζ inhibitor, administration of a T cell therapy, and administration of an antagonist of the PD1/PD-L1 axis. In some embodiments, the combination therapy comprises administration of a dgkα and/or dgkζ inhibitor, administration of a T cell therapy, and administration of a CTLA4 antagonist. In some embodiments, the combination therapy comprises administration of a dgkα and/or dgkζ inhibitor, administration of a T cell therapy, administration of an antagonist of the PD1/PD-L1 axis, and administration of a CTLA4 antagonist.

In some embodiments, the antagonist of the PD1/PD-L1 axis is nal Wu Liyou mab. In some embodiments, the antagonist of the checkpoint is ipilimumab. The checkpoint inhibitor may be any of those described in PCT/US202066198, the entire contents of which are incorporated by reference in their entirety.

D. Lymphocyte removal therapy

In some aspects, provided methods may further comprise administering one or more lymphocyte removal therapies, such as prior to or concurrent with the beginning of administration of the T cell therapy. In some embodiments, the lymphocyte removal therapy comprises administration of a phosphoramide, such as cyclophosphamide. In some embodiments, the lymphocyte removal therapy may comprise administration of fludarabine.

In some aspects, pretreatment of a subject with an immune clearance (e.g., lymphocyte clearance) therapy can improve the efficacy of Adoptive Cell Therapy (ACT). Pretreatment with lymphocyte scavengers (including a combination of cyclosporine and fludarabine) is effective in increasing the efficacy of metastatic Tumor Infiltrating Lymphocytes (TILs) in cell therapy, including improving the response and/or persistence of metastatic cells. See, e.g., dudley et al, science,298,850-54 (2002); rosenberg et al, CLIN CANCER RES,17 (13): 4550-4557 (2011). Similarly, in the case of car+ T cells, some studies have introduced lymphocyte scavengers, most commonly cyclophosphamide, fludarabine, bendamustine, or combinations thereof, sometimes with low dose irradiation. See, see Han et al Journal of Hematology & Oncology,6:47 (2013), kochenderfer et al Blood,119:2709-2720 (2012), kalos et al SCI TRANSL MED,3 (95): 95ra73 (2011), CLINICAL TRIAL Study Record nos.: NCT02315612, NCT01822652.

Such pretreatment may be targeted to reduce the risk of one or more of the various outcomes that may inhibit the therapeutic effect. These include the phenomenon known as "cytokine sink", i.e., T cells, B cells, NK cells compete with TIL for homeostasis and activate cytokines such as IL-2, IL-7 and/or IL-15, inhibit TIL by regulatory T cells, NK cells or other cells of the immune system, and affect negative regulators in the tumor microenvironment. Muranski et al, NAT CLIN PRACT Oncol. December, 3 (12): 668-681 (2006).

Thus in some embodiments, the methods provided further involve administering to the subject a lymphocyte removal therapy. In some embodiments, the method involves administering lymphocyte removal therapy to the subject prior to administering the dose of cells. In some embodiments, the lymphocyte removal therapy comprises a chemotherapeutic agent, such as fludarabine and/or cyclophosphamide. In some embodiments, the administration of the cell and/or the lymphocyte removal therapy is via an outpatient delivery.

In some embodiments, the method comprises administering a pretreatment agent, such as a lymphocyte scavenger or a chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, to the subject prior to administering the dose of cells. For example, the subject may administer the pretreatment agent at least 2 days, such as at least 3, 4, 5, 6, or 7 days, prior to the first or subsequent dose. In some embodiments, the subject may administer the pretreatment agent no more than 7 days, such as no more than 6, 5, 4, 3, or 2 days prior to administration of the dose of cells.

In some embodiments, the subject is pretreated with cyclophosphamide at a dose of between or about 20mg/kg and 100mg/kg, such as between or about 40mg/kg and 80 mg/kg. In some aspects, the subject is pretreated with or with about 60mg/kg cyclophosphamide. In some embodiments, the fludarabine may be administered in a single dose or in multiple doses, such as daily administration, every other day, or every third day. In some embodiments, the cyclophosphamide is administered once daily for one or two days.

In some embodiments, when the lymphocyte scavenger comprises fludarabine, the subject is administered fludarabine at a dose between or about 1mg/m² and 100mg/m², such as between or about 10mg/m² and 75mg/m², 15mg/m² and 50mg/m², 20mg/m² and 30mg/m², or 24mg/m² and 26mg/m². In some cases, 25mg/m² fludarabine is administered to the subject. In some embodiments, fludarabine may be administered in a single dose or in multiple doses, such as daily, every other day, or every third day. In some embodiments, fludarabine is administered once daily, such as for 1-5 days, e.g., for 3 to 5 days.

In some embodiments, the lymphocyte scavenger comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or administration regimen, such as those described above, and fludarabine at any dose or administration regimen, such as those described above. For example, in some aspects, 60mg/kg (2 g/m²) cyclophosphamide and 3 to 5 doses of 25mg/m² fludarabine are administered to the subject prior to the dose of cells.

In some embodiments, administration of the pretreatment agent prior to infusion of the dose of cells improves the outcome of the treatment. For example, in some aspects, the pretreatment improves the efficacy of the dose of treatment or increases the persistence of recombinant receptor-expressing cells (e.g., CAR-expressing cells, such as CAR-expressing T cells) in the subject. In some embodiments, the pretreatment treatment increases disease-free survival, such as the percentage of subjects that survive a given period of time after the dose of cells and that do not exhibit minimal residual or molecularly detectable disease. In some embodiments, the time to reach median disease-free survival is increased.

Once the cells are administered to a subject (e.g., human), the biological activity of the engineered cell population in certain aspects is measured by any of a number of known methods. Parameters assessed include specific binding of engineered T cells or natural T cells or other immune cells to antigen in vivo (e.g., by imaging) or in vitro (e.g., by ELISA or flow cytometry). In certain embodiments, the ability of an engineered cell to destroy a target cell can be measured using any suitable method known in the art, such as cytotoxicity assays described, for example, in Kochenderfer et al, J.Immunotherapy,32 (7): 689-702 (2009) and Herman et al, J.Immunol Methods,285 (1): 25-40 (2004). In certain embodiments, the biological activity of a cell may also be measured by assaying the expression and/or secretion of certain cytokines (such as CD107a, IFNγ, IL-2, and TNF). In some aspects, biological activity is measured by assessing clinical outcome (such as a decrease in tumor burden or burden). In some aspects, toxicity results, cell persistence, and/or expansion and/or presence or absence of a host immune response are evaluated.

In some embodiments, administration of a pretreatment agent prior to infusion of the dose of cells improves the outcome of the treatment, such as improving the efficacy of the dose of treatment or increasing the persistence of recombinant receptor-expressing cells (e.g., TCR or CAR-expressing cells) in the subject. Thus, in some embodiments, the pretreatment agent is administered in a method of combination therapy with a DGK inhibitor and cell therapy at a higher dose than in a method in the absence of a DGK inhibitor.

T cell therapy and engineered cells

In some embodiments, T cell therapies for use in accordance with the provided combination therapy methods include administering recombinant receptor expression engineered cells designed to recognize and/or specifically bind molecules associated with a disease or condition and cause a response, such as an immune response to such molecules when bound thereto. The receptor may include chimeric receptors, for example, chimeric Antigen Receptors (CARs) and other transgenic antigen receptors including transgenic T Cell Receptors (TCRs).

In some embodiments, the cells contain or are engineered to contain an engineered receptor, e.g., an engineered antigen receptor, such as a Chimeric Antigen Receptor (CAR) or a T Cell Receptor (TCR). Populations of these cells, compositions containing these cells and/or enriched for these cells (such as enriching or selecting a certain type of cell, such as T cells or CD8⁺ or CD4⁺ cells) are also provided. The compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Therapeutic methods for administering the cells and compositions to a subject (e.g., a patient) are also provided.

Thus, in some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thus express recombinant or genetically engineered products of these nucleic acids. In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by binding it to a stimulus that induces a response (such as proliferation, survival, and/or activation) (e.g., as measured by expression of a cytokine or activation marker), followed by transduction of the activated cell, and expansion in culture to an amount sufficient for clinical use.

A. Recombinant receptors

The cells typically express recombinant receptors, such as antigen receptors including non-functionalized TCR antigen receptors, e.g., chimeric Antigen Receptors (CARs) and other antigen binding receptors such as transgenic T Cell Receptors (TCRs). Receptors also include other chimeric receptors.

1. Chimeric Antigen Receptor (CAR)

In some embodiments, the engineered cells (such as T cells) used in the provided embodiments express a CAR that is specific for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or over-expressed on cells of a disease or condition (e.g., tumor or pathogenic cells) as compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or on engineered cells.

In particular embodiments, the recombinant receptor (such as a chimeric receptor) contains an intracellular signaling region comprising a cytoplasmic signaling domain or region (also interchangeably referred to as an intracellular signaling domain or region), such as a cytoplasmic (intracellular) region capable of inducing a T cell primary activation signal, e.g., a cytoplasmic signaling domain or region of a zeta chain of a T Cell Receptor (TCR) component (e.g., a CD 3-zeta (CD 3 zeta) chain, or a functional variant thereof, or a signaling portion thereof) and/or a cytoplasmic signaling domain or region comprising an immune receptor tyrosine activation motif (ITAM).

In some embodiments, the chimeric receptor further comprises an extracellular ligand binding domain that specifically binds to a ligand (e.g., antigen) antigen. In some embodiments, the chimeric receptor is a CAR that contains an extracellular antigen recognition domain that specifically binds an antigen. In some embodiments, the ligand (such as an antigen) is a cell surface expressed protein.

In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen (such as a peptide antigen of an intracellular protein), which is recognized on the cell surface in the case of a Major Histocompatibility Complex (MHC) molecule, similar to a TCR. In general, CARs containing antibodies or antigen binding fragments that exhibit TCR-like specificity for peptide-MHC complexes may also be referred to as TCR-like CARs. In some embodiments, the extracellular antigen binding domain specific for the MHC-peptide complex of the TCR-like CAR is linked in some aspects to one or more intracellular signaling components via a linker and/or transmembrane domain(s). In some embodiments, these molecules may generally mimic or approximate the signal through a native antigen receptor (such as a TCR), and optionally through the signal that the receptor binds to a co-stimulatory receptor.

Exemplary antigen receptors including CARs and methods for engineering and introducing these receptors into cells include those described, for example, in international patent application publication nos. WO200014257、WO2013126726、WO2012/129514、WO2014031687、WO2013/166321、WO2013/071154、WO2013/123061、WO2016/0046724、WO2016/014789、WO2016/090320、WO2016/094304、WO2017/025038、WO2017/173256,, US2002131960, US2013287748, US20130149337, US 6,451,995、7,446,190、8,252,592、8,339,645、8,398,282、7,446,179、6,410,319、7,070,995、7,265,209、7,354,762、7,446,191、8,324,353、8,479,118 and 9,765,342, and european patent application No. EP2537416, and/or by Sadelain et al, cancer discover, 3 (4): 388-398 (2013); davila et al, PLoS ONE 8 (4): e61338 (2013); turtle et al, curr. Opin. Immunol.,24 (5): 633-39 (2012); wu et al, cancer,18 (2): 160-75 (2012), each of which is incorporated by reference in its entirety. In some aspects, the antigen receptor includes a CAR as described in U.S. patent No. 7,446,190 and those described in international patent application publication No. WO/2014055668A1, the respective contents of which are incorporated by reference in their entirety. Examples of CARs include CARs as disclosed in any of the foregoing disclosures, such as WO2014031687, US 8,339,645, US 7,446,179, US2013/0149337, US patent 7,446,190, US patent 8,389,282, kochenderfer et al, nature REVIEWS CLINICAL Oncology,10,267-276 (2013), wang et al, j.immunother.35 (9): 689-701 (2012), and Brentjens et al, SCI TRANSL med.5 (177) (2013), each of which is incorporated by reference in its entirety. See also WO2014031687, US 8,339,645, US 7,446,179, US2013/0149337, US patent No. 7,446,190 and US patent No. 8,389,282, the respective contents of which are incorporated by reference in their entirety. The chimeric receptor, such as a CAR, typically comprises an extracellular antigen-binding domain, such as a portion of an antibody molecule, typically comprising a heavy chain Variable (VH) region and/or a light chain Variable (VL) region of an antibody, e.g., an scFv antibody fragment.

In some embodiments, the CAR is constructed to be specific for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker and/or an antigen intended to induce an inhibitory response, such as an antigen expressed by a normal or non-diseased cell type. Thus, a CAR typically comprises one or more antigen binding molecules, such as one or more antigen binding fragments, domains, or portions or one or more antibody variable domains, and/or antibody molecules, outside of its cell. In some embodiments, the CAR comprises one or more antigen-binding portions of an antibody molecule, such as a single chain antibody fragment (scFv) derived from a variable heavy chain (V_H) and a variable light chain (V_L) of a monoclonal antibody (mAb), or a single domain antibody (sdAb), such as sdFv, nanobody, V_H H, and V_NAR. In some embodiments, the antigen binding fragment comprises an antibody variable region linked by a flexible linker.

The antigen binding domain comprised in the CAR is an antibody fragment. An "antibody fragment" or "antigen-binding fragment" refers to a molecule that comprises, in addition to an intact antibody, a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ')₂, diabodies, linear antibodies, heavy chain variable (V_H) regions, single chain antibody molecules such as scFv and single domain antibodies comprising only the V_H region, and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibody is a single chain antibody fragment, such as an scFv, comprising a heavy chain variable (V_H) region and/or a light chain variable (V_L) region.

In certain embodiments, a multispecific binding molecule (e.g., a multispecific chimeric receptor, such as a multispecific CAR) can contain any multispecific antibody, including, for example, bispecific antibodies, multispecific single-chain antibodies (e.g., diabodies, triabodies, and tetrabodies), tandem diavs, and tandem triavs.

A single domain antibody (sdAb) is an antibody fragment that comprises all or part of an antibody heavy chain variable region or all or part of an antibody light chain variable region. In certain embodiments, the single domain antibody is a human single domain antibody.

Antibody fragments may be prepared by a variety of techniques including, but not limited to, proteolytic hydrolysis of intact antibodies and recombinant host cell production. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment comprising a non-naturally occurring structure, such as two or more antibody regions or chains linked by synthetic linkers (e.g., peptide linkers and/or those that may not be produced by enzymatic digestion of naturally occurring intact antibodies). In some aspects, the antibody fragment is an scFv.

In some embodiments, the antibody or antigen-binding fragment thereof is a single chain antibody fragment, such as a single chain variable fragment (scFv) or diabody or single domain antibody (sdAb). In some embodiments, the antibody or antigen binding fragment is a single domain antibody comprising only V_H domains. In some embodiments, the antibody or antigen binding fragment is an scFv comprising a heavy chain variable (V_H) region and a light chain variable (V_L) region.

In some embodiments, the receptor-targeted antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or over-expressed on cells of a disease or condition (e.g., tumor or pathogenic cells) as compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or on engineered cells.

In certain embodiments, the antigen or antigen comprises αvβ6 integrin (avb 6 integrin), B cell activator receptor (BAFF-R), B Cell Maturation Antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA 9, also known as CAIX or G250), cancer-testis antigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and rage-2), carcinoembryonic antigen (CEA), cyclin A2, C-C motif cytokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, carbonic anhydrase 9 (CA 9), and combinations thereof, CD24, CD30, CD33, CD38, CD44v6, CD44v7/8, CD70, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG 4), delta-like ligand 3 (DLL 3), epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa 2), and, Estrogen receptor, fc receptor-like 5 (FCRL 5; also known as Fc receptor analog 5 or FCRH 5), fetal acetylcholine receptor (fetal AchR), folic acid binding protein (FBP), folic acid receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD 2), ganglioside GD3, glycoprotein 100 (gp 100), phosphatidylinositol glycan-3 (GPC 3), G protein coupled receptor 5D (GPCR 5D), her2/neu (receptor tyrosine kinase erb-B2), her3 (erb-B3), her4 (erb-B4), erbB dimer, human high molecular weight-melanomA-Associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLa-A1), human leukocyte antigen A2 (HLa-A2), IL-22 receptor alpha (IL-22 Rα), IL-13 receptor alpha 2 (IL-13 Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine rich repeat 8 family member A (LRRC 8A), lewis (Lewis) Y, melanomA-Associated antigen (MAGE) -A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine Cytomegalovirus (CMV), mucin 1 (MUC 1), MUC16, natural killer group 2 member D (NKG 2D) ligand, melan A (MART-1), neural cell adhesion factor (NCAM), mucin 1 (MUC 1) carcinoembryonic antigen, melanoma preferential expression antigen (PRAME), progesterone receptor, prostate specific antigen, prostate Stem Cell Antigen (PSCA), prostate Specific Membrane Antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR 1), and, Survivin, trophoblast glycoprotein (TPBG also known as 5T 4), tumor-associated glycoprotein 72 (TAG 72), tyrosine kinase-associated protein 1 (TRP 1, also known as TYRP1 or gp 75), tyrosine kinase-associated protein 2 (TRP 2, also known as dopamine tautomerase, dopamine delta isomerase, or DCT), vascular Endothelial Growth Factor Receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR 2), wilms tumor 1 (WT-1), antigens specific for or expressing a pathogen, or antigens associated with a universal TAG and/or biotinylated molecules, and/or antibodies directed against a polypeptide selected from the group consisting of HIV, and combinations thereof, HCV, HBV, or other pathogen expressed molecules. In some embodiments, the receptor-targeted antigen includes an antigen associated with a B cell malignancy, such as any of the known B cell markers. In some embodiments, the antigen is or comprises CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30.

In some embodiments, the antigen is or includes an antigen that is specific for or expresses a pathogen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and/or a parasitic antigen.

Exemplary CAR T cell therapies for use in accordance with the methods provided herein are known in the art. CAR T cell therapies suitable for use in accordance with the methods provided herein include any described in Marofi et al, STEM CELL RES THER12:81 (2021), townsend et al, J Exp CLIN CANCER RES 37:163 (2018), ma et al, int J Biol Sci15 (12): 2548-2560 (2019), zhao and Cao, front Immunol 10:2250 (2019), and Han et al, JCancer (2): 326-334 (2021), each of which is incorporated herein by reference in its entirety.

Exemplary CAR T cell therapies targeting EGFR include those that were or are being studied in clinical trials NCT03179007, NCT01869166, NCT02331693, NCT03182816, NCT03152435, and NCT 03525782. CD 70-targeted CAR T cell therapies include those that were or are being studied in clinical trials NCT03125577 and NCT 028307242. Exemplary CART cell therapies targeting CD138 include those that were or are being studied in clinical trials NCT01886976 and NCT 03672318. Exemplary CAR T cell therapies targeting CD38 include those that were or are being studied in clinical trials NCT03464916, NCT03473496, NCT03473457, NCT03125577, NCT03222674, and NCT 032716322. Exemplary CAR T cell therapies targeting CD123 include those that were or are being studied in clinical trials NCT03473457, NCT03125577, NCT02937103, NCT03114670, NCT02159495, NCT03098355, NCT03222674, NCT03203369, and NCT 03190278. Exemplary CAR T cell therapies targeting CD133 include those that were or are being studied in clinical trials NCT03473457, NCT03356782, NCT02541370, and NCT 03423992. Exemplary CAR T cell therapies targeting GPC3 include those that were or are being studied in clinical trials NCT02905188、NCT02932956、NCT02715362、NCT03130712、NCT02395250、NCT02876978、NCT03198546、NCT02723942、NCT03084380、NCT03302403、NCT03146234 and NCT 02959151. Exemplary CAR T cell therapies targeting CD5 include those that were or are being studied in clinical trial NCT 03081910. Exemplary CAR T cell therapies targeting ROR1 include those that were or are being studied in clinical trial NCT 02706392. Exemplary CAR T cell therapies targeting Herin CAR-PD1 include those that were or are being studied in clinical trials NCT02873390 and NCT 02862028. Exemplary CAR T cell therapies targeting HER2 include those that were or are being studied in clinical trials NCT03500991、NCT03423992、NCT02713984、NCT01935843、NCT03267173、NCT02792114、NCT02442297、NCT00889954、NCT03423992、NCT01109095、NCT02706392、NCT00902044、NCT03389230、NCT02713984、NCT02547961 and NCT 01818323. exemplary CAR T cell therapies targeting EGFR806 include those that were or are being studied in clinical trial NCT 03179012. Exemplary CAR T cell therapies targeting NY-ESO-1 include those that were or are being studied in clinical trial NCT 03029273. Exemplary CAR T cell therapies targeting mesothelin include those that were or are being studied in clinical trials NCT02930993、NCT03182803、NCT03030001、NCT02706782、NCT01583686、NCT03356795、NCT03054298、NCT03267173、NCT02792114、NCT02959151、NCT02580747、NCT02414269、NCT02465983、NCT03182803 and NCT 03323944. Exemplary CAR T cell therapies targeting PSCA include those that were or are being studied in clinical trials NCT03198052, NCT02744287, and NCT 03267173. Exemplary CAR T cell therapies targeting MG7 include those that were or are being studied in clinical trial NCT 02862704. Exemplary CAR T cell therapies targeting MUC1 include those that were or are being studied in clinical trials NCT03179007, NCT02587689, NCT02617134, NCT03198052, NCT03356795, NCT03267173, NCT03222674, and NCT 03356782. Exemplary CAR T cell therapies targeting Claudin 18.2 include those that were or are being studied in clinical trials NCT03874897 and NCT 03159819. Exemplary CAR T cell therapies targeting EpCAM include those that were or are being studied in clinical trials NCT02915445, NCT03013712, NCT02729493, NCT02725125, NCT02728882, and NCT 02735291. Exemplary CAR T cell therapies targeting GD2 include those that were or are being studied in clinical trials NCT04099797、NCT03423992、NCT03356795、NCT02992210、NCT01953900、NCT02761915、NCT03373097、NCT02765243、NCT03423992、NCT03294954、NCT03356782 and NCT 02919046. Exemplary CAR T cell therapies targeting VEGFR2 include those that were or are being studied in clinical trial NCT 01218867. Exemplary CAR T cell therapies targeting AFP include those that were or are being studied in clinical trial NCT 03349255. Exemplary CAR T cell therapies targeting fibronectin 4/FAP include those that were or are being studied in clinical trial NCT 03932565. Exemplary CAR T cell therapies targeting FAP include those that were or are being studied in clinical trial NCT 01722149. Exemplary CAR T cell therapies targeting CEA include those that were or are being studied in clinical trials NCT02850536, NCT02349724, NCT03267173, NCT02959151, and NCT 01212887. Exemplary CAR T cell therapies targeting lewis Y include those that were or are being studied in clinical trial NCT 03851146. Exemplary CAR T cell therapies targeting glypican-3 include those that were or are being studied in clinical trial NCT 02932956. Exemplary CAR T cell therapies targeted to EGFRIII include those that were or are being studied in clinical trial NCT 01454596. Exemplary CAR T cell therapies targeting IL-13 ra 2 include those that were or are being studied in clinical trial NCT 02208362. Exemplary CAR T cell therapies targeting CD171 include those that were or are being studied in clinical trial NCT 02311621. Exemplary CAR T cell therapies targeting MUC16 include those that were or are being studied in clinical trial NCT 02311621. exemplary CAR T cell therapies targeting PSMA include those that were or are being studied in clinical trials NCT03356795, NCT03089203, NCT03185468, and NCT 01140373. Exemplary CAR T cell therapies targeting AFP include those that were or are being studied in clinical trial NCT 03349255. Exemplary CAR T cell therapies targeting AXL include those that were or are being studied in clinical trial NCT 03393936. Exemplary CAR T cell therapies targeting CD20 include those that were or are being studied in clinical trials NCT03893019 and NCT 04169932. Exemplary CAR T cell therapies targeting CD80/86 include those that were or are being studied in clinical trial NCT 03198052. Exemplary CAR T cell therapies targeting CD30 include those that were or are being studied in clinical trials NCT03383965, NCT04134325, and NCT 04008394. Exemplary CAR T cell therapies targeting c-MET include those that were or are being studied in clinical trials NCT03060356 and NCT 03638206. Exemplary CAR T cell therapies targeting DLL-3 include those that were or are being studied in clinical trial NCT 03392064. exemplary CAR T cell therapies targeting DR5 include those that were or are being studied in clinical trial NCT 03638206. Exemplary CAR T cell therapies targeted to EpHA2 include those that were or are being studied in clinical trials NCT02575261 and NCT 03423992. Exemplary CAR T cell therapies targeting FR-a include those that were or are being studied in clinical trial NCT 00019136. Exemplary CAR T cell therapies targeting gp100 include those that were or are being studied in clinical trial NCT 03649529. exemplary CAR T cell therapies targeting IL13Ra2 include those that were or are being studied in clinical trial NCT 02208362. Exemplary CAR T cell therapies targeting MAGE-A1/3/4 include those that were or are being studied in clinical trials NCT03356808 and NCT 03535246. Exemplary CAR T cell therapies targeting LMP1 include those that were or are being studied in clinical trial NCT 02980315. Exemplary CAR T cell therapies targeting egfrvlll include those that were or are being studied in clinical trials NCT03283631, NCT02844062, and NCT 03170141. exemplary CAR T cell therapies targeting PD-L1 CSR include those that were or are being studied in clinical trial NCT 02937844. Exemplary CAR T cell therapies targeting CD19 include those that were or are being studied in clinical trials NCT02644655、NCT03744676、NCT01087294、NCT03366350、NCT03790891、NCT03497533、NCT04007029、NCT03960840、NCT04049383、NCT04094766、NCT03366324、NCT02546739、NCT03448393、NCT03467256、NCT03488160、NCT04012879、NCT03016377、NCT03468153、NCT03483688、NCT03398967、NCT03229876、NCT03455972、NCT03423706、NCT03497533 and NCT04002401, which include FDA approved products(Li Jimai, lisocabtagene maraleucel), TECARTUS^TM (buckyolol, brexucabtagene autoleucel), KYMRIAH^TM (s Li Fuming, tisagenlecleucel), YESCARTA^TM (alzem, axicabtagene ciloleucel). Exemplary CAR T cell therapies targeting BCMA include those that were or are being studied in clinical trials NCT03448978、NCT04182581、NCT03271632、NCT03473496、NCT03430011、NCT03455972、NCT02954445、NCT03322735、NCT03338972、NCT03318861、NCT02215967、NCT03093168、NCT03274219、NCT03302403、NCT03492268、NCT03288493、NCT03070327、NCT03196414、NCT03448978、NCT02958410、NCT03287804、NCT03473496、NCT03380039、NCT03430011、NCT03361748、NCT03455972、NCT02546167、NCT03271632 and NCT03548207 (CARVYKTI^TM (sidaopranluki, ciltacabtagene autoleucel)). Exemplary CAR T cell therapies targeting BCMA also include FDA-approved products(Ai Dika Bajin, idecabtagene vicleucel) and CARVYKTI^TM (Sida-based alendronate). Exemplary CAR T cell therapies targeting CD33 also include those that were or are being studied in clinical trials NCT03473457, NCT02958397, NCT03126864, and NCT 03222674. Exemplary CAR T cell therapies targeting GAPs also include those that were or are being studied in clinical trial NCT 02932956. Exemplary CAR T cell therapies targeted to Zeushield also include those that were or are being studied in clinical trial NCT 03060343. Exemplary CAR T cell therapies targeting DLL3 also include those that were or are being studied in clinical trial NCT 03392064.

In some embodiments, the CAR is an anti BCMACAR specific for BCMA, e.g., human BCMA. Chimeric antigen receptors contain anti-BCMA antibodies, which include mouse anti-human BCMA antibodies and human anti-human BCMA antibodies as well as cells expressing these chimeric receptors, which have been described previously. See Carpenter et al ,Clin Cancer Res.,2013,19(8):2048-2060、US 9,765,342、WO 2016/090320、WO2016090327、WO2010104949A2、WO2016/0046724、WO2016/014789、WO2016/094304、WO2017/025038 and WO2017173256, the respective contents of which are incorporated by reference in their entirety.

In some embodiments, the antigen binding domain of anti BCMACAR is a single chain antibody fragment, such as a single chain variable fragment (scFv) or diabody or single domain antibody (sdAb). In some embodiments, the antigen binding domain is a single domain antibody (sdAb). In some embodiments, the antigen binding domain is a single domain antibody comprising only the V_H region. In some embodiments, the antigen binding domain is a single domain antibody comprising only the V_H region as set forth in SEQ ID NO 113.

In some embodiments, the antibody BCMACAR contains an antigen-binding domain, such as an scFv, that contains a heavy chain variable (V_H) region and/or a light chain variable (V_L) region derived from an antibody described in WO 2016/090320 or WO2016090327, each of which is incorporated by reference in its entirety. In some embodiments, the antigen binding domain is an antibody fragment comprising a heavy chain variable (V_H) region and a light chain variable (V_L) region. In some aspects, the V_H region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a V_H region amino acid sequence set forth in any one of SEQ ID NO:30、32、34、36、38、40、42、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、181、183、185 and 187, and/or the V_L region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a V_H region amino acid sequence set forth in any one of SEQ ID NO:31、33、35、37、39、41、43、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、182、184、186 and 188.

In some embodiments, the antigen binding domain of anti BCMACAR (such as an scFv) contains V_H as set forth in SEQ ID No. 30 and V_L as set forth in SEQ ID No. 31. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 32 and V_L as set forth in SEQ ID NO. 33. in some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 34 and V_L as set forth in SEQ ID NO. 35. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO:36 and V_L as set forth in SEQ ID NO: 37. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO:38 and V_L as set forth in SEQ ID NO: 39. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 40 and V_L as set forth in SEQ ID NO. 41. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 42 and V_L as set forth in SEQ ID NO. 43. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO:77 and V_L as set forth in SEQ ID NO: 78. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO:79 and V_L as set forth in SEQ ID NO: 80. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 81 and V_L as set forth in SEQ ID NO. 82. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 83 and V_L as set forth in SEQ ID NO. 84. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO:85 and V_L as set forth in SEQ ID NO: 86. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 87 and V_L as set forth in SEQ ID NO. 88. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 89 and V_L as set forth in SEQ ID NO. 90. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 91 and V_L as set forth in SEQ ID NO. 92. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 93 and V_L as set forth in SEQ ID NO. 94. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 95 and V_L as set forth in SEQ ID NO. 96. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 97 and V_L as set forth in SEQ ID NO. 98. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in EQ ID NO:99 and V_L as set forth in SEQ ID NO: 100. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 101 and V_L as set forth in SEQ ID NO. 102. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 103 and V_L as set forth in SEQ ID NO. 104. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 105 and V_L as set forth in SEQ ID NO. 106. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 107 and V_L as set forth in SEQ ID NO. 106. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 30 and V_L as set forth in SEQ ID NO. 108. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 109 and V_L as set forth in SEQ ID NO. 110. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO:111 and V_L as set forth in SEQ ID NO: 112. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO:181 and V_L as set forth in SEQ ID NO: 182. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 183 and V_L as set forth in SEQ ID NO. 184. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO:185 and V_L as set forth in SEQ ID NO: 186. In some embodiments, the antigen binding domain (such as scFv) contains V_H as set forth in SEQ ID NO. 187 and V_L as set forth in SEQ ID NO. 188. In some embodiments, the V_H or V_L has a sequence that exhibits at least 85% sequence identity to any one of the aforementioned V_H or V_L sequences, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and maintaining amino acid sequence binding to BCMA. In some embodiments, the V_H region is the amino terminus of the V_L region. In some embodiments, the V_H region is the carboxy terminus of the V_L region. In some embodiments, the variable heavy and variable light chains are linked by a linker. In some embodiments, the linker is as set forth in SEQ ID NO 70, 72, 73, 74 or 189.

In some embodiments, the antibody BCMACAR is any one of those set forth in SEQ ID NOS.126-177.

In some embodiments, the anti-BCMACAR contains the V_H and the V_L of the anti-BCMA antibody cd115d.3. In some embodiments, the antibody BCMACAR contains V_H as set forth in SEQ ID NO. 30 and V_L as set forth in SEQ ID NO. 31. In some embodiments, the anti-BCMACAR comprises a scFv having the sequence set forth in SEQ ID NO. 249. In some embodiments, the antibody BCMACAR contains the sequence set forth as SEQ ID NO. 152 (see WO 2016/094304). In some embodiments, the anti BCMACAR is a CAR of ABECMA (Ai Jiwei, lam, idecabtagene vicleucel (ide-cel)). In some embodiments, the T cell therapy is ABECMA (Ai Jiwei am (ide-cel)). In some embodiments of the present invention, in some embodiments,

In some embodiments, the anti BCMACAR contains an anti-BCMA single domain antibody. In some embodiments, the anti BCMACAR contains two anti-BCMA single domain antibodies. In some embodiments, the anti BCMACAR is a CAR of Carvykti (sidaorensai, ciltacabtagene autoleucel (cilta-cel)). In some embodiments, the T cell therapy is Carvykti (sidaky-alendronate (cilta-cel)).

In some embodiments, the antibody BCMACAR contains V_H as set forth in SEQ ID NO. 36 and V_L as set forth in SEQ ID NO. 37. In some embodiments, the anti-BCMACAR comprises a scFv comprising the sequence set forth as SEQ ID NO. 250. In some embodiments, the antibody BCMACAR contains the sequence set forth as SEQ ID NO. 160.

In some embodiments, the CAR is an anti-CD 19CAR specific for CD19 (e.g., human CD 19). In some embodiments, the antibody or antigen binding fragment (e.g., scFv or V_H domain) specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen binding fragment is derived from or is a variant of an antibody or antigen binding fragment that specifically binds CD19.

In some embodiments, the antigen is CD19. In some embodiments, the scFv comprises V_H and V_L derived from an antibody or antibody fragment specific for CD19. In some embodiments, the antibody or antibody fragment that binds CD19 is a mouse-derived antibody, such as FMC63 and SJ25C1. In some embodiments, exemplary antibodies or antibody fragments include those described in U.S. patent publication nos. WO 2014/031687, US2016/0152723, and WO 2016/033570, the respective contents of which are incorporated by reference in their entirety.

In some embodiments, the antigen binding domain comprises V_H and/or V_L derived from FMC63, which in some aspects may be an scFv. In some embodiments, the scFv and/or V_H domain is derived from FMC63. FMC63 is typically a mouse monoclonal IgG1 antibody raised against Nalm-1 and Nalm-16 cells expressing human CD19 (Ling, N.R., et al (1987) Leucocyte typing III.302). The FMC63 antibody comprises CDRH1 and H2 as set forth in SEQ ID NO. 44, 45, respectively, and CDRH3 as set forth in SEQ ID NO. 46, 47 or 66, and CDRL1 as set forth in SEQ ID NO. 48, and CDR L2 as set forth in SEQ ID NO. 49, 50 or 67, and CDR L3 as set forth in SEQ ID NO. 51, 52 or 68. The FMC63 antibody comprises a heavy chain variable region (V_H) comprising amino acid sequence SEQ ID NO. 53 and a light chain variable region (V_L) comprising amino acid sequence SEQ ID NO. 54. In some embodiments, the scFv comprises a variable light chain comprising the CDRL1 sequence of SEQ ID NO. 48, the CDRL2 sequence of SEQ ID NO. 49, and the CDRL3 sequence of SEQ ID NO. 51, and/or a variable heavy chain comprising the CDRH1 sequence of SEQ ID NO. 44, the CDRH2 sequence of SEQ ID NO. 45, and the CDRH3 sequence of SEQ ID NO. 46. In some embodiments, the scFv comprises a heavy chain variable region of FMC63 as set forth in SEQ ID NO. 53 and a light chain variable region of FMC63 as set forth in SEQ ID NO. 54. In some embodiments, the variable heavy chain and the variable light chain are linked by a linker. In some embodiments, the linker is as set forth in SEQ ID NO 70, 72, 73, 74 or 189. In some embodiments, the scFv comprises V_H, a linker, and V_L in that order. In some embodiments, the scFv comprises V_L, a linker, and V_H in that order. In some embodiments, the scFv is encoded by a nucleotide sequence as set forth in SEQ ID NO:69 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 69. In some embodiments, the scFv comprises an amino acid sequence as set forth in SEQ ID NO. 55 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 55. In some embodiments, the anti-CD 19 CAR is(Li Jimai, am, lisocabtagene maraleucel). In some embodiments, the T cell therapy is(Li Jimai Lun race).

In some embodiments, the antigen binding domain of the anti-CD 19 CAR comprises V_H and/or V_L derived from SJ25C1, which in some aspects may be an scFv. SJ25C1 is a mouse monoclonal IgG1 antibody raised against Nalm-1 and Nalm-16 cells expressing human CD19 (Ling, N.R., et al (1987) Leucocyte typing III.302). The SJ25C1 antibody comprises CDRH1, H2 and H3 respectively listed as SEQ ID NO. 59-61, and CDRL1, L2 and L3 sequences respectively listed as SEQ ID NO. 56-58. The SJ25C1 antibody comprises a heavy chain variable region (V_H) comprising the amino acid sequence SEQ ID NO. 62 and a light chain variable region (V_L) comprising the amino acid sequence SEQ ID NO. 63. In some embodiments, the scFv comprises a variable light chain comprising the CDRL1 sequence of SEQ ID NO:56, the CDRL2 sequence of SEQ ID NO:57, and the CDRL3 sequence of SEQ ID NO:58, and/or a variable heavy chain comprising the CDRH1 sequence of SEQ ID NO:59, the CDRH2 sequence of SEQ ID NO:60, and the CDRH3 sequence of SEQ ID NO: 61. In some embodiments, the scFv comprises a heavy chain variable region of SJ25C1 as set forth in SEQ ID NO. 62 and a light chain variable region of SJ25C1 as set forth in SEQ ID NO. 63. In some embodiments, the variable heavy chain and the variable light chain are linked by a linker. In some embodiments, the linker is as set forth in SEQ ID NO. 64. In some embodiments, the scFv comprises V_H, a linker, and V_L in that order. In some embodiments, the scFv comprises V_L, a linker, and V_H in that order. In some embodiments, the scFv comprises an amino acid sequence as set forth in SEQ ID NO. 65 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 65.

In some embodiments, the anti-CD 19 CAR is a CAR of TECARTUS^TM (brix, brexucabtagene autoleucel). In some embodiments, the T cell therapy is TECARTUS^TM (brix).

In some embodiments, the anti-CD 19 CAR is a CAR of KYMRIAH^TM (st Li Fuming, tisagenlecleucel). In some embodiments, the T cell therapy is KYMRIAH^TM (se Li Fuming).

In some embodiments, the anti-CD 19 CAR is a CAR of YESCARTA^TM (alemtuquor, axicabtagene ciloleucel). In some embodiments, the T cell therapy is YESCARTA^TM (aliskiren).

In some embodiments, the antigen is CD20. In some embodiments, the scFv comprises V_H and V_L derived from an antibody or antibody fragment specific for CD20. In some embodiments, the antibody or antibody fragment that binds CD20 is an antibody that is or is derived from rituximab, such as rituximab scFv.

In some embodiments, the antigen is CD22. In some embodiments, the scFv comprises V_H and V_L derived from an antibody or antibody fragment specific for CD22. In some embodiments, the antibody or antibody fragment that binds CD22 is an antibody that is or is derived from m971, such as an m971 scFv.

In some embodiments, the antigen is ROR1. In some embodiments, the scFv comprises V_H and V_L derived from an antibody or antibody fragment specific for ROR1. In some embodiments, the ROR1 binding antibody or antibody fragment is or contains V_H and V_L from antibodies or antibody fragments as listed in international patent application publication nos. WO 2014/031687, WO 2016/115559 and WO 2020/160050 (the respective contents of which are incorporated by reference in their entirety).

In some embodiments, the antigen is GPRC5D. In some embodiments, the scFv comprises V_H and V_L derived from an antibody or antibody fragment specific for GPRC5D. In some embodiments, the antibody or antibody fragment that binds GPRC5D is or contains V_H and V_L from antibodies or antibody fragments as listed in international patent application publication nos. WO 2016/090329, WO 2016/090312, and WO 2020/092854, the respective contents of which are incorporated by reference in their entirety.

In some embodiments, the antigen is FcRL5. In some embodiments, the scFv comprises V_H and V_L derived from an antibody or antibody fragment specific for FcRL5. In some embodiments, the antibody or antibody fragment that binds FcRL5 is or contains V_H and V_L from an antibody or antibody fragment as listed in international patent application publication nos. WO 2016/090337 and WO 2017/096120 (the respective contents of which are incorporated by reference in their entirety).

In some embodiments, the antigen is mesothelin. In some embodiments, the scFv comprises V_H and V_L derived from antibodies or antibody fragments specific for mesothelin. In some embodiments, the antibodies or antibody fragments that bind mesothelin are or contain V_H and V_L from antibodies or antibody fragments as listed in US2018/0230429 (the contents of which are incorporated by reference in their entirety).

In some embodiments, the antibody is an antigen binding fragment, such as an scFv, that includes one or more linkers connecting two antibody domains or regions, such as a heavy chain variable (V_H) region and a light chain variable (V_L) region. Accordingly, such antibodies include single chain antibody fragments, such as scFv and diabodies, particularly human single chain antibody fragments, typically comprising a linker(s) linking two antibody domains or regions, such as the V_H and V_L regions. The linker is typically a peptide linker, e.g., a flexible and/or soluble peptide linker, such as a glycine and serine rich linker. The linkers therein are those rich in glycine and serine and/or in some cases threonine. In some embodiments, the linker further comprises charged residues such as lysine and/or glutamic acid, which may improve solubility. In some embodiments, the linker further comprises one or more prolines.

In some aspects, the glycine and serine (and/or threonine) rich linker comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of such amino acids. In some embodiments, it comprises at least or about 50%, 55%, 60%, 70% or 75% glycine, serine and/or threonine. In some embodiments, the linker consists essentially entirely of glycine, serine, and/or threonine. The length of the linker is typically between about 5 and about 50 amino acids, typically between or about 10 and or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having a different number of repeats of the sequence GGGGGGS (4 GS; SEQ ID NO: 19) or GGGS (3 GS; SEQ ID NO: 71), such as between 2, 3, 4 and 5 repeats of such sequences. Exemplary linkers include those having or consisting of the sequences set forth in SEQ ID NO:72 (GGGGSGGGGSGGGGS), SEQ ID NO:189 (ASGGGGSGGRASGGGGS), SEQ ID NO:73 (GSTSGSGKPGSGEGSTKG), or SEQ ID NO:74 (SRGGGGSGGGGSGGGGSLEMA).

In some embodiments, the recombinant receptor, such as a CAR, such as an antibody portion of the recombinant receptor (e.g., CAR), further comprises a spacer, which may be or comprise at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, an IgG1 hinge region, a C_H1/C_L, and/or an Fc region. In some embodiments, the recombinant receptor further comprises a spacer and/or a hinge region. In some embodiments, the constant region or portion is a human IgG, such as IgG4 or IgG1. In some aspects, a portion of the constant region serves as a spacer region between an antigen-recognition component (e.g., scFv) and a transmembrane domain. The spacer may have a length that provides increased reactivity of the cell after antigen binding, as compared to the absence of the spacer.

Exemplary spacers (e.g., hinge regions) include those described in international patent application publication No. WO 2014031687. In some examples, the spacer is or is about 12 amino acids or no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and include any integer between the endpoints of any of the listed ranges. In some embodiments, the spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. In some embodiments, the spacer is a spacer having at least a particular length, such as a length of at least 100 amino acids, such as a length of at least 110, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids. Exemplary spacers include an individual IgG4 hinge, an IgG4 hinge linked to C_H 2 and C_H 3 domains, or an IgG4 hinge linked to a C_H 3 domain. Exemplary spacers include an individual IgG4 hinge, an IgG4 hinge linked to C_H 2 and C_H 3 domains, or an IgG4 hinge linked to a C_H 3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al, clin.cancer Res.,19:3153 (2013), hudecek et al (2015) Cancer Immunol Res.3 (2): 125-135, international patent application publication No. WO2014031687, U.S. patent No. 8,822,647, or published application No. US 2014/0271635. In some embodiments, the spacer comprises the sequence of an immunoglobulin hinge region (C_H and C_H regions). In some embodiments, one or more of the hinges (C_H and C_H) is derived in whole or in part from IgG4 or IgG2. In some cases, the hinge regions (C_H and C_H 3) are derived from IgG4. In some aspects, one or more of the hinge regions (C_H and C_H) are chimeric and contain sequences derived from IgG4 and IgG 2. In some examples, the spacer contains an IgG4/2 chimeric hinge, an IgG2/4C_H 2, and an IgG 4C_H region.

In some embodiments, the spacer, which may be a constant region of an immunoglobulin or a portion thereof, is a human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (as set forth in SEQ ID NO: 1). In some embodiments, the spacer has a sequence encoded by a nucleotide sequence set forth in SEQ ID NO. 2. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO. 3. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO. 4. In some embodiments, the encoded spacer is or comprises a sequence as set forth in SEQ ID NO. 29. In some embodiments, the constant region or portion is a constant region or portion of IgD. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO. 5. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO. 125.

In some embodiments, the spacer may be derived in whole or in part from IgG4 and/or IgG2 and may contain mutations, such as one or more single amino acid mutations in one or more domains. In some examples, the amino acid modification is substitution of proline (P) for serine (S) in the hinge region of IgG 4. In some embodiments, the amino acid modification is substitution of glutamine (Q) for asparagine (N) to reduce glycosylation heterogeneity, such as an N177Q mutation at position 177 of the C_H 2 region of the IgG4 Fc full-length sequence or an N176Q mutation at position 176 of the CH2 region of the IgG4 Fc full-length sequence.

Other exemplary spacer regions include hinge regions derived from CD8a, CD28, CTLA4, PD-1, or fcyriiia. In some embodiments, the spacer comprises a truncated extracellular domain or hinge region of CD8a, CD28, CTLA4, PD-1, or fcyriiia. In some embodiments, the spacer is a truncated CD28 hinge region. In some embodiments, there is a short oligopeptide or polypeptide linker, e.g., a linker between 2 and 10 amino acids in length, such as one containing multiple alanine or alanine and arginine, e.g., alanine triplets (AAA) or RAAA (SEQ ID NO: 180), and forms a linkage between the scFv and the spacer region of the CAR. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO. 114. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO. 116. In some embodiments, the spacer has a sequence as set forth in any one of SEQ ID NOS.117-119. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO. 120. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO. 122. In some embodiments, the spacer has a sequence as set forth in SEQ ID NO. 124.

In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs 1, 3, 4, 5 or 29, 114, 116, 117, 118, 119, 120, 122, 124 or 125.

Such antigen recognition domains are typically linked to one or more intracellular signaling components, such as signaling components that mimic stimulation and/or activation by an antigen receptor complex (such as a TCR complex, in the case of a CAR) and/or signaling via another cell surface receptor. Thus, in some embodiments, the antigen binding component (e.g., an antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to an extracellular domain. In one embodiment, a transmembrane domain is used that is naturally associated with one domain in a receptor (e.g., CAR). In some cases, the transmembrane domains are selected or modified by amino acid substitutions to avoid binding of these domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. The transmembrane regions include those derived from (i.e., at least including) the alpha, beta, or zeta chain 、CD28、CD3ε、CD45、CD4、CD5、CD8、CD8a、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD 134、CD137(4-1BB)、CD154、CTLA-4 of the T cell receptor or the transmembrane region of PD-1. Or alternatively in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain consists essentially of hydrophobic residues, such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine occur at each end of the synthetic transmembrane domain. In some embodiments, the connection is through a linker, spacer and/or transmembrane domain(s). Exemplary transmembrane domains are or include the sequences set forth in SEQ ID NO. 8, 115, 121, 123, 178 or 179.

Intracellular signaling domains include those that mimic or approximate signals through natural antigen receptors, signals through binding of such receptors to co-stimulatory receptors, and/or signals through co-stimulatory receptors alone. In some embodiments, there is a short oligopeptide or polypeptide linker, e.g., a linker between 2 and 10 amino acids in length (such as containing glycine and serine, e.g., glycine-serine duplex) and forming a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

The receptor (e.g., CAR) typically comprises at least one or more intracellular signaling components. In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as a TCR CD3 chain (e.g., a cd3ζ chain) that mediates T cell activation and/or activation and cytotoxicity. Thus, in some aspects, the antigen binding portion is linked to one or more cell signaling molecules. In some embodiments, the cell signaling molecule comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and/or other CD transmembrane domain. In some embodiments, the receptor (e.g., CAR) further comprises a portion of one or more additional molecules, such as Fc receptor gamma, CD8, CD4, CD25, or CD 16. For example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3- ζ or Fc receptor γ and CD8, CD4, CD25, or CD 16.

In some embodiments, upon attachment of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor stimulates and/or activates at least one normal effector function or response of an immune cell (e.g., a T cell engineered to express the CAR). For example, in some cases, the CAR induces a function of the T cell such as cytolytic activity or T helper activity (such as secretion of cytokines or other factors). In some embodiments, a truncated portion of the intracellular signaling region of the antigen receptor component or co-stimulatory molecule is used in place of the intact immunostimulatory chain (e.g., if it transduces an effector function signal). In some embodiments, the one or more intracellular signaling domains comprise cytoplasmic sequences of T Cell Receptors (TCRs), and in some aspects co-receptor sequences that cooperate with these receptors in the natural environment to initiate signal transduction upon antigen receptor binding, and/or any derivative or variant of these molecules, and/or any synthetic sequence having the same function.

In the case of native TCRs, complete activation typically requires not only signaling through the TCR, but also a co-stimulatory signal. Thus, in some embodiments, to facilitate complete activation, components for generating secondary or co-stimulatory signals are also included in the CAR. In other embodiments, the CAR does not include a component for generating a co-stimulatory signal. In some aspects, the additional CAR is expressed in the same cell and provides a component for generating a secondary or co-stimulatory signal.

T cell stimulation and/or activation is described in certain aspects as being mediated by two classes of cytoplasmic signaling sequences, those that initiate antigen dependent primary stimulation and/or activation by a TCR (primary cytoplasmic signaling region, domain or sequence) and those that function in an antigen independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling region, domain or sequence). In some aspects, the CAR includes one or both of these signal components.

In some aspects, the CAR comprises a primary cytoplasmic signaling region, domain, or sequence that modulates primary activation of the TCR complex. The primary cytoplasmic signaling region, domain or sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include those derived from TCR ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD8, CD22, CD79a, CD79b and CD66 d. In some embodiments, the cytoplasmic signaling molecule(s) in the CAR contain a cytoplasmic signaling domain derived from cd3ζ, portion or sequence thereof. In some embodiments, the CAR comprises a signaling region and/or transmembrane portion of a co-stimulatory receptor, such as CD28, 4-1BB, OX40 (CD 134), CD27, DAP10, DAP12, ICOS, and/or other co-stimulatory receptor. In some aspects, the same CAR comprises a primary cytoplasmic signaling region and a costimulatory signaling component.

In some embodiments, one or more different recombinant receptors may contain one or more different intracellular signaling regions or domains. In some embodiments, the primary cytoplasmic signaling region is contained within one CAR, while the co-stimulatory component is provided by another receptor (e.g., another CAR that recognizes another antigen). In some embodiments, the CAR comprises an activated or stimulated CAR, and a co-stimulated CAR, both expressed on the same cell (see WO 2014/055668).

In some aspects, the cell comprises one or more stimulating or activating CARs and/or co-stimulating CARs. In some embodiments, the cells further include an inhibitory CAR (iCAR, see Fedorov et al, sci.tranl.medicine, 5 (215) (2013), such as a CAR that recognizes an antigen other than that associated with and/or specific for a disease or condition, thereby eliminating or inhibiting an activation signal delivered by a disease-targeted CAR by binding the inhibitory CAR to its ligand, e.g., reducing off-target effects.

In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3- ζ) intracellular domain. In some embodiments, the intracellular signaling domain comprises chimeric CD28 and CD137 (4-1 BB, TNFRSF 9) co-stimulatory domains linked to a cd3ζ intracellular domain.

In some embodiments, the CAR encompasses one or more (e.g., two or more) co-stimulatory domains and a primary cytoplasmic signaling region in the cytoplasmic portion. Exemplary CARs include intracellular components such as one or more intracellular signaling regions or domains of CD3- ζ, CD28, CD137 (4-1 BB), OX40 (CD 134), CD27, DAP10, DAP12, NKG2D, and/or ICOS. In some embodiments, the chimeric antigen receptor contains an intracellular signaling region or domain of a T cell co-stimulatory molecule, e.g., from CD28, CD137 (4-1 BB), OX40 (CD 134), CD27, DAP10, DAP12, NKG2D, and/or ICOS, in some cases, between the transmembrane domain and the intracellular signaling region or domain. In some aspects, the T cell costimulatory molecule is one or more of CD28, CD137 (4-1 BB), OX40 (CD 134), CD27, DAP10, DAP12, NKG2D, and/or ICOS.

In some cases, the CARs are referred to as first, second, and/or third generation CARs. In some aspects, the first generation CAR is a CAR that provides only a CD3 chain-induced signal upon antigen binding, in some aspects the second generation CAR is a CAR that provides such a signal and a co-stimulatory signal, such as a CAR that includes an intracellular signaling domain from a co-stimulatory receptor (such as CD28 or CD 137), and in some aspects the third generation CAR is a CAR that includes multiple co-stimulatory domains of different co-stimulatory receptors.

In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or antibody fragment. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv and the intracellular domain comprises ITAM. In some aspects, the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD 3-zeta (CD 3 zeta) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain that connects an extracellular domain and an intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD 28. In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule. The extracellular domain and the transmembrane domain may be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane domain are connected by a spacer, such as any of those described herein. In some embodiments, the receptor contains an extracellular portion of a molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule, or a functional variant thereof, such as between a transcellular domain and an intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

For example, in some embodiments, the CAR contains an antibody (e.g., an antibody fragment), a transmembrane domain (which is or contains a transmembrane portion of CD28 or a functional variant thereof), and an intracellular signaling domain (which contains a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3 zeta or a functional variant thereof). In some embodiments, the CAR contains an antibody (e.g., an antibody fragment), a transmembrane domain (which is or contains a transmembrane portion of CD28 or a functional variant thereof), and an intracellular signaling domain (which contains a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of CD3 zeta or a functional variant thereof). In some such embodiments, the receptor further comprises a spacer comprising a portion of an Ig molecule (such as a human Ig molecule, such as an Ig hinge, e.g., an IgG4 hinge), such as a hinge-only spacer.

In some embodiments, the transmembrane domain of the recombinant receptor (e.g., CAR) is or comprises a transmembrane domain of human CD28 (e.g., accession number P10747.1) or CD8a (accession number P01732.1) or a variant thereof, such as a transmembrane domain comprising an amino acid sequence as set forth in SEQ ID No. 8, 115, 178 or 179 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, and in some embodiments, the transmembrane domain of a portion containing the recombinant receptor comprises an amino acid sequence as set forth in SEQ ID No. 9 or an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In some embodiments, the intracellular signaling component(s) (e.g., CAR) of the recombinant receptor contain an intracellular co-stimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain having a LL to GG substitution at positions 186-187 of the native CD28 protein. For example, the intracellular signaling domain may comprise an amino acid sequence as set forth in SEQ ID No. 10 or 11 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 10 or 11. In some embodiments, the intracellular domain comprises an intracellular co-stimulatory signaling domain of 4-1BB (e.g., accession number Q07011.1), or a functional variant or portion thereof, such as the amino acid sequence set forth in SEQ ID NO:12 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

In some embodiments, the intracellular signaling domain of the recombinant receptor (e.g., CAR) comprises a human cd3ζ costimulatory signaling domain, or a functional variant thereof, such as the 112AA cytoplasmic domain of isoform 3 of human cd3ζ (accession No. P20963.2) or a CD3 ζ signaling domain as described in U.S. patent No. 7,446,190 or U.S. patent No. 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID No. 13, 14, or 15 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 13, 14, or 15.

In some aspects, the spacer contains only the hinge region of IgG, such as only the hinge of IgG4 or IgG1, such as the hinge contains only the spacer as set forth in SEQ ID No. 1 or SEQ ID No. 125. In other embodiments, the spacer is or comprises an Ig hinge, e.g., an IgG 4-derived hinge, optionally linked to a CH2 and/or CH3 domain. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO. 4. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked only to a CH3 domain, such as set forth in SEQ ID NO. 3. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker (such as a known flexible linker). In some embodiments, the spacer is a CD8a hinge (such as set forth in any one of SEQ ID NOS: 117-119), an FcgammaRIIIa hinge (such as set forth in SEQ ID NO: 124), a CTLA4 hinge (such as set forth in SEQ ID NO: 120), or a PD-1 hinge (such as set forth in SEQ ID NO: 122).

For example, in some embodiments, the CAR comprises an antibody (such as an antibody fragment, comprising an scFv), a spacer (such as a spacer comprising part of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as a spacer comprising an Ig hinge), a transmembrane domain comprising all or part of a CD 28-derived transmembrane domain, a CD 28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an antibody or fragment (such as an scFv), a spacer (such as any of the spacers comprising an Ig hinge), a CD 28-derived transmembrane domain, a 4-1 BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.

The recombinant receptor (such as a CAR) expressed by cells administered to a subject typically recognizes or specifically binds to a molecule expressed in, associated with, and/or specific for the disease or condition being treated or cells thereof. Upon specific binding to the molecule (e.g., antigen), the receptor typically delivers an immunostimulatory signal (such as an ITAM transduction signal) into the cell, thereby promoting an immune response against the disease or condition. For example, in some embodiments, the cell expresses a CAR that specifically binds to an antigen expressed by a cell or tissue of a disease or condition or a cell or tissue associated with the disease or condition. Non-limiting exemplary CAR sequences are set forth in SEQ ID NOS.126-177.

In some embodiments, the encoded CAR sequence may further comprise a signal sequence or signal peptide that directs or delivers the CAR to the surface of a CAR-expressing cell. In some embodiments, the signal peptide is derived from a transmembrane protein. In some examples, the signal peptide is derived from CD8a, CD33, or IgG. Exemplary signal peptides include the sequences set forth in SEQ ID NOS.21, 75 and 76, or variants thereof.

In some embodiments, the CAR comprises an anti-CD 19 antibody (such as an antibody fragment, comprising an scFv), a spacer (such as a spacer comprising part of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as a spacer comprising an Ig hinge or any of the other spacers described herein), a transmembrane domain comprising all or part of a CD 28-derived transmembrane domain, a CD 28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an anti-CD 19 antibody or fragment (such as an scFv), a spacer (such as any of the spacers containing an Ig hinge or other spacers described herein), a CD 28-derived transmembrane domain, a 4-1 BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain. In some embodiments, the CAR construct further comprises a T2A ribosome-hopping element and/or a tgfr sequence, e.g., downstream of the CAR.

In some embodiments, the CAR comprises an anti-BCMA antibody or fragment (such as any of the anti-human BCMA antibodies (including sdabs and scFv described herein), a spacer (such as a spacer comprising an Ig hinge or any of the other spacers described herein), a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an anti-BCMA antibody or fragment (such as any of the anti-human BCMA antibodies (comprising sdabs and scFv described herein), a spacer (such as any of the spacers containing an Ig hinge or other spacers described herein), a CD28 transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR construct further comprises a T2A ribosome-hopping element and/or a tgfr sequence, e.g., downstream of the CAR. In some embodiments, the CAR comprises an anti-BCMA antibody or fragment (such as any of the anti-human BCMA antibodies (including sdabs and scFv described herein), a hinge region, and a transmembrane domain from a CD8 a, 4-1BB intracellular signaling domain, and a CD3 zeta signaling domain.

2. Chimeric autoantibody receptors (CAAR)

In some embodiments, the recombinant receptor is a chimeric autoantibody receptor (CAAR). In some embodiments, the CAAR binds (e.g., specifically binds or recognizes) an autoantibody. In some embodiments, cells expressing CAAR (such as T cells engineered to express CAAR) may be used to bind and kill cells expressing autoantibodies, but not cells expressing normal antibodies. In some embodiments, the cells expressing the CAAR may be used to treat an autoimmune disease (such as an autoimmune disease) associated with expression of an autoantigen. In some embodiments, cells expressing CAAR may target B cells that ultimately produce and present autoantibodies on their cell surface, which are labeled as disease-specific targets for therapeutic intervention. In some embodiments, cells expressing CAAR can be used to effectively target and kill pathogenic B cells by targeting pathogenic B cells using antigen-specific chimeric autoantibody receptors. In some embodiments, the recombinant receptor is CAAR, such as any of those described in U.S. patent application publication No. US 2017/0051035.

In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and one or more intracellular signaling domains or domains (also referred to interchangeably as cytoplasmic signaling domains or regions). In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling region, a signaling domain capable of stimulating and/or causing a primary activation signal in a T cell, a signaling domain of a T Cell Receptor (TCR) component (e.g., an intracellular signaling domain or region of the zeta chain of the CD 3-zeta (CD 3 zeta) chain or a functional variant or signaling portion thereof), and/or a signaling domain comprising an immune receptor tyrosine based activation motif (ITAM).

In some embodiments, the autoantibody binding domain comprises an autoantigen or fragment thereof. The choice of autoantigen may depend on the type of autoantibody to which it is directed. For example, an autoantigen may be selected because it recognizes an autoantibody on a target cell (such as a B cell) associated with a particular disease state (e.g., an autoimmune disease, such as an autoantibody-mediated autoimmune disease). In some embodiments, the autoimmune disease comprises pemphigus vulgaris (pemphigus vulgaris, PV). Exemplary antigens include desmosomal protein 1 (Dsg 1) and Dsg3.

3.TCRS

In some embodiments, an engineered cell (such as a T cell) is provided that expresses a T Cell Receptor (TCR) or antigen binding portion thereof that recognizes a peptide epitope of a target polypeptide or an antigen of a T cell epitope, such as a tumor, virus, or autoimmune protein. In some aspects, the TCR is or comprises a recombinant TCR.

In some embodiments, a "T cell receptor" or "TCR" is a molecule containing variable alpha and beta chains (also referred to as TCR alpha and TCR beta, respectively) or variable gamma and delta chains (also referred to as TCR alpha and TCR beta, respectively), or antigen-binding portions thereof, which is capable of specifically binding peptides bound to MHC molecules. In some embodiments, the TCR is in the αβ form. Generally, TCRs in the form of αβ and γδ are generally similar in structure, but T cells expressing them may have different anatomical locations or functions. TCRs may be present on the cell surface or in soluble form. Typically, TCRs appear on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens that bind to Major Histocompatibility Complex (MHC) molecules.

The term "TCR" is understood to encompass a full TCR, as well as antigen-binding portions or antigen-binding fragments thereof, unless otherwise indicated. In some embodiments, the TCR is an intact or full length TCR, including TCRs in αβ form or γδ form. In some embodiments, the TCR is an antigen-binding portion that is smaller than a full-length TCR, but that binds to a specific peptide that binds to an MHC molecule (such as to an MHC-peptide complex). In some cases, the antigen binding portion or fragment of the TCR may contain only a partial domain of the full or complete TCR, but still be able to bind to a peptide epitope, such as an MHC-peptide complex, that binds to the full TCR. In some cases, the antigen binding portion contains a variable domain of a TCR, such as the variable alpha and beta chains of a TCR, sufficient to form a binding site for binding to a particular MHC-peptide complex. Typically, the variable chain of a TCR contains complementarity determining regions involved in recognition of peptides, MHC and/or MHC-peptide complexes.

In some embodiments, the variable domain of the TCR contains highly variable loops or Complementarity Determining Regions (CDRs), which are typically the primary contributors to antigen recognition and binding capacity and specificity. In some embodiments, the CDRs of a TCR, or a combination thereof, form all or substantially all of the antigen binding sites of a given TCR molecule. The various CDRs within the variable region of the TCR chain are typically separated by a Framework Region (FR) which generally exhibits less variability between TCR molecules than the CDRs (see, e.g., jores et al, proc. Nat' lAcad. Sci. U.S. A.87:9138,1990; chothia et al, EMBO J.7:3745,1988; see also Lefranc et al, dev. Comp. Immunol.27:55,2003). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or the most important of the three CDRs on a given TCR variable region for antigen recognition and/or interaction with the processed peptide portion of the peptide-MHC complex. In some cases, CDR1 of the alpha chain may interact with the N-terminal portion of certain antigenic peptides. In some cases, CDR1 of the β chain may interact with the C-terminal portion of the peptide. In some cases, CDR2 contributes the largest or the predominant CDR to interact with or recognize the MHC portion of the MHC-peptide complex. In some embodiments, the variable region of the β -strand may contain another highly variable region (CDR 4 or HVR 4) that is generally involved in superantigen binding rather than antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

In some embodiments, the TCR may also contain constant domains, transmembrane domains, and/or short cytoplasmic tails (see, e.g., janeway et al, immunology: the immunone SYSTEMIN HEALTH AND DISEASE, 3 rd edition, current Biology Publications, p.4:33,1997). In some aspects, each chain of the TCR can have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with a constant protein that is involved in a CD3 complex that mediates signal transduction.

In some embodiments, the TCR chain comprises one or more constant domains. For example, the extracellular portion of a given TCR chain (e.g., an alpha-chain or beta-chain) may contain two immunoglobulin-like domains, such as variable domains (e.g., V.alpha. Or V.beta.; typically amino acids 1 through 116, based on Kabat numbering, kabat et al ,"Sequences of Proteins of Immunological Interest,USDept.Health and Human Services,Public Health Service National Institutes of Health,1991, th edition) and constant domains near the cell membrane (e.g., an alpha-chain constant domain or C.alpha., typically positions 117 through 259 of the chain, based on Kabat numbering or beta chain constant domain or C.beta., typically positions 117 through 295 of the chain, based on Kabat). For example, in some cases, the extracellular portion of a TCR formed by two chains contains two membrane proximal constant domains, and two membrane distal variable domains, each of which contains CDRs. The constant domain of the TCR may contain a short linking sequence in which the cysteine residues form a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, the TCR may have additional cysteine residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domain.

In some embodiments, the TCR chain comprises a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain comprises a cytoplasmic tail. In some cases, the structure allows the TCR to bind to other molecules such as CD3 and its subunits. For example, TCRs containing constant domains with transmembrane regions can anchor proteins to the cell membrane and bind to a constant subunit of a CD3 signaling element or complex. The intracellular tail of the CD3 signaling subunit (e.g., the cd3γ, cd3δ, cd3ε, and cd3ζ chains) contains one or more immunoreceptor tyrosine-based activation motifs or ITAMs that are involved in the signaling capacity of the TCR complex.

In some embodiments, the TCR may be a heterodimer of two chains α and β (or optionally γ and δ) or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (an alpha and beta chain or a gamma and delta chain), such as linked by one or more disulfide bonds.

In some embodiments, the TCRs may be produced from known TCR sequence(s), such as sequences of vα, β chains, which are readily available in substantially full length coding sequences. Methods for obtaining full length TCR sequences (including V chain sequences) from cellular sources are well known. In some embodiments, the nucleic acid encoding the TCR may be obtained from a variety of sources, such as by Polymerase Chain Reaction (PCR) amplification or synthesis of TCR-encoding nucleic acid isolated within or from a given cell(s).

In some embodiments, the recombinant receptor comprises a recombinant TCR and/or a TCR cloned from a naturally occurring T cell. In some embodiments, high affinity T cell clones directed against a target antigen (e.g., a cancer antigen) are identified, isolated from a patient, and introduced into cells. In some embodiments, TCR clones of target antigens have been generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system or HLA). See, for example, tumor antigens (see, e.g., parkhurst et al (2009) CLIN CANCER Res.15:169-180 and Cohen et al (2005) J Immunol. 175:5799-5808). In some embodiments, phage display is used to isolate TCRs against target antigens (see, e.g., varela-Rohena et al (2008) Nat Med.14:1390-1395 and Li (2005) Nat Biotechnol.23:349-354).

In some embodiments, the TCR is obtained from a biological source, such as from a cell, such as from a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cells may be obtained from cells isolated in vivo. In some embodiments, the TCR is a thymus-selected TCR. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cell may be a cultured T cell hybridoma or clone. In some embodiments, the TCR, or antigen-binding portion thereof, can be synthetically produced by knowledge of TCR sequences.

In some embodiments, the TCR is produced by a TCR identified or selected from a candidate TCR library screened for a target polypeptide antigen or a target T cell epitope thereof. TCR libraries can be generated by expanding libraries of vα and vβ from T cells isolated from a subject, including cells present in PBMCs, spleen, or other lymphoid organs. In some cases, T cells may be expanded from Tumor Infiltrating Lymphocytes (TILs). In some embodiments, the TCR library can be generated from CD4⁺ or CD8⁺ cells. In some embodiments, the TCR can be amplified from a T cell source (i.e., a normal TCR library) of a normal healthy subject. In some embodiments, the TCR can be amplified from a T cell source of the diseased subject (i.e., a diseased TCR library). In some embodiments, degenerate primers are used to amplify the V.alpha.and V.beta.gene libraries, such as by RT-PCR in samples obtained from humans (such as T cells). In some embodiments, scTv libraries can be assembled from naive vα and vβ libraries, wherein the amplification products are cloned or assembled for separation by adaptors. Depending on the subject and the source of the cells, the library may be HLA allele specific. Or alternatively, in some embodiments, a TCR library can be generated by mutational formation or diversification of a parent or scaffold TCR molecule. In some aspects, the TCR is subject to directed evolution, such as by mutation (e.g., of the alpha or beta chain). In some aspects, specific residues within the CDRs of the TCR are altered. In some embodiments, the selected TCRs may be modified by affinity maturation. In some embodiments, antigen-specific T cells may be selected, such as by screening, to evaluate CTL activity against the peptide. In some aspects, TCRs (e.g., present on antigen-specific T cells) may be selected, such as by binding activity (e.g., a particular affinity or avidity for an antigen).

In some embodiments, the TCR, or antigen-binding portion thereof, is modified or engineered. In some embodiments, directed evolution methods are used to produce TCRs with altered properties, such as higher affinity for a particular MHC-peptide complex. In some embodiments, the directed evolution method is achieved by a display method including, but not limited to, yeast display (Holler et al, (2003) Nat Immunol,4,55-62; holler et al, (2000) Proc NATL ACAD SCI U S A,97,5387-92), phage display (Li et al, (2005) Nat Biotechnol,23,349-54) or T cell display (Chervin et al, (2008) J Immunol Methods,339,175-84). In some embodiments, the display methods involve engineering or modifying a known parent or reference TCR. For example, in some cases, a wild-type TCR can be used as a template for producing a mutated TCR in which one or more residues of the CDRs are mutated and mutants are selected that have the desired altered properties (such as higher affinity for the desired target antigen).

Exemplary TCR T cell therapies for use according to the methods provided herein are known in the art. TCR T Cell therapies suitable for use in accordance with the methods provided herein include any of those described in Zhao and Cao, front Immunol 10:2250 (2019), ping et al, protein Cell 9 (3): 254-266 (2018), and Zhang and Wang, technology CANCER RES TREAT 18:1533033819831068 (2019), the respective contents of which are incorporated by reference in their entirety.

Exemplary TCR T cell therapies targeting PRAME include those that were or are being studied in clinical trials NCT03503968 and NCT 02743611. Exemplary TCR T cell therapies targeting MAGE-A3/A6 include those that were or are being studied in clinical trial NCT 03139370. Exemplary TCR T cell therapies targeting CEA include those that were or are being studied in clinical trial NCT 00923806. Exemplary TCR T cell therapies targeting MAGE-A3/12 include those that were or are being studied in clinical trial NCT 01273181. Exemplary TCR T cell therapies targeting MAGE-a10 include those that were or are being studied in clinical trials NCT02592577, NCT03391791 and NCT 02989064. Exemplary TCR T cell therapies targeting NY-ESO-1 include those that were or are being studied in clinical trials NCT01343043、NCT03029273、NCT03462316、NCT01892293、NCT01352286、NCT01567891、NCT01350401、NCT02588612、NCT03691376 and NCT 03168438. Exemplary TCR T cell therapies targeting AFP include those that were or are being studied in clinical trial NCT 03132792. Exemplary TCR T cell therapies targeting HA-1 include those that were or are being studied in clinical trial NCT 03326921. Exemplary TCR T cell therapies targeting WT1 include those that were or are being studied in clinical trials NCT02550535 and NCT 02770820. Exemplary TCR T cell therapies targeting Gp100 include those that were or are being studied in clinical trials NCT00923195 and NCT 02889861. Exemplary TCR T cell therapies targeting CMV include those that were or are being studied in clinical trial NCT 02988258. Exemplary TCR T cell therapies targeting MART-1 include those that were or are being studied in clinical trial NCT 00091104. Exemplary TCR T cell therapies targeting HBV include those that were or are being studied in clinical trial NCT 02719782. Exemplary TCR T cell therapies targeting P53 include those that were or are being studied in clinical trial NCT 00393029. Exemplary TCR T cell therapies targeting HPV-16E6 include those that were or are being studied in clinical trials NCT03578406 and NCT 02280811. Exemplary TCR T cell therapies targeting HPV-16E7 include those that were or are being studied in clinical trial NCT 02858310. Exemplary TCR T cell therapies targeting SL9 include those that were or are being studied in clinical trial NCT 00991224. Exemplary TCR T cell therapies targeting tgfβii include those that were or are being studied in clinical trial NCT 03431311. Exemplary TCR T cell therapies targeted MCPyV include those that were or are being studied in clinical trial NCT 03747484. exemplary TCR T cell therapies targeting TRAIL include those that were or are being studied in clinical trial NCT 00923390. Exemplary TCR T cell therapies targeting EBV include those that were or are being studied in clinical trial NCT 03648697. Exemplary TCR T cell therapies targeting KRAS include those that were or are being studied in clinical trials NCT03190941 and NCT 03745326.

In some embodiments, the peptide used to produce or produce the target polypeptide of the TCR of interest is known or can be readily identified conventionally. In some embodiments, peptides suitable for use in producing a TCR or antigen-binding portion can be determined based on the presence of HLA restriction motifs in a target polypeptide of interest (such as the target polypeptide described below). In some embodiments, the polypeptides are routinely identified using in silico predictive models. In some embodiments, such models for predicting MHC class I binding sites include, but are not limited to ProPred1 (Singh and Raghava (2001) Bioinformation 17 (12): 1236-1237) and SYFPEITHI (see Schulter et al, (2007) Immunoinformatics Methods in Molecular Biology,409 (1): 75-93 2007). In some embodiments, the MHC restriction epitope is HLA-A0201, which is expressed in about 39-46% of the total caucasian population, and thus represents a suitable choice of MHC antigen for the preparation of a TCR or other MHC-peptide binding molecule.

The cleavage sites for the HLA-A 0201-binding motif, proteasome and immunoproteasome are known using in silico prediction models. For predicting MHC class I binding sites, these models include, but are not limited to, proPred1 (Singh and Raghava, proPred: prediction of HLA-DR binding sites. BIOINFORMATICS17 (12): 1236-1237 2001, described in more detail) and SYFPEITHI (see Schulter et al) ,SYFPEITHI,Database for Searching and T-Cell Epitope Prediction.in Immunoinformatics Methods in Molecular Biology,vol 409(1):75-93 2007).

In some embodiments, the TCR, or antigen-binding portion thereof, can be a recombinantly produced native protein, or a mutant form thereof, in which one or more characteristics (such as binding properties) have been altered. In some embodiments, TCRs may be derived from various animal species (such as humans, mice, rats, or other mammals). TCRs may be in cell-bound or soluble form. In some embodiments, for the purposes of the methods provided, the TCR is in the form of cell binding expressed on the cell surface.

In some embodiments, the TCR is a full length TCR. In some embodiments, the TCR is an antigen-binding moiety. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR has a structure as described in WO 03/020763, WO 04/033685, WO 2011/044186.

In some embodiments, the TCR comprises a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR comprises a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR is capable of forming a TCR complex with CD 3. In some embodiments, any of the TCRs (including dTCR or scTCR) may be linked to a signaling domain that produces an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the cell surface.

In some embodiments, the dTCR comprises a first polypeptide in which the sequence corresponding to the TCR α chain variable region sequence is fused to the N-terminus of the sequence corresponding to the TCR α chain constant region extracellular sequence, and a second polypeptide in which the sequence corresponding to the TCR β chain variable region sequence is fused to the N-terminus of the sequence corresponding to the TCR β chain constant region extracellular sequence, the first and second polypeptides being linked by a disulfide bond. In some embodiments, the bond may correspond to a native interchain disulfide bond present in a native dimeric αβ TCR. In some embodiments, the interchain disulfide linkage is not present in a native TCR. For example, in some embodiments, one or more cysteines may be incorporated into an extracellular sequence constant region of a dTCR polypeptide pair. In some cases, both natural and non-natural disulfide bonds may be desired. In some embodiments, the TCR comprises a transmembrane sequence anchored to a membrane.

In some embodiments, the dTCR comprises a TCR a chain (comprising a variable a domain, a constant a domain, and a first dimerization motif linked to the C-terminus of the constant a domain), and a TCR β chain (comprising a variable β domain, a constant β domain, and a first dimerization motif linked to the C-terminus of the constant β domain), wherein the first and second dimerization motifs readily interact to form a covalent bond between an amino acid in the first dimerization motif and an amino acid in the second dimerization motif, linking the TCR a chain and the TCR β chain together.

In some embodiments, the TCR is a scTCR. Typically, scTCRs can be produced using suitable known methods, see, for example, soo Hoo, W.F. et al, PNAS (USA) 89,4759 (1992), W lfing, C. And Pluckthun, A., J.mol.biol.242,655 (1994), kurucz, I. et al, PNAS (USA) 90 3830 (1993), international publication PCT Nos. WO 96/13593, WO 96/18105, WO99/60120, WO99/18129, WO 03/020763, WO2011/044186, and Schluetter, C.J. et al, J.mol.biol.256,859 (1996). In some embodiments, the scTCR contains an incorporated unnatural inter-chain disulfide bond to facilitate TCR chain binding (see, e.g., international publication PCT No. WO 03/020763). In some embodiments, the scTCR is a non-disulfide linked truncated TCR in which a heterologous leucine zipper fused to its C-terminus facilitates chain binding (see, e.g., international publication PCT No. WO 99/60120). In some embodiments, the scTCR comprises a TCR a variable domain covalently linked to a TCR β variable domain via a peptide linker (see, e.g., international publication PCT No. WO 99/18129).

In some embodiments, the scTCR comprises a first fragment comprising an amino acid sequence corresponding to a TCR α chain variable region, a second fragment comprising an amino acid sequence corresponding to a TCR β chain variable region sequence, fused to the N-terminus of the amino acid sequence corresponding to a TCR β chain constant domain extracellular sequence, and a linker sequence connecting the C-terminus of the first fragment to the N-terminus of the second fragment.

In some embodiments, the scTCR comprises a first fragment comprising an a-chain variable region sequence fused to the N-terminus of an a-chain extracellular constant domain sequence, and a second fragment comprising a β -chain variable region sequence fused to the N-terminus of a β -chain extracellular constant and transmembrane sequence, and, optionally, a linker sequence linking the C-terminus of the first fragment and the N-terminus of the second fragment.

In some embodiments, the scTCR comprises a first fragment comprising a TCR β chain variable region sequence fused to the N-terminus of a β chain extracellular constant domain sequence, and a second fragment comprising an α chain variable region sequence fused to the N-terminus of an α chain extracellular constant and transmembrane sequence, and, optionally, a linker sequence linking the C-terminus of the first fragment and the N-terminus of the second fragment.

In some embodiments, the linker of the scTCR that connects the first and second TCR fragments may be any linker capable of forming a single polypeptide chain while retaining TCR binding specificity. In some embodiments, the linker sequence may, for example, have the formula-P-AA-P-, wherein P is proline and AA represents an amino acid sequence, wherein the amino acids are glycine and serine. In some embodiments, the first and second fragments pair such that their variable region sequences are oriented for such binding. Thus, in some cases, the linker is of sufficient length to span the distance between the C-terminus of the first fragment and the N-terminus of the second fragment, and vice versa, but not so long as to block or reduce binding of the scTCR to the target ligand. In some embodiments, the linker may contain from 10 to 45 or about 10 to 45 amino acids, such as from 10 to 30 amino acids or from 26 to 41 amino acid residues, e.g., 29, 30, 31, or 32 amino acids. In some embodiments, the linker has the formula-PGGG- (SGGGG) 5-P-, wherein P is proline, G is glycine and S is serine (SEQ ID NO: 16). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 17).

In some embodiments, the scTCR comprises a covalent disulfide bond that connects a residue of an immunoglobulin region of a constant domain of an alpha chain to a residue of an immunoglobulin region of a constant domain of a beta chain. In some embodiments, there are no interchain disulfide bonds in the native TCR. For example, in some embodiments, one or more cysteines may be incorporated into constant regions of extracellular sequences of first and second fragments of scTCR polypeptides. In some cases, both natural and non-natural disulfide bonds may be desired.

In some embodiments of dTCR or scTCR containing an introduced interchain disulfide bond, no native disulfide bond is present. In some embodiments, one or more native cysteines forming a native interchain disulfide bond are substituted with another residue, such as serine or alanine. In some embodiments, the introduced disulfide bond may be formed by mutating non-cysteine residues on the first and second fragments to cysteine. Exemplary unnatural disulfide bonds for TCRs are described in published International PCT publication No. WO 2006/000830.

In some embodiments, the TCR, or antigen-binding fragment thereof, exhibits an affinity for the target antigen with a equilibrium binding constant at or between about 10^-5 and 10^-12 M, and all individual values and ranges therein. In some embodiments, the target antigen is an MHC-peptide complex or ligand.

In some aspects, the TCR, or antigen-binding fragment thereof, binds to a peptide epitope derived from HPV16E 6 protein, HPV16E7 protein, and/or to a peptide epitope expressed on HPV-infected cells. In some embodiments, the TCR, or antigen-binding fragment thereof, binds or recognizes a peptide epitope of HPV16E 6 or E7 in the context of an MHC molecule. In most cervical cancer cases, HPV is a pathogenic organism and is associated with anal, vaginal, exogenous, penile, and oropharyngeal cancers, among others. Typically, the HPV genome comprises an early region comprising six open reading frames (E1, E2, E4, E5, E6 and E7) encoding proteins involved in cell transformation and replication, and a late region comprising two open reading frames (L1 and L2) encoding proteins of viral plasmids. In general, E6 and E7 are oncogenes that can affect cell cycle regulation and promote the formation of cancer. For example, the E6 gene product may cause degradation of p53 and the E7 gene product may cause inactivation of retinoblastoma (Rb).

In some aspects, the TCR, or antigen-binding fragment thereof, recognizes or binds an HPV 16E6 or E7 peptide epitope in the context of an MHC molecule (such as an MHC class I molecule). In some aspects, the MHC class I molecule is a Human Leukocyte Antigen (HLA) -A2 molecule, including any one or more subtypes thereof, such as HLA-a 0201, 0202, 0203, 0206, or 0207. In some cases, the frequency of occurrence of subtypes may vary from population to population. For example, in some embodiments, more than 95% of the population of HLA-A2 positive caucasians are HLA-A x 0201, while in the chinese population, the frequency is reported to be about 23% HLA-A x 0201, 45% HLA-A x 0207, 8% HLA-A x 0206, and 23% HLA-A x 0203. In some embodiments, the MHC molecule is HLA-A x 0201.

In some embodiments, the TCR, or antigen-binding fragment thereof, recognizes or binds to an epitope or region of HPV 16E 6 or HPV 16E7, such as a peptide epitope comprising an amino acid sequence as set forth in any one of SEQ ID NOs 192-199 and as set forth in table 1 below. In some embodiments, the TCR, or antigen-binding fragment thereof, has antigen specificity for HPV 16E7 protein or a portion of HPV 16E7 protein. In some embodiments, the TCR or antigen binding fragment thereof recognizes or binds to a peptide epitope derived from HPV 16E7, which is or comprises E7 (11-19) YMLDLQPET (SEQ ID NO: 196).

In some embodiments, the TCR, or antigen-binding fragment thereof, recognizes or binds to an HPV 16 epitope, which includes any of those described in US20190062398A1, US20190225692A1, US20190321401A1, and WO2019070541A1, the respective contents of which are incorporated by reference in their entirety. In some embodiments, the TCR, or antigen-binding epitope thereof, comprises a TCR sequence (e.g., a combination of alpha and beta chains) found in US20190062398A1, US20190225692A1, US20190321401A1, and WO2019070541A1, which recognizes or binds to HPV 16E7 protein or a portion of HPV 16E7 protein. In some embodiments, the TCR, or antigen-binding fragment thereof, comprises a TCR sequence (e.g., a combination of alpha and beta chains) found in US20190062398A1, US20190225692A1, US20190321401A1, and WO2019070541A1, which recognizes or binds to HPV 16E7 (11-19).

In some embodiments, the TCR, or a functional variant thereof, comprises an alpha chain comprising a variable alpha (vα) chain having CDR1, CDR2, and CDR3 contained in a vα chain as set forth in SEQ ID No. 202, and a beta chain comprising a variable beta (vβ) chain having CDR1, CDR2, and CDR3 contained in a vβ chain as set forth in SEQ ID No. 208. In some embodiments, the TCR or functional variant thereof comprises an alpha chain, comprising a V alpha chain having CDR1, CDR2 and CDR3 as set forth in SEQ ID NOS 203, 204 and 205, respectively, and a V beta chain having CDR1, CDR2 and CDR3 as set forth in SEQ ID NOS 209, 210 and 211, respectively. In some embodiments, the TCR or functional variant thereof comprises an alpha chain comprising a V alpha chain as set forth in SEQ ID NO. 202 and a beta chain comprising a V beta chain as set forth in SEQ ID NO. 208. In some embodiments, the TCR or functional variant thereof comprises an alpha chain as set forth in SEQ ID NO. 201 and a beta chain as set forth in SEQ ID NO. 207. In some embodiments, the TCR or functional variant thereof contains an alpha chain encoded by the nucleotide sequence set forth in SEQ ID NO:200 and a beta chain encoded by the nucleotide sequence set forth in SEQ ID NO: 206. In some embodiments, the TCR or functional variant thereof is encoded by the nucleotide sequence set forth in SEQ ID NO. 212.

In some embodiments, the TCR, or a functional variant thereof, comprises an alpha chain comprising a variable alpha (vα) chain having CDR1, CDR2, and CDR3 contained in the vα chain as set forth in SEQ ID No. 215, and a beta chain comprising a variable beta (vβ) chain having CDR1, CDR2, and CDR3 contained in the vβ chain as set forth in SEQ ID No. 218. In some embodiments, the TCR or functional variant thereof comprises an alpha chain comprising a V alpha chain having CDR1, CDR2 and CDR3 as set forth in SEQ ID NOS 219, 220 and 221, respectively, and a V beta chain having CDR1, CDR2 and CDR3 as set forth in SEQ ID NOS 222, 223 and 224, respectively. In some embodiments, the TCR or functional variant thereof comprises an alpha chain comprising a V alpha chain as set forth in SEQ ID NO. 215 and a beta chain comprising a V beta chain as set forth in SEQ ID NO. 218. In some embodiments, the TCR or functional variant thereof comprises an alpha chain as set forth in SEQ ID NO:214 and a beta chain as set forth in SEQ ID NO: 217. In some embodiments, the TCR or functional variant thereof contains an alpha chain encoded by the nucleotide sequence set forth in SEQ ID NO:213 and a beta chain encoded by the nucleotide sequence set forth in SEQ ID NO: 216.

In some embodiments, the TCR, or a functional variant thereof, comprises an alpha chain comprising a variable alpha (vα) chain having CDR1, CDR2, and CDR3 contained in the vα chain as set forth in SEQ ID No. 227, and a beta chain comprising a variable beta (vβ) chain having CDR1, CDR2, and CDR3 contained in the vβ chain as set forth in SEQ ID No. 230. In some embodiments, the TCR or functional variant thereof comprises an alpha chain comprising a V alpha chain having CDR1, CDR2 and CDR3 as set forth in SEQ ID NOS 231, 232 and 233, respectively, and a V beta chain having CDR1, CDR2 and CDR3 as set forth in SEQ ID NOS 234, 235 and 236, respectively. In some embodiments, the TCR or functional variant thereof comprises an alpha chain comprising a V alpha chain as set forth in SEQ ID NO:227 and a beta chain comprising a V beta chain as set forth in SEQ ID NO: 230. In some embodiments, the TCR or functional variant thereof comprises an alpha chain as set forth in SEQ ID NO:226 and a beta chain as set forth in SEQ ID NO: 229. In some embodiments, the TCR or functional variant thereof comprises an alpha chain encoded by a nucleotide sequence set forth in SEQ ID NO:225 and a beta chain encoded by a nucleotide sequence set forth in SEQ ID NO: 228.

In some embodiments, the TCR, or a functional variant thereof, comprises an alpha chain comprising a variable alpha (vα) chain having CDR1, CDR2, and CDR3 comprised in a vα chain as set forth in SEQ ID No. 239, and a beta chain comprising a variable beta (vβ) chain having CDR1, CDR2, and CDR3 comprised in a vβ chain as set forth in SEQ ID No. 242. In some embodiments, the TCR or functional variant thereof comprises an alpha chain comprising a V alpha chain having CDR1, CDR2 and CDR3 as set forth in SEQ ID NOS 243, 244 and 245, respectively, and a V beta chain having CDR1, CDR2 and CDR3 as set forth in SEQ ID NOS 246, 247 and 248, respectively. In some embodiments, the TCR or functional variant thereof comprises an alpha chain comprising a V alpha chain as set forth in SEQ ID NO. 239 and a beta chain comprising a V beta chain as set forth in SEQ ID NO. 242. In some embodiments, the TCR or functional variant thereof comprises an alpha chain as set forth in SEQ ID NO. 238 and a beta chain as set forth in SEQ ID NO. 241. In some embodiments, the TCR or functional variant thereof contains an alpha chain encoded by the nucleotide sequence set forth in SEQ ID NO. 237 and a beta chain encoded by the nucleotide sequence set forth in SEQ ID NO. 240.

In some embodiments, one or more nucleic acids encoding TCRs (such as the α and β chains) may be amplified by PCR, cloning, or other suitable methods and cloned into a suitable expression vector or vectors. The expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or expression or both, such as plasmids and viruses.

In some embodiments, the vector may be a pUC series (FERMENTAS LIFE SCIENCES), pBluescript series (Stratagene, la Jolla, calif.), pET series (Novagen, madison, wis.), pGEX series (PHARMACIA BIOTECH, uppsala, sweden) or pEX series (Clontech, palo Alto, calif.). In some cases, phage vectors such as λg10, λgt11, λ ZapII (Stratagene), λembl4, and λnm1149 may also be used. In some embodiments, plant expression vectors may be used, including pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, a viral vector, such as a retroviral vector, is used.

In some embodiments, the recombinant expression vector may be prepared using standard recombinant DNA techniques. In some embodiments, the virus may contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific for the type of host (e.g., bacteria, fungi, plants or animals) into which the vector is to be introduced, and depending on the case where the vector is DNA-or RNA-based. In some embodiments, the vector may contain a non-native promoter operably linked to a nucleotide sequence encoding the TCR or antigen-binding portion (or other MHC-peptide binding molecule). In some embodiments, the promoter may be a non-viral promoter or a viral promoter, such as a Cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and promoters found in the long-terminal repeat of murine stem cell viruses. Other known promoters are also contemplated.

In some embodiments, to generate a vector encoding a TCR, the α and β chains are PCR amplified from total cDNA isolated from T cell clones expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α and β chains are cloned into the same vector. In some embodiments, the α and β chains are cloned into different vectors. In some embodiments, the alpha and beta strands produced are introduced into a retroviral (e.g., lentiviral) vector.

4. Multi-targeting

In some embodiments, the cells and methods include a multi-targeting strategy, such as expressing two or more genetically engineered receptors on the cell, each recognizing the same or different antigens, and typically each including a different intracellular signaling component. These multi-targeting strategies are described, for example, in PCT publication No. WO 2014055668 A1 (describing a combination of activating and co-stimulating CARs, e.g., targeting two different antigens that are present alone on off-target cells (e.g., normal cells), but together on cells of a disease or condition to be treated) and Fedorov et al, sci.trans.medicine, 5 (215) (2013) (describing cells expressing activating and inhibiting CARs, such as binding of an activating CAR to one antigen expressed on both normal or non-diseased cells and cells of a disease or condition to be treated, and binding of the inhibiting CAR to another antigen expressed on only normal cells or cells not desired for treatment).

For example, in some embodiments, the cell comprises a receptor that expresses a first genetically engineered antigen receptor (e.g., CAR or TCR) that is generally capable of inducing an activation signal to the cell upon specific binding to an antigen recognized by the first receptor (e.g., first antigen). In some embodiments, the cell further comprises a second genetically engineered antigen receptor (e.g., CAR or TCR), e.g., a chimeric co-stimulatory receptor, which is generally capable of inducing a co-stimulatory signal to the immune cell upon specific binding to a second antigen recognized by the second receptor. In some embodiments, the first antigen and the second antigen are the same. In some embodiments, the first antigen and the second antigen are different.

In some embodiments, the first and/or second genetically engineered antigen receptor (e.g., CAR or TCR) is capable of inducing an activation signal to a cell. In some embodiments, the receptor induces intracellular signaling components containing ITAM or ITAM-like motifs. In some embodiments, the first receptor-induced activation involves a change in intracellular signal transduction or protein expression, resulting in initiation of an immune response, such as ITAM phosphorylation and/or initiation of an ITAM-mediated signal transduction cascade, formation of an immune synapse and/or aggregation of molecules near a binding receptor (e.g., CD4 or CD8, etc.), activation of one or more transcription factors (such as NF- κb and/or AP-1) and/or induction of expression of factors (such as cytokines), proliferation, and/or survival.

In some embodiments, the first and/or second receptor comprises an intracellular signaling domain of a co-stimulatory receptor (such as CD28, CD137 (4-1 BB), OX40 and/or ICOS). In some embodiments, the first and second receptors comprise intracellular signaling domains of different co-stimulatory receptors. In one embodiment, the first receptor comprises a CD28 costimulatory signaling region and the second receptor comprises a 4-1BB costimulatory signaling region, or vice versa.

In some embodiments, the first and/or second receptor comprises an intracellular signaling domain comprising an ITAM or ITAM-like motif and an intracellular signaling domain of a co-stimulatory receptor.

In some embodiments, the first receptor comprises an intracellular signaling domain comprising an ITAM or ITAM-like motif and the second receptor comprises an intracellular signaling domain of a co-stimulatory receptor. The co-stimulatory signal is combined with the activation signal induced in the same cell as a signal that leads to an immune response, such as a strong and sustained immune response (such as increased gene expression), secretion of cytokines and other factors, and T-cell mediated effector functions (such as cell killing).

In some embodiments, neither the first receptor alone nor the second receptor alone is capable of inducing a strong immune response. In some aspects, if only one receptor is attached, the cell will be resistant or unresponsive to the antigen, or inhibited, and/or not be induced to proliferate or secrete factors or produce effector function. However, in some such embodiments, when multiple receptors are linked, such as when encountering cells expressing the first and second antigens, a desired response is achieved, such as complete immune activation or stimulation, e.g., as indicated by secretion of one or more cytokines, proliferation, maintenance, and/or production of immune effector functions (such as cytotoxic killing of target cells).

In some embodiments, the cell expressing the recombinant receptor further comprises an inhibitory CAR (iCAR, see Fedorov et al, sci.tranl.medicine, 5 (215) (2013), such as a CAR that recognizes antigens other than those associated with and/or specific for a disease or condition, thereby eliminating or inhibiting an activation signal delivered by the disease-targeted CAR (e.g., reducing off-target effects) by binding the inhibitory CAR to its ligand.

In some embodiments, both receptors induce activation and inhibition signals, respectively, to the cell such that binding of one receptor to its antigen activates the cell or induces a response, while binding of the second inhibitory receptor to its antigen induces inhibition or attenuation of the signal of the response. Examples are combinations of activating CAR and inhibiting CAR or iCAR. For example, such a strategy can be used in which an activating CAR binds to an antigen that is expressed in a disease or condition but is also expressed on normal cells, as well as inhibits binding of a receptor to a separate antigen that is expressed on normal cells but is not expressed on cells of the disease or condition.

In some aspects, the chimeric receptor is or includes a CAR (e.g., iCAR) that inhibits and includes an intracellular component that attenuates or inhibits an immune response, such as ITAM-and/or co-stimulation in a cell that promotes the response. Exemplary such intracellular signaling components are those found on immune checkpoint molecules including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, EP2/4 adenosine receptor (including A2 AR). In some aspects, the engineered cells comprise an inhibitory CAR comprising a signaling domain of such an inhibitory molecule or a signaling domain derived from such an inhibitory molecule, such that it is useful for inhibiting a cellular response, e.g., a cellular response induced by activating and/or co-stimulating the CAR.

In some embodiments, a multi-targeting strategy is used where an antigen associated with a particular disease or condition is expressed on non-diseased cells and/or expressed only transiently (e.g., upon stimulation associated with genetic engineering) or permanently on the engineered cells themselves. In this case, the specificity, selectivity and/or effectiveness can be increased by requiring the ligation of two separate and single unique antigen receptors.

In some embodiments, the plurality of antigens (e.g., the first and second antigens) are expressed on the cell, tissue, or disease or condition being targeted, such as on a cancer cell. In some aspects, the cell, tissue, disease or condition is multiple myeloma or multiple myeloma cells. In some embodiments, one or more of the plurality of antigens is also typically expressed on cells that are not desired to be targeted with cell therapy, such as normal or non-diseased cells or tissues, and/or engineered cells themselves. In these embodiments, specificity and/or therapeutic effects are achieved by requiring the attachment of multiple receptors to effect a cellular response.

B. cells and preparation of cells for genetic engineering

Among the cells expressing the receptor and administered by the methods provided are engineered cells. The genetic engineering generally involves introducing a nucleic acid encoding a recombinant or engineered component into a composition containing cells, such as by retroviral transduction, transfection, or transformation.

In some embodiments, the nucleic acid is heterologous, i.e., is not normally present in the cell or in a sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, e.g., is not normally found in the cell being engineered and/or the organism from which the cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acids not found in nature, including nucleic acids comprising chimeric combinations of nucleic acids encoding different domains from a plurality of different cell types.

The cells are typically eukaryotic cells, such as mammalian cells, and are typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of innate immunity or adaptive immunity, e.g., bone marrow or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as pluripotent and multipotent stem cells, including induced pluripotent stem cells (ipscs). The cells are typically primary cells, such as cells isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subpopulations of T cells or other cell types, such as whole T cell populations, CD4⁺ cells, CD8⁺ cells, and subpopulations thereof, such as those defined by function, activation status, maturity, differentiation potential, expansion, recycling, localization and/or persistence capabilities, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. For the subject to be treated, the cells may be allogeneic and/or autologous. Among these methods are off-the-shelf methods. In some aspects, such as for off-the-shelf technology, the cells are multifunctional and/or pluripotent, such as stem cells, such as induced multifunctional stem cells (ipscs). In some embodiments, the methods comprise isolating cells from a subject, preparing, processing, culturing, and/or engineering them, and introducing them into the same subject before or after cryopreservation.

Among the subtypes and subpopulations of T cells and/or CD4⁺ and/or CD8⁺ T cells are naive T (T_N) cells, effector T cells (T_EFF), memory T cells and subtypes thereof, such as stem cell memory T (T_SCM), central memory T (T_CM), effector memory T (T_EM) or terminally differentiated effector memory T cells, tumor Infiltrating Lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells and delta/gamma T cells.

In some embodiments, the cell is a Natural Killer (NK) cell. In some embodiments, the cell is a monocyte or granulocyte, e.g., a bone marrow cell, macrophage, neutrophil, dendritic cell, mast cell, eosinophil, and/or basophil.

In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thus express recombinant or genetically engineered products of these nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., is not normally present in the cell or in a sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, e.g., is not normally found in the cell being engineered and/or the organism from which the cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acids not found in nature, including nucleic acids comprising chimeric combinations of nucleic acids encoding different domains from a plurality of different cell types.

In some embodiments, the genes and/or gene products (and/or their expression) in the provided cells and/or compositions containing the cells are reduced, deleted, eliminated, knocked out or disrupted. In some aspects, these genes and/or gene products comprise one or more of the genes encoding TCR α (TRAC) and/or TCR β (TRBC) (or products thereof), e.g., reducing or preventing expression of endogenous TCRs and/or chains thereof in the cell (e.g., T cell). In some embodiments, the cell (e.g., T cell) expresses an engineered TCR (e.g., any as described in section II-A-3). In some embodiments, reducing or preventing endogenous TCR expression may result in a reduced risk or chance of mismatch between the chains of the engineered TCR and the endogenous TCR, thereby creating a new TCR that may result in a higher risk of undesired or unexpected antigen recognition and/or side effects, and/or may reduce the desired expression level of the endogenous TCR. In some aspects, reducing or preventing endogenous TCR expression can increase expression of the engineered TCR in the cell, such as by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more, as compared to a cell that does not reduce or prevent TCR expression. For example, in some cases, suboptimal expression of an engineered or recombinant TCR may occur due to competition with endogenous TCRs and/or TCRs with mismatched chains, as a result of the involvement of invariant CD3 signaling molecules that allow the complex to be expressed on the cell surface. In some embodiments, the reduction, deletion, elimination, knockout or disruption is achieved by gene editing, such as using Zinc Finger Nucleases (ZFNs), TALENs, or CRISPR/Ca systems with engineered single guide RNAs (grnas) that cleave TCR genes. In some embodiments, reduced expression of an endogenous TCR is performed using an inhibitory nucleic acid molecule against a target nucleic acid encoding a specific TCR (e.g., TCR- α and TCR- β). In some embodiments, the inhibitory nucleic acid is or contains or encodes a small interfering RNA (siRNA), shRNA that is adapted to a microrna, short hairpin RNA (shRNA), hairpin siRNA, microrna (miRNA-precursor), or microrna (miRNA). Exemplary methods for reducing or preventing endogenous TCR expression are known in the art, see, e.g., U.S. patent No. 9,273,283, U.S. publication nos. US2011/0158957, US2014/0301990, US2015/0098954, US2016/0208243, US2016/272999 and US2015/056705, international PCT publications nos. WO2014/191128、WO2015/136001、WO2015/161276、WO2016/069283、WO2016/016341、WO2017/193107、WO2017/093969、WO2019/070541 and WO2019/195492, and Osborn et al (2016) mol. Ter.24 (3): 570-581.

In some of the embodiments provided herein, homology Directed Repair (HDR) can be used to target integration of a particular portion of a template polynucleotide containing a transgene, e.g., a nucleic acid sequence encoding any of the provided recombinant receptors (e.g., recombinant T Cell Receptors (TCRs)), at a particular location in the genome (e.g., the TRAC, TRBC1, and/or TRBC2 loci). In some embodiments, a template polynucleotide comprising a nucleic acid sequence (e.g., transgene) encoding a recombinant T Cell Receptor (TCR) or antigen-binding fragment or chain thereof is introduced into a cell, e.g., an immune cell having genetic disruption at a target site within a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene. In some embodiments, a nucleic acid sequence or transgene encoding a recombinant TCR or antigen-binding fragment or chain thereof is targeted for integration at or near a target site via Homology Directed Repair (HDR). In certain embodiments, the portion of the coding sequence integrated within the TRAC and/or TRBC gene at or near the target site, such as, for example, a portion of the coding sequence downstream or 3' downstream of the target site.

In some embodiments, the preparation of the engineered cells includes one or more culturing and/or preparation steps. Cells for introducing nucleic acid encoding a transgenic receptor (such as a CAR) can be isolated from a sample (such as a biological sample, e.g., a sample obtained from or derived from a subject). In some embodiments, the subject from which the cells are isolated is a subject suffering from a disease or condition or in need of or to whom a cell therapy is to be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention (such as adoptive cell therapy to isolate, treat, and/or engineer cells).

Accordingly, in some embodiments, the cell is a primary cell, e.g., a primary human cell. The samples include tissues, fluids, and other samples taken directly from the subject, as well as samples produced by one or more processing steps such as isolation, centrifugation, genetic engineering (e.g., transduction with viral vectors), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source or processed. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including treated samples derived therefrom.

In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is derived from a apheresis or leukopenia product. Exemplary samples include whole blood, peripheral Blood Mononuclear Cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemia, lymphomas, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and/or cells derived therefrom. In the case of cell therapy, the sample includes, for example, adoptive cell therapy, samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from a cell line, e.g., a T cell line. The cells in some embodiments are obtained from a heterologous source, e.g., from mice, rats, non-human primates, and pigs.

In some embodiments, the separation of cells includes one or more preparation steps and/or non-affinity based cell separation steps. In some examples, the cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, e.g., to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to a particular reagent. In some examples, the cells are isolated based on one or more characteristics, such as density, adhesion characteristics, size, sensitivity, and/or resistance to a particular component.

In some examples, cells from the circulating blood of the subject are obtained, for example, by apheresis or leukopenia. The sample contains lymphocytes in some aspects, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and/or platelets, and in some aspects contains cells other than erythrocytes and platelets.

In some embodiments, blood cells collected from the subject are washed, e.g., the plasma fraction is removed and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with Phosphate Buffered Saline (PBS). In some embodiments, the wash liquor lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, the washing step is accomplished in a semi-automated "flow" centrifuge (e.g., cobe2991 cell processor, baxter) according to manufacturer's instructions. In some aspects, the washing step is accomplished by Tangential Flow Filtration (TFF) according to manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing, such as, for example, ca⁺⁺/Mg⁺⁺ -free PBS. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

In some embodiments, the methods include density-based cell separation methods, such as the preparation of leukocytes from peripheral blood by lysing erythrocytes and by Percoll or Ficoll gradient centrifugation.

In some embodiments, the isolation method comprises isolating different cell types based on the expression or presence of one or more specific molecules (such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids) in the cell. In some embodiments, any known isolation method based on the markers may be used. In some embodiments, the separation is an affinity or immunoaffinity based separation. For example, the isolating includes in some aspects isolating cells and cell populations based on the expression or level of expression of one or more markers (typically cell surface markers) by the cells, e.g., by incubation with antibodies or binding partners that specifically bind these markers, and then isolating cells that bind the antibodies or binding partners from those cells that do not bind the antibodies or binding partners, typically by a washing step.

These separation steps may be based on positive selection, wherein cells that have bound the reagent are retained for further use, and/or negative selection, wherein cells that have not bound the antibody or binding partner are retained. In some examples, both fragments are reserved for further use. In some aspects, negative selection may be particularly useful when no antibodies can specifically recognize cell types in a heterogeneous population, such that isolation is preferably based on markers expressed by cells of an undesired population.

The isolation does not require 100% enrichment or removal of specific cell populations or cells expressing specific markers. For example, positive selection or enrichment of a particular type of cell (such as those expressing a marker) refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal or clearance of a particular type of cell (such as those expressing a marker) refers to a reduction in the number or percentage of such cells, but does not require complete removal of all of these cells.

In some examples, multiple rounds of separation steps are performed, wherein portions from positive or negative selections of one step are subjected to another separation step, such as subsequent positive or negative selections. In some examples, a single isolation step may simultaneously clear cells expressing multiple markers, such as by incubating the cells with multiple antibodies or binding partners, each antibody or binding partner specific for a marker targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on the multiple cell types.

For example, in some aspects, a particular subpopulation of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., ,CD28⁺、CD62L⁺、CCR7⁺、CD27⁺、CD127⁺、CD4⁺、CD8⁺、CD45RA⁺ and/or CD45RO⁺ T cells, is isolated by positive or negative selection techniques.

For example, CD3⁺、CD28⁺ T cells can use anti-CD 3/anti-CD 28 conjugated magnetic beads (e.g.,M-450CD3/CD28T Cell Expander).

In some embodiments, the separation is performed by enriching for a particular cell population by positive selection or by clearing for a particular cell population by negative selection. In some embodiments, positive or negative selection is accomplished by incubating the cells with one or more antibodies or other binding reagents that specifically bind to one or more surface markers expressed on the positive or negative selected cells (marker⁺) or expressed at a relatively high level (marker high), respectively.

In some embodiments, T cells are isolated from a PBMC sample by negative selection for a marker expressed on non-T cells (such as B cells, monocytes or other leukocytes such as CD 14). In some aspects, the CD4⁺ or CD8⁺ selection step is used to isolate CD4⁺ helper and CD8⁺ cytotoxic T cells. These CD4⁺ and CD8⁺ populations can be further sorted into subpopulations by positive or negative selection of markers expressed or expressed to a relatively high degree on one or more naive, memory and/or effector T cell subpopulations.

In some embodiments, the CD8⁺ cells are further enriched or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulations. In some embodiments, enrichment of central memory T (T_CM) cells is performed to increase therapeutic efficacy, such as to improve long-term survival, expansion, and/or implantation after administration, which in some aspects is particularly intense in these subpopulations. See Terakura et al, blood.1:72-82 (2012), wang et al, J Immunother.35 (9): 689-701 (2012). In some embodiments, combining TCM-enriched CD8⁺ T cells and CD4⁺ T cells further enhances therapeutic efficacy.

In embodiments, memory T cells are present in both CD62L⁺ and CD 62L-subpopulations of CD8⁺ peripheral blood lymphocytes. PBMCs may enrich or clear CD62L-CD8⁺ and/or CD62L⁺CD8⁺ portions, such as with anti-CD 8 and anti-CD 62L antibodies.

In some embodiments, enrichment of central memory T (T_CM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3 and/or CD 127, in some aspects, on negative selection of cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, the isolation of the population of CD8⁺ enriched for T_CM cells is performed by clearing cells expressing CD4, CD14, CD45RA and positive selection or enrichment of cells expressing CD 62L. In one aspect, central memory T (T_CM) cells are enriched by first subjecting them to negative selection based on expression of CD14 and CD45RA, and positive selection based on CD62L, using a negative cell fraction selected based on CD4 expression. This selection is performed simultaneously in some aspects and sequentially in any order in other aspects. In some aspects, the same selection step based on CD4 expression used to prepare the population or subpopulation of CD8⁺ cells is also used to generate the population or subpopulation of CD4⁺ cells such that both positive and negative portions from the CD 4-based isolation are retained and used in subsequent steps of the method, optionally after one or more further positive or negative selection steps.

In a specific example, a sample of PBMCs or other leukocyte samples is subjected to selection of CD4⁺ cells, wherein both negative and positive portions are retained. The negative portion is then subjected to a negative selection based on the expression of CD14 and CD45RA or CD19, and a positive selection based on a marker characterized by central memory T cells (such as CD62L or CCR 7), wherein the positive and negative selections are performed in either order.

CD4⁺ T helper cells are sorted into naive, central memory and effector cells by recognizing cell populations with cell surface antigens. CD4⁺ lymphocytes can be obtained by standard methods. In some embodiments, the naive CD4⁺ T lymphocytes are CD45RO-, CD45RA⁺、CD62L⁺、CD4⁺ T cells. In some embodiments, the central memory CD4⁺ cells are CD62L⁺ and CD45RO⁺. In some embodiments, effector CD4⁺ cells are CD 62L-and CD45RO-.

In one example, to enrich for CD4⁺ cells by negative selection, monoclonal antibody cocktail typically includes antibodies directed against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as magnetic or paramagnetic beads, such that positive and/or negative selection is performed for cell separation. For example, in some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used (at Methods in Molecular Medicine,vol.58:Metastasis Research Protocols,Vol.2:Cell Behavior In vitro and In vivo,p 17-25S.A.Brooks and U.S. Schumacher,Isolated or isolated cells and cell populations are reviewed in Humana Press inc., totowa, NJ).

In some aspects, the cell sample or cell composition to be isolated is incubated with a small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material (e.g., particles) is typically directly or indirectly attached to a binding partner (e.g., an antibody) that specifically binds to a molecule (e.g., a surface marker present on a single cell, multiple cells, or cell population that is desired to be isolated (e.g., desired for negative or positive selection).

In some embodiments, the magnetic particles or beads comprise magnetically responsive material bound to a specific binding member (such as an antibody or other binding partner). There are many known magnetically responsive materials in magnetic separation methods. Suitable magnetic particles include those as described in Molday, U.S. Pat. No.4,452,773 and in european patent specification EP 452342B (which is incorporated herein by reference). Colloid-sized particles such as those described in U.S. patent No.4,795,698 to Owen and U.S. patent No. 5,200,084 to Liberti et al are other examples.

The incubation is typically performed under conditions in which antibodies or binding partners or molecules (such as secondary antibodies or other reagents) that specifically bind to these antibodies or binding partners attached to the magnetic particles or beads specifically bind to cell surface molecules (if present on cells within the sample).

In some aspects, the sample is placed in a magnetic field, and those cells to which magnetically responsive or magnetizable particles are attached will be attracted by the magnet and separated from unlabeled cells. For positive selection, cells attracted to the magnet were retained, and for negative selection, cells not attracted (unlabeled cells) were retained. In some aspects, a combination of positive and negative selections is performed during the same selection step, wherein the positive and negative portions are retained and further processed or subjected to further separation steps.

In certain embodiments, the magnetically responsive particles are coated in a primary or other binding partner, secondary antibody, lectin, enzyme or streptavidin. In certain embodiments, the magnetic particles are attached to the cells via a coating of a primary antibody specific for one or more markers. In certain embodiments, cells are labeled with a primary antibody or binding partner instead of beads, and then magnetic particles coated with a cell type specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

In some embodiments, the magnetically responsive particles are attached to cells that are subsequently incubated, cultured, and/or engineered, in some aspects, the particles are attached to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competitive non-labeled antibodies and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, affinity-based selection is via Magnetically Activated Cell Sorting (MACS) (Miltenyi Biotec, auburn, CA). Magnetically Activated Cell Sorting (MACS) systems enable high purity selection of cells of magnetized particles attached thereto. In certain embodiments, MACS operates in a mode in which non-target and target species are eluted sequentially after the application of an externally applied magnetic field. That is, cells attached to the magnetized particles are fixed in place, while unattached species are eluted. Then, after the first elution step is completed, the species that will be trapped in the magnetic field and that will be eluted is prevented from being released in some way so that it can be eluted and recovered. In certain embodiments, non-target cells are labeled and cleared from a heterogeneous cell population.

In certain embodiments, the separation (isolation) or isolation (separation) is performed using a system, device, or instrument that performs one or more of the steps of isolation, cell preparation, separation, processing, incubation, culture, and/or formulation of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, e.g., minimizing errors, user operations, and/or contamination. In one embodiment, the system is a system as described in PCT publication No. WO2009/072003 or US20110003380 A1.

In some embodiments, the system or apparatus performs one or more of the separation, processing, engineering, and formulation steps, e.g., all, in an integrated or self-contained system and/or in an automated or programmable manner. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus that allows a user to program, control, evaluate, isolate, engineer, and formulate the results of the steps and/or adjust various aspects thereof.

In some aspects, the isolation and/or other steps are performed using a clinic macs system (Miltenyi Biotec), e.g., automated cell isolation at the clinical scale level in closed and sterile systems. The assembly may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. The integrated computer controls in some aspects all components of the instrument and directs the system to perform the iterative process in accordance with the standardized procedure. The magnetic separation unit in some aspects includes a movable permanent magnet and a bracket for a selection column. The peristaltic pump, together with the pinch valve, controls the flow rate of the whole tube set, ensuring a controlled flow of buffer through the system and a continuous suspension of cells.

The CliniMACS system uses in some aspects antibody-coupled magnetizable particles that are provided in sterile, pyrogen-free solutions. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to the tube set, which in turn is connected to a bag containing buffer and a cell collection bag. The tube set consists of a pre-assembled sterile tube containing a front column and a separation column and is intended for single use only. After the separation procedure is initiated, the system automatically applies the cell sample to the separation column. The labeled cells remain within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population markers used in the methods described herein are not retained in the column. In some embodiments, the population of cells used in the methods described herein is labeled and retained in a column. In some embodiments, after removal of the magnetic field, the population of cells used in the methods described herein is eluted from the column and collected in a cell collection bag.

In certain embodiments, the separation and/or other steps are performed using the CLINIMACS PRODIGY system (Miltenyi Biotec). The CLINIMACS PRODIGY system, in some aspects, configures a cell handling unit that allows for automatic washing and fractionation of cells by centrifugation. The CLINIMACS PRODIGY system may also include an onboard camera and image recognition software that determines the optimal cell-classification endpoint by identifying the macroscopic layer of the source cell product. For example, peripheral blood is automatically separated into red blood cells, white blood cells, and plasma layers. The CLINIMACS PRODIGY system may also include an integrated cell culture chamber that completes cell culture protocols such as, for example, cell differentiation and expansion, antigen loading, and long-term cell culture. The input port allows sterile removal and replenishment of the medium, and the cells can be monitored using an integrated microscope. See, for example, klebanoff et al, JImmunother.35 (9): 651-660 (2012), terakura et al, blood.1:72-82 (2012), and Wang et al, J Immunother.35 (9): 689-701 (2012).

In some embodiments, the population of cells described herein is collected and enriched (or cleared) via flow cytometry, wherein cells stained for a plurality of cell surface markers are carried in a fluid stream. In some embodiments, the population of cells described herein is collected and enriched (or cleared) via preparative scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or cleared) by using microelectromechanical systems (MEMS) chips (see, e.g., WO 2010/033140, cho et al, lab Chip 10,1567-1573 (2010), and Godin et al, J Biophoton.1 (5): 355-376 (2008)) in combination with a FACS-based detection system. In both cases, the cells are labeled with a variety of markers, allowing for the isolation of well-defined T cell subsets in high purity.

In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate isolation for positive and/or negative selection. For example, the separation may be based on binding to a fluorescently labeled antibody. In some examples, isolating cells based on antibodies or other binding partners specific for one or more cell surface markers is performed in a fluid stream, such as by Fluorescence Activated Cell Sorting (FACS), which includes preparation scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in conjunction with a flow cytometry detection system. This method allows positive and negative selection based on multiple markers simultaneously.

In some embodiments, the methods of preparation include steps for freezing (e.g., cryopreserving) cells before or after isolation, incubation, and/or engineering. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and to some extent monocytes from the cell population. In some embodiments, the cells are suspended in a frozen solution, for example, after a washing step to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be used. One embodiment involves the use of PBS or other suitable cell freezing medium containing 20% DMSO and 8% Human Serum Albumin (HSA). It was then diluted 1:1 with medium to give final DMSO and HAS concentrations of 10% and 4%, respectively. Cells are typically frozen to-80 ℃ at a rate of 1 ℃ per minute and stored in the gas phase of a liquid nitrogen storage tank.

In some embodiments, the cells are incubated and/or cultured prior to or in combination with genetic engineering. The incubation step may include culturing, incubating, stimulating, activating, and/or proliferating. Incubation and/or engineering may be performed in a culture vessel (such as a unit, chamber, well, column, tube, set of tubes, valve, vial, petri dish, bag, or other vessel for culturing or incubating cells). In some embodiments, the composition or cell is incubated in the presence of a stimulating condition or agent. These conditions include conditions designed to induce proliferation, expansion, activation and/or survival of cells in a cell population to mimic antigen exposure and/or to sensitize cells for genetic engineering, such as for the introduction of recombinant antigen receptors.

The conditions may include one or more specific media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cell.

In some embodiments, the stimulating condition or agent comprises one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent initiates or initiates a TCR/CD3 intracellular signaling cascade in the T cell. These agents may include antibodies, such as those specific for TCRs (e.g., anti-CD 3). In some embodiments, the stimulation conditions include one or more agents, e.g., ligands, capable of stimulating a co-stimulatory receptor, e.g., anti-CD 28. In some embodiments, these reagents and/or ligands may be bound to a solid support such as a bead and/or one or more cytokines. Optionally, the amplification method may further comprise adding anti-CD 3 and/or anti-CD 28 antibodies to the medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulatory agent includes IL-2, IL-15 and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/mL.

In some aspects, incubation is performed according to techniques such as those described in U.S. Pat. No. 6,040,177, klebanoff to Riddell et al, J Immunother.35 (9): 651-660 (2012), terakura et al, blood.1:72-82 (2012), and/or Wang et al, J Immunother.35 (9): 689-701 (2012).

In some embodiments, the T cells are expanded by adding feeder cells, such as non-dividing Peripheral Blood Mononuclear Cells (PBMCs), to the culture starting composition (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded), and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can comprise gamma irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays ranging from about 3000 to 3600 rads to prevent cell division. In one aspect, the feeder cells are added to the culture medium prior to the addition of the T cell population.

In some embodiments, the stimulation conditions include a temperature suitable for growth of human T lymphocytes, e.g., at least about 25 degrees celsius, typically at least about 30 degrees celsius, and typically at or about 37 degrees celsius. Optionally, the incubating may further comprise adding non-dividing EBV-transformed lymphoblastic-Like Cells (LCLs) as feeder cells. The LCL may be irradiated with gamma rays ranging from about 6000 to 10000 rad. The LCL feeder cells may be provided in any suitable amount in some aspects, such as a ratio of LCL feeder cells to naive T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells (such as antigen-specific CD4⁺ and/or CD8⁺ T cells) are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, by isolating T cells from an infected subject and stimulating the cells with the same antigen in vitro, antigen-specific T cell lines or clones can be generated that are directed against cytomegalovirus antigens.

C. nucleic acids, vectors and methods for genetic engineering

In some embodiments, the cells (e.g., T cells) are genetically engineered to express a recombinant receptor. In some embodiments, the engineering is performed by introducing a nucleic acid molecule encoding the recombinant receptor. Nucleic acid molecules encoding the recombinant receptor and vectors or constructs comprising the nucleic acids and/or nucleic acid molecules are also provided.

In some cases, the nucleic acid sequence encoding a recombinant receptor (e.g., chimeric Antigen Receptor (CAR)) contains a signal sequence encoding a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. In some embodiments, the signal peptide is derived from a transmembrane protein. In some embodiments, the signal peptide is derived from CD8a, CD33, or IgG. Non-limiting illustrative examples of signal peptides include, for example, the CD33 signal peptide set forth in SEQ ID NO. 21, the CD8a signal peptide set forth in SEQ ID NO. 75, or the signal peptide set forth in SEQ ID NO. 76, or modified variants thereof.

In some embodiments, the nucleic acid molecule encoding the recombinant receptor comprises at least one promoter operably linked to control expression of the recombinant receptor. In some examples, the nucleic acid molecule contains two, three, or more promoters operably linked to control expression of the recombinant receptor. In some embodiments, the nucleic acid molecule may contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific for the type of host (e.g., bacterial, fungal, plant, or animal) into which the nucleic acid molecule is introduced, and depending on whether the nucleic acid molecule is DNA-or RNA-based. In some embodiments, the nucleic acid molecule may contain regulatory/control elements such as promoters, enhancers, introns, polyadenylation signals, kozak consensus sequences (Kozak consensus sequence), and splice acceptors or donors. In some embodiments, the nucleic acid molecule may contain a non-native promoter operably linked to a nucleotide sequence encoding a recombinant receptor and/or one or more additional polypeptides. In some embodiments, the promoter is selected from the group consisting of RNA pol I, pol II, or pol III promoters. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., CMV, SV40 early region, or adenovirus major late promoter). In another embodiment, the promoter is recognized by RNA polymerase III (e.g., U6 or H1 promoter). In some embodiments, the promoter may be a non-viral promoter or a viral promoter, such as a Cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and promoters found in the long-terminal repeat of murine stem cell viruses. Other known promoters are also contemplated.

In some embodiments, the promoter is or includes a constitutive promoter. Exemplary constitutive promoters include, for example, the simian virus 40 early promoter (SV 40), the cytomegalovirus immediate early promoter (cytomegalovirus immediate-early promoter, CMV), the human ubiquitin C promoter (UBC), the human elongation factor 1 alpha promoter (EF 1 alpha), the mouse phosphoglycerate kinase 1 Promoter (PGK), and the chicken β -actin promoter coupled to the CMV early enhancer (CAGG). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises an MND promoter (a synthetic promoter containing the U3 region of the modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer) (see Challita et al (1995) J.Virol.69 (2): 748-755). In some embodiments, the promoter is a tissue specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter.

In another embodiment, the promoter is a regulated promoter (e.g., an inducible promoter). In some embodiments, the promoter is an inducible promoter or a repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, or a doxycycline operator sequence, or an analog thereof, or is capable of binding or recognizing by a Lac inhibitor or a tetracycline inhibitor or analog thereof. In some embodiments, the nucleic acid molecule does not include a regulatory element (e.g., a promoter).

In some embodiments, the nucleic acid molecule encoding a recombinant receptor (e.g., CAR or other antigen receptor) further comprises a nucleic acid sequence encoding a marker, and/or the cell expressing the CAR or other antigen receptor further comprises a marker, e.g., a surrogate marker, such as a cell surface marker, that can be used to confirm transduction or engineering of the cell to express a receptor, such as a truncated form of the cell surface receptor, such as truncated EGFR (tgfr). In some embodiments, the one or more markers are transduction markers, surrogate markers, and/or selection markers.

In some embodiments, the marker is a transduction marker or a surrogate marker. The transduction markers or surrogate markers can be used to detect cells into which a nucleic acid molecule (e.g., a nucleic acid molecule encoding a recombinant receptor) has been introduced. In some embodiments, the transduction markers may indicate or confirm modification of the cell. In some embodiments, the surrogate marker is a protein that is co-expressed with the recombinant receptor (e.g., CAR) at the cell surface. In particular embodiments, such surrogate markers are surface proteins that have been modified to have little or no activity. In certain embodiments, such surrogate markers are encoded on the same nucleic acid molecule encoding the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operably linked to a nucleic acid sequence encoding a marker, optionally isolated by an Internal Ribosome Entry Site (IRES) or a nucleic acid encoding a self-cleaving peptide or a ribosome-jump-causing peptide, such as a 2A sequence, such as T2A, P2A, E a or F2A. Exogenous marker genes can be used in conjunction with engineered cells in some cases to allow detection or selection of cells and also to promote cell suicide in some cases.

Exemplary surrogate markers may include truncated forms of a cell surface polypeptide, such as those that are nonfunctional and do not conduct or are incapable of conducting a signal (or the full length form of a cell surface polypeptide typically conducts a signal), and/or truncated forms that are not or cannot internalize. Exemplary truncated cell surface polypeptides include truncated growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER 2), truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequences as set forth in SEQ ID NO:7 or 28), or Prostate Specific Membrane Antigen (PSMA), or modified forms thereof. tEGFR may contain cetuximab by antibodyOr other therapeutic anti-EGFR antibodies or binding molecules, which can be used to recognize or select cells that have been engineered with the tgfr construct and the encoded foreign protein, and/or eliminate or isolate cells expressing the encoded foreign protein. See U.S. patent No. 8,802,374 and Liu et al, nature biotech.2016april;34 (4): 430-434). In some aspects, the markers (e.g., surrogate markers) include all or a portion (e.g., a truncated form) of CD34, NGFR, CD19, or truncated CD19 (e.g., truncated non-human CD 19) or an epidermal growth factor receptor (e.g., tgfr). In some embodiments, the marker is or comprises a fluorescent protein, such as Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (EGFP), such as superfolder GFP (sfGFP), red Fluorescent Protein (RFP), such as tdTomato, mCherry, mStrawberry, asRed, dsRed or DsRed2, cyan Fluorescent Protein (CFP), blue-green fluorescent protein (BFP), enhanced Blue Fluorescent Protein (EBFP), and Yellow Fluorescent Protein (YFP), and variants thereof, including species variants, monomeric variants, and codon optimized and/or enhanced variants of the fluorescent protein. In some embodiments, the marker is or includes an enzyme, such as luciferase, lacZ gene from E.coli, alkaline phosphatase, secreted Embryonic Alkaline Phosphatase (SEAP), chloramphenicol Acetyl Transferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), beta-galactosidase, chloramphenicol Acetyl Transferase (CAT), beta-Galactosidase (GUS), or variants thereof.

In some embodiments, the marker is a selectable marker. In some embodiments, the selectable marker is or comprises a polypeptide that confers resistance to an exogenous agent or drug. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the selectable marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selectable marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin (Blasticidin) resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a gecomycin resistance gene, or a modified form thereof.

In some aspects, the markers (e.g., surrogate markers) include all or a portion (e.g., truncated form) of CD34, NGFR, or an epidermal growth factor receptor (e.g., tgfr). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence (e.g., T2A). For example, the marker, and optionally the linker sequence, may be any as disclosed in PCT publication No. WO 2014031687. For example, the marker may be truncated EGFR (tgfr), optionally linked to a linker sequence, such as a T2A cleavable linker sequence. Exemplary polypeptides of truncated EGFR (e.g., tEGFR) comprise an amino acid sequence set forth in SEQ ID NO:7 or 28, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:7 or 28. Exemplary T2A linker sequences comprise the amino acid sequence set forth in SEQ ID NO. 6 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 6.

In some embodiments, the nucleic acid molecule encoding the CAR construct further comprises a sequence encoding a T2A ribosome hopping element and/or a tgfr sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, the sequence encodes a T2A ribosome jump element as set forth in SEQ ID NO. 6, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 6. In some embodiments, T cells expressing an antigen receptor (e.g., CAR) can also be generated to express truncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g., by introducing a construct encoding the CAR and EGFRt, isolated by T2A ribosomal switch to express two proteins from the same construct), which can then be used as a marker for detecting the cells (see, e.g., U.S. patent No. 8,802,374). In some embodiments, the sequence encodes a tEGFR sequence set forth in SEQ ID NO 7 or 28, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 7 or 28.

In some embodiments, a single promoter may direct expression of an RNA containing two or three genes separated from each other by a sequence encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin) in a single Open Reading Frame (ORF) (e.g., encoding a molecule involved in regulating a metabolic pathway and encoding the recombinant receptor). The ORF thus encodes a single polypeptide which is processed into separate proteins during translation (in the case of 2A) or after translation. In some cases, the peptide (such as T2A) may cause ribosomes to jump at the C-terminus of the 2A element (ribosome jump) to synthesize a peptide bond, resulting in separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe. Genetic VACCINES AND Ther.2:13 (2004) and deFelipe et al Traffic 5:616-626 (2004)). Many 2A elements are known in the art. Examples of 2A sequences useful in the methods and nucleic acids described herein are not limited to 2A sequences from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 27), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 26), racecar virus (Thosea asigna virus, T2A, e.g., SEQ ID NO:6 or 23), and porcine circovirus 1 (porcine teschovirus-1, P2A, e.g., SEQ ID NO:24 or 25), as described in U.S. patent publication No. 20070116690.

In some embodiments, the marker is a molecule, e.g., a cell surface protein, not naturally found on a T cell or not naturally found on the surface of a T cell or a portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., a molecule that the immune system of the host into which the cell will adoptively transfer does not recognize as "self.

In some embodiments, the marker is not therapeutically functional and/or does not produce any effect other than use as a genetically engineered marker (e.g., for selection of successfully engineered cells). In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, such as a ligand of a cell encountered in vivo, such as a co-stimulatory or immune checkpoint molecule, to enhance and/or attenuate the response of the cell upon adoptive transfer and encountering the ligand.

The introduction of the nucleic acid molecule encoding the recombinant receptor into the cell may be performed using any of a number of known vectors. The vectors include viral and nonviral systems, including lentiviral and gamma retroviral systems, as well as transposon-based systems, such as PiggyBac or Sleeping Beauty based gene transfer systems. Exemplary methods include methods for transferring nucleic acids encoding the receptor, including transduction via a virus (e.g., retrovirus or lentivirus), transposon, and electroporation.

In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by binding it to a stimulus that induces a response (such as proliferation, survival, and/or activation) (e.g., as measured by expression of a cytokine or activation marker), followed by transduction of the activated cell, and expansion in culture to an amount sufficient for clinical use.

In some cases, overexpression of a stimulus (e.g., a lymphokine or cytokine) can be toxic to a subject. Thus, in some cases, the engineered cells include gene fragments that result in the cells being susceptible to negative selection in vivo, such as when administered in adoptive immunotherapy. For example, in some aspects, the cells are engineered such that they can be eliminated due to changes in the condition in the patient to whom they are administered. A negative selectable phenotype may be caused by gene insertion that confers sensitivity to the agent (e.g., compound) administered. Negative selectable genes include the herpes simplex virus type I thymidine kinase (HSV-ITK) gene which confers ganciclovir sensitivity (Wigler et al, cell 2:223, 1977), the cellular hypoxanthine phosphoribosyl transferase (HPRT) gene, the cellular adenine phosphoribosyl transferase (APRT) gene, bacterial cytosine deaminase (Mullen et al, proc. Natl. Acad. Sci. USA.89:33 (1992)).

In some embodiments, recombinant nucleic acid is transferred into cells using recombinant infectious viral particles, such as, for example, vectors derived from simian virus 40 (SV 40), adenovirus, adeno-associated virus (AAV). In some embodiments, recombinant lentiviral vectors or retroviral vectors (such as gamma-retroviral vectors) are used to transfer recombinant nucleic acids into T cells (see, e.g., koste et al (2014) GENE THERAPY 2014Apr 3.doi:10.1038/gt.2014.25; carlens et al (2000) Exp Hematol (10): 1137-46; alonso-Camino et al (2013) Mol Ther Nucl Acids 2, e93; park et al, trends Biotechnol.2011November 29 (11): 550-557).

In some embodiments, the retroviral vector has a Long Terminal Repeat (LTR), e.g., a retroviral vector derived from moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine Stem Cell Virus (MSCV), spleen Focus Forming Virus (SFFV), or adeno-associated virus (AAV). Most retroviruses are derived from murine retroviruses. In some embodiments, the retrovirus includes those derived from any avian or mammalian cell source. The retroviruses are typically amphotropic (amphotropic), meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740;6,207,453;5,219,740; MILLER AND Rosman (1989) BioTechniques 7:980-990; miller, A.D. (1990) Human GENE THERAPY 1:5-14; scarpa et al (1991) Virology 180:849-852; burns et al (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-LAWRIE AND TEMIN (1993) Cur. Opin. Genet. Development. 3:102-109).

Methods of lentiviral transduction are known. Exemplary Methods are described, for example, in Wang et al (2012) J.Immunother.35 (9): 689-701, cooper et al (2003) blood.101:1637-1644, verhoeyen et al (2009) Methods Mol biol.506:97-114, and CAVALIERI et al (2003) blood.102 (2): 497-505.

In some embodiments, the recombinant nucleic acid is transferred into T cells via electroporation (see, e.g., chicaybam et al, (2013) PLoS ONE 8 (3): e60298 and Van Tedeloo et al (2000) GENE THERAPY (16): 1431-1437). In some embodiments, the recombinant nucleic acid is transferred into T cells via transposition (see, e.g., manuri et al (2010) Hum Gene Ther 21 (4): 427-437; shalma et al (2013) Molec Ther Nucl Acids, e74; and Huang et al (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, john Wiley & Sons, new York. N.Y.), protoplast fusion, cationic liposome-mediated transfection, tungsten particle-promoted microprojectile bombardment (Johnston, nature,346:776-777 (1990)), and strontium phosphate DNA co-precipitation (Brash et al, mol. Cell biol.,7:2031-2034 (1987)).

Other methods and vectors for transferring nucleic acids encoding recombinant products are those described in, for example, international patent application publication No. WO2014055668 and U.S. Pat. No. 7,446,190.

In some embodiments, the cells (e.g., T cells) can be transfected during or after expansion, e.g., with a T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR). Such transfection of the gene for introduction of the desired receptor may be performed using, for example, any suitable retroviral vector. The genetically modified cell population may then be released from the primary stimulus (e.g., a CD3/CD28 stimulus) and subsequently stimulated with a second type of stimulus (e.g., via a de novo introduced receptor). Such a second type of stimulus may include antigenic stimulus in the form of a peptide/MHC molecule, a cognate (cross-linked) ligand of a genetically introduced receptor (e.g. the natural ligand of a CAR) or any ligand (such as an antibody) that binds directly within the framework of a new receptor (e.g. by recognizing a constant region within the receptor). See, for example, cheadle et al, "CHIMERIC ANTIGEN receptors for T-cell based therapy" Methods Mol biol.2012;907:645-66 or Barrett et al, CHIMERIC ANTIGEN Receptor Therapy for Cancer Annual Review of Medicine Vol.65:333-347 (2014).

In some cases, vectors may be used that do not require activation of cells (e.g., T cells). In some such cases, the cells may be selected and/or transduced prior to activation. Thus, the cells may be engineered before or after cell culture, and in some cases simultaneously with or during at least a portion of the culture.

In some aspects, the cells are further engineered to promote expression of cytokines or other factors. Among other nucleic acids, for example, genes for introduction are those that enhance therapeutic effects, such as by promoting viability and/or function of the transferred cells, genes that provide genetic markers for selection and/or evaluation of cells, such as evaluating survival or localization in vivo, genes that enhance safety, for example, by making cells susceptible to negative selection in vivo, as described by Lupton s.d. et al, mol.and Cell biol.,11:6 (1991), and Riddell et al, human GENE THERAPY 3:319-338 (1992), see also PCT/US91/08442 and PCT/US94/05601 disclosures of Lupton et al, wherein the use of bifunctional selectable fusion genes derived from fusion dominant positive selectable markers with negative selectable markers is described. See, e.g., riddell et al, U.S. patent No. 6,040,177, columns 14-17.

Exemplary therapeutic results and methods of evaluating same

In some embodiments of the methods, compositions, combinations, uses, kits and articles of manufacture provided herein, the provided combination therapies produce one or more therapeutic results, such as features related to any one or more therapy or therapy-related parameters, as described below. In some embodiments, the method is any method as described in section I. In some embodiments, the methods further comprise evaluating exposure, persistence, and proliferation of T cells (e.g., T cells administered based on T cell therapy). In some embodiments, the exposure or sustained expansion and/or persistence of cells (e.g., cells for immunotherapy, such as T cell therapy administration), and/or changes in cellular phenotype or cellular functional activity in the methods provided herein can be measured by evaluating characteristics of T cells in vitro or ex vivo. In some embodiments, these assays can be used to determine or confirm the function of T cells (e.g., T cell therapies) prior to, during, or after administration of the combination therapies provided herein.

In some embodiments, the combination therapy may further comprise one or more screening steps to identify subjects treated with the combination therapy and/or continuing the combination therapy, and/or steps to evaluate treatment outcome and/or monitor treatment outcome. In some embodiments, the treatment outcome evaluation step may include the step of evaluating and/or monitoring the treatment and/or identifying further or remaining steps for administration of the therapy and/or the subject for repeat therapy. In some embodiments, the screening step and/or treatment outcome evaluation may be used to determine the dose, frequency, duration, timing, and/or order of the combination therapies provided herein.

In some embodiments, any of the screening steps and/or treatment outcome evaluations described herein can be performed prior to, during, or subsequent to one or more steps of administering the provided combination therapies (e.g., administering T cell therapies (e.g., TCR or CAR expressing T cells) and/or administering DGK inhibitors). In some embodiments, the evaluation is made before, during, or after any one of the methods provided herein. In some embodiments, the evaluation is made prior to performing the methods provided herein. In some embodiments, the evaluation is made after performing one or more steps of the methods provided herein. In some embodiments, the evaluation is performed prior to one or more steps of administering the provided combination therapy, e.g., screening and identifying patients who are suitable and/or sensitive to receiving the combination therapy. In some embodiments, the evaluating is performed during, or subsequent to one or more steps of administering the provided combination therapy, e.g., evaluating mid-term or final treatment outcome (e.g., determining treatment efficacy and/or determining whether to continue or repeat treatment and/or determining whether to administer the remaining steps of the combination therapy).

In some embodiments, the therapeutic result includes improved immune function, e.g., immune function of T cells and/or endogenous T cells in vivo for cell-based therapy administration. In some embodiments, exemplary therapeutic results include, but are not limited to, enhanced T cell proliferation, enhanced T cell functional activity, altered expression of immune cell phenotype markers, such as those features associated with engineered T cells (e.g., TCR or CAR-T cells administered to the subject). In some embodiments, exemplary therapeutic results include reduced disease burden (e.g., tumor burden), improved clinical results, and/or enhanced therapeutic efficacy of the therapy.

In some embodiments, the screening step and/or treatment outcome evaluation comprises evaluating survival and/or function of T cells for cell-based therapy administration. In some embodiments, the screening step and/or treatment outcome evaluation comprises evaluating the level of a cytokine or growth factor. In some embodiments, the screening step and/or treatment outcome evaluation comprises evaluating disease burden and/or improvement, e.g., evaluating tumor burden and/or clinical outcome. In some embodiments, one of the screening steps and/or treatment outcome evaluations may include any of the evaluation methods and/or assays described herein and/or known, and may be performed one or more times at one or more steps (e.g., before, during, or after) of the combination therapy. Exemplary parameter sets associated with therapeutic results that can be evaluated in some embodiments of the methods provided herein include peripheral blood immune cell population profiles and/or tumor burden.

In some embodiments, the method affects the efficacy of a cell therapy in a subject. In some embodiments, the persistence, expansion, and/or presence of cells expressing a recombinant receptor (e.g., expressing a TCR or CAR) in a subject is greater after administration of a dose of cells in a method comprising a DGK inhibitor (e.g., any of those described in section I-B) than is achieved via a subject not administered a DGK inhibitor. In some embodiments, the expansion and/or persistence in a subject administered a T cell therapy (e.g., a T cell expressing a TCR or CAR) is assessed compared to a method of administering a T cell therapy to a subject in the absence of a DGK inhibitor. In some embodiments, the method results in the administered T cells exhibiting increased or prolonged expansion and/or persistence in the subject as compared to a method of administering T cell therapy to the subject in the absence of the DGK inhibitor.

In some embodiments, administration of the DGK inhibitor reduces disease burden, e.g., tumor burden, compared to methods in which a dose of cells expressing the recombinant receptor is administered to a subject in the absence of the DGK inhibitor. In some embodiments, administration of the DGK inhibitor reduces bone marrow blast in the subject as compared to a method of administering a dose of cells expressing the recombinant receptor to the subject in the absence of the DGK inhibitor. In some embodiments, administration of the DGK inhibitor results in improved clinical outcomes, such as Objective Remission Rate (ORR), progression Free Survival (PFS), and total survival (OS), compared to methods of administering a dose of cells expressing the recombinant receptor to a subject in the absence of the DGK inhibitor.

In some embodiments, the subject may be screened prior to one or more steps of administering the combination therapy. For example, prior to administration of a combination therapy, a subject may be screened for disease and/or disease burden characteristics (e.g., tumor burden) to determine suitability, responsiveness, and/or susceptibility to administration of the combination therapy. In some embodiments, the screening step and/or treatment outcome evaluation may be used to determine the dose, frequency, duration, timing, and/or order of the combination therapies provided herein.

In some embodiments, the subject may be screened after one or more steps of administration of the combination therapy to determine and identify subjects who received the remaining steps of the combination therapy and/or monitor the efficacy of the therapy. In some embodiments, the number, level or amount of T cells administered and/or proliferation and/or activity of T cells administered is assessed prior to and/or after administration of the DGK inhibitor.

In some embodiments, the change and/or alteration, e.g., increase, decrease, or decrease, in the level, value, or measurement of the parameter or result is determined or evaluated as compared to the level, value, or measurement of the same parameter or result at a different evaluation time point, a different condition, reference point, and/or a different subject. For example, in some embodiments, a multiplicative change (e.g., an increase or decrease) in a particular parameter (e.g., the number of engineered T cells in a sample) can be determined as compared to the same parameter under different conditions (e.g., prior to administration of a DGK inhibitor). In some embodiments, the level, value or measurement of two or more parameters is determined and the relative levels are compared. In some embodiments, the level, value, or measurement of the determined parameter is compared to the level, value, or measurement of a control sample or untreated sample. In some embodiments, the level, value or measurement of the determined parameter is compared to the level of a sample from the same subject but at a different time point. The values obtained in the quantification of the individual parameters may be combined for disease assessment purposes, for example, by using multiparameter analysis to form arithmetic or logical operations on the levels, values or measured values of the parameters. In some embodiments, a ratio of two or more specific parameters may be calculated.

A.T cell exposure, persistence and proliferation

In some embodiments, the parameters associated with the therapy or treatment outcome (including parameters useful for evaluating the screening step and/or evaluating the treatment outcome and/or monitoring the treatment outcome) are or include evaluating the exposure, persistence, and proliferation of T cells (e.g., T cells for administration based on T cell therapy). In some embodiments, increased exposure or sustained expansion and/or persistence of cells (e.g., cells administered for immunotherapy (e.g., T cell therapy in the methods provided herein), and/or changes in cell phenotype or cell functional activity) can be measured by evaluating characteristics of T cells in vitro or ex vivo. In some embodiments, these assays can be used to determine or confirm the function of T cells for immunotherapy (e.g., T cell therapy) before or after one or more steps of administering the combination therapies provided herein.

In some embodiments, the DGK inhibitor is administered for the purpose of promoting exposure of the subject to cells (e.g., T cells for T cell-based therapy administration), such as by promoting expansion and/or persistence thereof over time. In some embodiments, the T cell therapy exhibits increased or prolonged expansion and/or persistence in the subject as compared to a method of administering the T cell therapy to the subject in the absence of the DGK inhibitor.

In some embodiments, provided methods increase the subject's exposure to administered cells (e.g., increased cell number or duration over time) and/or improve the efficacy and therapeutic outcome of immunotherapy (e.g., T cell therapy). In some aspects, the methods have the advantage of greater and/or longer exposure to cells expressing recombinant receptors (e.g., cells expressing TCR or CAR) compared to other methods, improving treatment outcome. These results may include survival and remission of the patient, even in individuals with severe tumor burden.

In some embodiments, administration of the DGK inhibitor may increase the maximum exposure, total exposure, and/or duration of exposure of cells in the subject (e.g., T cells for T cell based therapy administration) compared to T cells administered alone in the absence of the DGK inhibitor.

In some embodiments, the presence and/or amount of cells expressing a recombinant receptor (e.g., cells expressing a TCR or CAR for administration of T cell-based therapies) in the subject is detected after administration of the T cells and before, during, and/or after administration of the DGK inhibitor. In some aspects, quantitative PCR (qPCR) is used to assess the amount of cells expressing a recombinant receptor (e.g., cells expressing a TCR or CAR for administration of T cell-based therapies) in a blood or serum or organ or tissue sample (e.g., a disease site, e.g., a tumor sample) of a subject. In some aspects, persistence is quantified as the number of copies of DNA or plasmid encoding the receptor (e.g., TCR or CAR) per microgram of DNA (e.g., total DNA obtained from the sample), or as the number of cells expressing the receptor (e.g., expressing TCR or CAR) per microliter of sample (e.g., blood or serum), or the total number of Peripheral Blood Mononuclear Cells (PBMC) or leukocytes or T cells per microliter of sample.

In some embodiments, the cells are detected in the subject at or at least 4, 7, 10, 14, 18, 21, 24, 27, or 28 days after administration of the T cells (e.g., TCR or CAR-expressing T cells). In some aspects, the cells are detected at or at least 2, 4, or 6 weeks or 3, 6, or 12, 18, or 24, or 30, or 36 months or 1,2, 3, 4, 5, or more years after administration of the T cells.

In some embodiments, cells expressing the receptor (e.g., cells expressing the TCR or CAR) achieve higher persistence after administration of the T cells (e.g., T cells expressing the TCR or CAR) and/or DGK inhibitor than alternative methods (such as those involving administration of immunotherapy alone, e.g., T cells administered in the absence of a DGK inhibitor, e.g., T cells expressing the TCR or CAR).

Exposure (e.g., the number of cells (e.g., T cells for T cell therapy administration)) indicates expansion and/or persistence, which can be described in terms of the maximum number of cells exposed by the subject, the duration of detectable cells or cells above a certain number or percentage, the area under the curve of the number of cells over time, and/or combinations thereof, and indicators thereof. These results can be evaluated using known methods, such as qPCR to detect a comparison of the copy number of nucleic acid encoding the recombinant receptor to the total amount of nucleic acid or DNA in a particular sample (e.g., blood, serum, plasma, or tissue, such as a tumor sample), and/or flow cytometry assays that detect cells expressing the receptor, typically using antibodies specific for the receptor. Cell-based assays may also be used to detect the number or percentage of functional cells, such as cells that are capable of binding and/or neutralizing and/or inducing a response (e.g., a cytotoxic response against a disease or condition or cells expressing an antigen recognized by a receptor).

In some aspects, increased exposure of the subject to the cells comprises increased expansion of the cells. In some embodiments, the cells expressing the receptor (e.g., cells expressing the TCR or CAR) are expanded in the subject after administration of the T cells (e.g., T cells expressing the TCR or CAR) and/or administration of the DGK inhibitor. In some aspects, the method results in greater expansion of cells as compared to other methods, such as methods involving administering T cells without administration of a DGK inhibitor, e.g., T cells expressing a TCR or CAR.

In some aspects, the methods result in high in vivo proliferation of the administered cells, e.g., as measured by flow cytometry. In some aspects, a peak ratio of cells is detected. For example, in some embodiments, the peak or maximum level following administration of a T cell (e.g., a T cell expressing a TCR or CAR) and/or DGK inhibitor expresses the recombinant receptor (e.g., TAR or CAR) at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cell at the blood or disease site of the subject or a leukocyte fraction thereof (e.g., PBMC fraction or T cell fraction).

In some embodiments, the method results in a maximum concentration of at least 100, 500, 1000, 1500, 2000, 5000, 10,000, or 15,000 copies of the receptor or nucleic acid encoding the receptor (e.g., TCR or CAR) per microgram of DNA in the blood or serum or other bodily fluid or organ or tissue of the subject, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 receptor-expressing cells (e.g., TCR or CAR-expressing cells) per total Peripheral Blood Mononuclear Cells (PBMC), total monocytes, total T cells, or total microliter. In some embodiments, the cells expressing the receptor are detected in the blood of the subject as at least 10, 20, 30, 40, 50, or 60% of total PBMCs, and/or are maintained at the level for at least 1,2,3,4,5, 6,7,8, 9, 10, 11, 12, 24, 36, 48, or 52 weeks after administration of the T cells (e.g., TCR or CAR-expressing T cells) and/or DGK inhibitor, or for 1,2,3,4, or 5 or more years after the administration.

In some aspects, the method results in at least a 2-fold, at least a 4-fold, at least a 10-fold, or at least a 20-fold increase in the copy number of a nucleic acid encoding the recombinant receptor (e.g., TCR or CAR) per microgram of DNA (e.g., serum, plasma, blood, or tissue (e.g., tumor sample)) in the subject.

In some embodiments, the cells expressing the receptor are detectable in the serum, plasma, blood, or tissue (e.g., tumor sample) of the subject, e.g., by a specific method, such as qPCR or a flow cytometry-based detection method, for at least 20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59 or 60 or more days after administration of the T cells (e.g., T cells expressing the TCR or CAR) or after administration of the DGK inhibitor, or at least at or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or more weeks after administration of the T cells (e.g., T cells expressing the TCR or CAR) and/or the DGK inhibitor.

In some aspects, at least about 1x10², at least about 1x10³, at least about 1x10⁴, per microliter, At least about 1x10⁵ or at least about 1x10⁶ or at least about 5x10⁶ or at least about 1x10⁷ or at least about 5x10⁷ or at least about 1x10⁸ cells expressing a recombinant receptor (e.g., expressing a TCR or CAR), and/or at least 10 per microliter, 25. 50, 100, 200, 300, 400 or 500 or 1000 cells expressing the receptor (e.g., at least 10 per microliter) are detectable or present in the subject or in a liquid, plasma, serum, tissue or compartment thereof, such as in a blood thereof (e.g., peripheral blood) or in a disease site (e.g., tumor). In some embodiments, the number or concentration of T cells (e.g., TCR or CAR-expressing T cells) is detectable in the subject at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3, 4, 5, 6, 7, 8,9,10,11, or 12 months, or at least 2 or 3 years after administration of the DGK inhibitor. The cell number can be detected by flow cytometry-based or quantitative PCR-based methods and extrapolated to total cell number using known methods. See, e.g., brentjens et al, SCI TRANSL Med.2013 5 (177), park et al, molecular Therapy (4): 825-833 (2007), savoldo et al, JCI 121 (5): 1822-1826 (2011), davila et al, (2013) PLoS ONE 8 (4): e61338, davila et al, oncoimmunology (9): 1577-1583 (2012), Langers, blood 2011 117:72-82, jensen et al Biol Blood Marrow Transplant 2010September;16 (9): 1245-1256, brentjens et al, blood 201118 (18): 4817-4818).

In some aspects, the copy number (e.g., vector copy number) of nucleic acid encoding the recombinant receptor per 100 cells (e.g., in peripheral blood or bone marrow or other compartments) is at least 0.01, at least 0.1, at least 1 or at least 10, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at least about 6 weeks, or at least about 2, 3, 4, 5, 6, 7,8,9, 10, 11, or 12 months, or at least 2 or 3 years after administration of the cells (e.g., T cells expressing a TCR or CAR) and/or DGK inhibitor (as measured by immunohistochemistry, PCR, and/or flow cytometry). In some embodiments, the copy number of the vector expressing the receptor (e.g., TCR or CAR) per microgram of genomic DNA is at least 100, at least 1000, at least 5000, or at least 10,000, or at least 15,000, or at least 20,000, about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks after administration of the T cell (e.g., TCR or CAR expressing T cell) or DGK inhibitor, or at least 2, 3, 4, 5, 6, 7,8,9, 10, 11, or 12 months, or at least 2 or 3 years after said administration.

In some aspects, the time period after administration of the cell (e.g., after starting administration of the T cell (e.g., TCR or CAR-expressing T cell) and/or DGK inhibitor) for at least about 3 months, at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, or more than 3 years, by the receptor expressed by the cell (e.g., TCR or CAR) in the subject, its plasma, serum, blood, tissue, and/or disease site (e.g., tumor site) can be detected by quantitative PCR (qPCR) or by flow cytometry.

In some embodiments, the cells expressing the receptor (e.g., TCR or CAR) are greater in area under the concentration curve of the subject's liquid, plasma, serum, blood, tissue, organ, and/or disease site (e.g., tumor site) after administration of the T cells (e.g., TCR or CAR-expressing T cells) and/or DGK inhibitor compared to the area under the concentration curve (AUC) obtained via an alternative dosing regimen in which the subject is administered T cells (e.g., TCR or CAR-expressing T cells) without administration of the DGK inhibitor.

In some aspects, the methods result in vivo hyperproliferation of the administered cells, e.g., as measured by flow cytometry. In some aspects, a peak ratio of cells is detected. For example, in some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells express a recombinant receptor, e.g., a TCR or CAR, in the blood, plasma, serum, tissue, and disease site of a subject, or a leukocyte fraction thereof (e.g., PBMC fraction or T cell fraction), at a peak or maximum level following administration of a T cell (e.g., a T cell expressing a TCR or CAR) and/or a DGK inhibitor.

In some aspects, increased or prolonged expansion and/or persistence of a cellular dose in a subject administered a DGK inhibitor is associated with the benefit of tumor-related outcome in the subject. In some embodiments, the tumor-associated outcome comprises a reduction in tumor burden or a reduction in bone marrow blasts in the subject. In some embodiments, the tumor burden is reduced or reduced by at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent following administration of the method. In some embodiments, the disease burden, tumor size, tumor volume, tumor mass, and/or tumor burden or volume (bulk) is reduced by at least or about 50%, 60%, 70%, 80%, 90% or more after administration of a cell dose as compared to a subject treated with a method that does not involve administration of a DGK inhibitor.

B.T cell functional Activity

In some embodiments, the parameters associated with the therapy or treatment outcome (including parameters that may be used in the screening step and/or evaluating the treatment outcome and/or monitoring the treatment outcome) include one or more activities, phenotypes, proliferation, or functions of the T cells. In some embodiments, any of the assays known in the art for evaluating the activity, phenotype, proliferation, and/or function of T cells (e.g., T cells for T cell therapy administration) may be used. The biological activity of the engineered cell population is measured in some embodiments (e.g., by any of a number of known methods) before and/or after administration of the cells and/or DGK inhibitor. Parameters assessed include specific binding of engineered T cells or natural T cells or other immune cells to antigen, either in vivo (e.g., by imaging) or ex vivo (e.g., by ELISA or flow cytometry). In certain embodiments, the ability of the engineered cells to destroy target cells may be described using any suitable method known, such as cytotoxicity assays, e.g., in Kochenderfer et al, J.Immunotherapy,32 (7): 689-702 (2009) and Herman et al, J.Immunol Methods,285 (1): 25-40 (2004).

In some embodiments, T cells, such as T cells expressing a recombinant receptor (e.g., TCR or CAR), can be evaluated prior to and/or after administration of the cells and/or DGK inhibitor to assess or determine whether the T cells are characterized by depletion. In some cases, depletion can be assessed by monitoring loss of T cell function, such as a decrease or decrease in antigen-specific or antigen receptor-driven activity, such as the ability to produce cytokines or drive a decrease or decrease in cytolytic activity against the antigen of interest. In some cases, depletion can also be assessed by monitoring the expression of T cell (e.g., CD4 and/or CD4T cell) surface markers associated with the depletion phenotype. The depletion markers include inhibitory receptors such as PD-1, CTLA-4, LAG-3 and TIM-3.

In some embodiments, such reduced or decreased activity is observed over time following administration to a subject and/or prolonged exposure to an antigen.

In particular embodiments, provided methods (i) achieve the increase in antigen-specific or antigen receptor-driven activity and (ii) prevent, inhibit, or delay the occurrence of and/or reverse the depletion phenotype. In some embodiments, the number, duration, and/or frequency are effective (i) to achieve the increase in antigen-specific or antigen receptor-driven activity and (ii) to prevent, inhibit, or delay the occurrence of the depletion phenotype. In other embodiments, the amount, duration, and/or frequency are effective (i) to achieve the increase in antigen-specific or antigen receptor-driven activity and (ii) to prevent, inhibit, or delay the occurrence of a depletion phenotype and to reverse the depletion phenotype.

In some embodiments, the depletion phenotype associated with a T cell or T cell population comprises an increase in the level or extent of surface expression on a T cell, or an increase in the percentage of the T cell population that exhibits surface expression of one or more depletion markers (optionally 2, 3, 4, 5, or 6 depletion markers), as compared to a reference T cell population under the same conditions, or a decrease in the level or extent of activity exhibited by the T cell or T cell population when exposed to an antigen or antigen receptor specific agent as compared to a reference T cell population under the same conditions. In some embodiments, the increase in level, degree, or percentage is greater than or about 1.2-fold, or about 1.5-fold, or about 2.0-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 6-fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold, or more. In other embodiments, the reduction in level, degree, or percentage is greater than or about 1.2-fold, or about 1.5-fold, or about 2.0-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 6-fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold, or more.

In certain embodiments, the biological activity of a cell is determined by determining the expression and/or secretion of one or more cytokines (such as CD107a, IFNγ, IL-2, GM-CSF, and TNF α) and/or by determining cytolytic activity.

In some embodiments, assays for activity, phenotype, proliferation, and/or function of T cells (e.g., T cells for T cell therapy administration) include, but are not limited to, ELISPOT, ELISA, cell proliferation, cytotoxic lymphocyte (CTL) assays, binding to T cell epitopes, antigens, or ligands, or intracellular cytokine staining, proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays. In some embodiments, the proliferation response of T cells can be measured, for example, by incorporating³ H-thymine, brdU (5-bromo-2 '-deoxyuracil) or 2' -deoxy-5-ethyluracil (EdU) into their DNA or performing a dye dilution assay using a dye such as carboxyfluorescein diacetate succinimidyl (CFSE), CELLTRACE VIOLET, or membrane dye PKH 26.

In some embodiments, assessing the activity, phenotype, proliferation, and/or function of a T cell (e.g., a T cell for administration of a T cell therapy) comprises measuring cytokine production from the T cell, and/or measuring cytokine production from a biological sample (e.g., plasma, serum, blood, and/or a tissue sample, e.g., a tumor sample) from a subject. In some cases, these measured cytokines may include, but are not limited to, interleukin-2 (IL-2), interferon-gamma (IFNgamma), interleukin-4 (IL-4), TNF-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and/or TGF-beta (TGF beta). Assays for measuring cytokines are well known and include, but are not limited to, ELISA, intracellular cytokine staining, flow microbead arrays, RT-PCR, ELISPOT, flow cytometry, and bioassays, wherein the reaction of cells to relevant cytokines is detected in the presence of a test sample for determining responsiveness (e.g., proliferation).

In some embodiments, assessing the activity, phenotype, proliferation, and/or function of a T cell (e.g., a T cell for administration of a T cell therapy) comprises assessing a cell phenotype (e.g., expression of a particular cell surface marker). In some embodiments, the T cells (e.g., T cells for T cell therapy administration) are evaluated for expression of a T cell activation marker, a T cell depletion marker, and/or a T cell differentiation marker. In some embodiments, the cell phenotype is assessed prior to administration. In some embodiments, the cell phenotype is assessed during or after administration of the cell therapy and/or DGK inhibitor. T cell activation markers, T cell depletion markers and/or T cell differentiation markers for evaluation include markers of any known specific subset of T cells, e.g., CD25, CD38, human leukocyte antigen-DR (HLA-DR), CD69, CD44, CD137, KLRG1, CD62L ^{Low and low}、CCR7 ^{Low and low}, CD71, CD2, CD54, CD58, CD244, CD160, programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 protein (LAG-3), T-cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T Lymphocyte Attenuators (BTLA) and/or T-cell immunoglobulins and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) (see, e.g., liu et al, CELL DEATH AND DISEASE (2015) 6, e 1792). In some embodiments, the depletion marker is one or more of PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA, and TIGIT. In some embodiments, the cell surface markers evaluated are CD25, PD-1 and/or TIM-3. In some embodiments, the cell surface marker evaluated is CD25.

In some aspects, detecting the expression level comprises performing an in vitro assay. In some embodiments, the in vitro assay is an immunoassay, an aptamer-based assay, a histological or cytological assay, or an mRNA expression level assay. In some embodiments, the one or more parameters of each of the one or more factors, effectors, enzymes, and/or surface markers are detected by an enzyme-linked immunosorbent assay (ELISA), immunoblot, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry assay, surface Plasmon Resonance (SPR), chemiluminescent assay, lateral flow immunoassay, inhibition assay, or affinity assay. In some embodiments, detection of the cytokine and/or surface marker is determined using an agent that specifically binds to at least one biomarker. In some cases, the binding agent is an antibody or antigen binding fragment thereof, an aptamer, or a nucleic acid probe.

In some embodiments, administration of the DGK inhibitor increases the level of circulating CAR T cells.

C. Disease burden

In some embodiments, the parameters associated with the therapy or treatment outcome (including parameters that may be evaluated for the screening step and/or evaluating the treatment outcome and/or monitoring the treatment outcome) include tumor or disease burden. Administration of immunotherapy such as T cell therapy (e.g., TCR or CAR expressing T cells) and/or DGK inhibitors may reduce or prevent the burden or expansion of a disease or condition in a subject. For example, when the disease or condition is a tumor, the method generally reduces tumor size, volume, metastasis, the percentage of blast cells or molecularly detectable B-cell malignancy in the bone marrow and/or improves prognosis or survival or other symptoms associated with tumor burden.

In some aspects, the enlargement or burden of a disease or condition in a subject is generally reduced or prevented according to the provided methods and/or according to the provided administration of the article or composition. For example, when the disease or condition is a tumor, the method generally reduces tumor size, volume, metastasis, the percentage of blast cells or molecularly detectable B-cell malignancy in the bone marrow and/or improves prognosis or survival or other symptoms associated with tumor burden.

In some embodiments, the provided methods result in a reduced tumor burden in the treated subject as compared to alternative methods of administering immunotherapy, such as T cell therapy (e.g., TCR or CAR expressing T cells), without administration of a DGK inhibitor. It is not necessary to actually reduce the tumor burden in all subjects receiving the combination therapy, but the tumor burden is reduced on average in the treated subjects, such as based on clinical data, wherein most subjects treated with the combination therapy exhibit reduced tumor burden, such as at least 50%, 60%, 70%, 80%, 90%, 95% or more of the subjects treated with the combination therapy exhibit reduced tumor burden.

The disease burden may encompass the total number of disease cells in the subject or an organ, tissue or body fluid of the subject, such as a tumor or an organ or tissue of another location (e.g., which may indicate metastasis). For example, in the case of certain hematological malignancies, tumor cells can be detected and/or quantified in the blood, lymph, or bone marrow. In some embodiments, the disease burden may include tumor mass, the number or extent of metastases, and/or the percentage of blast cells present in bone marrow.

In some embodiments, the subject suffers from lymphoma or leukemia. The extent of disease burden can be determined by evaluating leukemia cells remaining in blood or bone marrow. In some embodiments, the subject suffers from non-hodgkin lymphoma (NHL), acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), or diffuse B-cell lymphoma (DLBCL). In some embodiments, the subject suffers from MM or DBCBL.

In some aspects, the response rate in a subject (such as a subject with NHL) is based on Lugano criteria. (Cheson et al, (2014) JCO.,32 (27): 3059-3067; johnson et al, (2015) Radiology 2:323-338; cheson, B.D. (2015) Chin. Clin. Oncol.4 (1): 5). In some aspects, the response assessment uses any of clinical, hematological, and/or molecular methods. In some aspects, the reaction evaluated using the Lugano standard involves using Positron Emission Tomography (PET) -Computed Tomography (CT) and/or CT (as the case may be). PET-CT evaluation may further include the use of Fluorodeoxyglucose (FDG) for FDG-avid lymphoma. In some aspects, 5 minutes may be used when PET-CT is to be used to evaluate the response of FDG-avid histology. In some aspects, 5 minutes includes criteria of 1, no above background uptake, 2, uptake < mediastinum, 3, uptake > mediastinum but < liver, 4, uptake micro > liver, 5, uptake significantly above liver and/or neo lesions, X, the newly ingested region being unlikely to be associated with lymphoma.

In some aspects, the complete reaction described using the Lugano standard involves a complete metabolic reaction and a complete radioreaction at various measurable sites. In some aspects, these sites include the extralymphoid sites of lymphoid tuberculosis, where CR is described as1, 2, or 3 minutes with or without residual mass in 5 minutes when PET-CT is used. In some aspects, there is high physiological uptake or spleen or bone marrow activation (e.g., with chemotherapy or myeloid colony stimulating factors) at the Waldeyer ring or extralymph node site, which uptake may be greater than normal longitudinal membrane and/or liver. In this case, if the uptake of the initially involved site is not greater than that of the surrounding normal tissue, a complete metabolic response can be inferred even if the tissue has a high physiological uptake. In some aspects, CT is used to evaluate the response in lymph nodes, where CR is described as a disease without extra-lymph sites and the target lymph node/lymph node mass must degenerate to 1.5cm or less in the longest transverse diameter (LDi) of the lesion. Further evaluation sites include bone marrow, where PET-CT based evaluation should indicate evidence of lack of FDG-avid disease in bone marrow and CT based evaluation should indicate normal morphology, if indeterminate, should be IHC negative. Further sites may include assessment of organ enlargement, which should be restored to normal. In some aspects, unmeasured lesions and new lesions are evaluated, which should be absent in the case of CR (Cheson et al, (2014) JCO.,32 (27): 3059-3067; johnson et al, (2015) Radiology 2:323-338; cheson, B.D. (2015) chip. Clin. Oncol.4 (1): 5).

In some aspects, the Partial Reactions (PR) described using the Lugano standard involve partial metabolic and/or radioreactions at various measurable sites. In some aspects, these sites include lymph nodes and extra-lymph parts, where PR is described as 4 or 5 minutes when PET-CT is used, with reduced uptake compared to baseline and any size of residual tumor. In the middle, this finding may indicate a reactive disease. At the end of the treatment, this finding may indicate residual disease. In some aspects, CT is used to evaluate the response in lymph nodes, where PR is described as up to 6 measurable lymph nodes and the extra-lymph node SPD decreases by ≡50%. If the lesion is too small to measure on CT, the default value is 5mm by 5mm, if the lesion is no longer visible, the value is 0mm by 0mm, if the lymph node is >5mm by 5mm, but less than normal, the actual measurement is used for calculation. Further evaluation sites include bone marrow, where PET-CT based evaluation should indicate that residual uptake is higher than normal bone marrow uptake, but reduced compared to baseline (allowing diffuse uptake compatible with chemotherapy-responsive changes). In some aspects, if there is a sustained change in bone marrow foci in the case of lymph node response, further evaluation should be considered using MRI or biopsy or interval scan. In some aspects, the further location may include an assessment of organ enlargement, wherein the spleen must be reversed by >50% from normal length. In some aspects, unmeasured lesions and new lesions are evaluated, which in the case of PR should be absent/normal, reversing, but not increasing. nonreactive/Stable Disease (SD) or Progressive Disease (PD) can also be measured using PET-CT and/or CT-based evaluation. (Cheson et al, (2014) JCO.,32 (27): 3059-3067; johnson et al, (2015) Radiology 2:323-338; cheson, B.D. (2015) Chin. Clin. Oncol.,4 (1): 5).

In some aspects, progression Free Survival (PFS) is described as the length of time during and after treatment of a disease, such as a B cell malignancy, a subject suffers from such disease but does not worsen. In some aspects, an Objective Response (OR) is described as a measurable response. In some aspects, the Objective Response Rate (ORR) is described as the proportion of patients who obtain CR or PR. In some aspects, total survival (OS) is described as the length of time that a subject diagnosed with a disease, such as a B-cell malignancy, remains alive, starting from the date of diagnosis or initiation of treatment of the disease. In some aspects, event-free survival (EFS) is described as the length of time that a subject remains free of treatment for certain complications or events intended to be prevented or delayed after B-cell malignancy treatment has ended. These events may include recurrence of the B cell malignancy or appearance of certain symptoms, such as bone pain caused by spread of the B cell malignancy to the bone, or death.

In some embodiments, the measurement of duration of response (DOR) includes a time from the recording of tumor response to disease progression. In some embodiments, the parameter used to evaluate the response may include a persistent response, e.g., a response that persists after a period of time from the initiation of treatment. In some embodiments, a sustained response is indicated by a response rate of about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months after initiation of the treatment. In some embodiments, the reaction lasts for greater than 3 months or greater than 6 months.

In some aspects, RECIST criteria are used to determine objective tumor response. (Eisenhauer et al, european Journal of Cancer 45 (2009) 228-247). In some aspects, RECIST criteria are used to determine objective tumor response of a target lesion. In some aspects, a complete response as determined using RECIST criteria is described as the disappearance of all target lesions and any pathological lymph nodes (whether target or non-target) must be reduced to <10mm on the short axis. In other aspects, the partial response determined using RECIST criteria is described as a reduction in the sum of diameters of the target lesions by at least 30%, with reference to the sum of baseline diameters. In other aspects, progressive Disease (PD) is described as an increase in target lesion diameter sum of at least 20% with the sum of the minimum in the study as a reference (including the baseline sum if the minimum in the study). In addition to a relative increase of 20%, the sum must also indicate an absolute increase of at least 5mm (in some aspects, the appearance of one or more new lesions is also considered to be progressive). In other aspects, stable Disease (SD) is described as neither shrinking enough to meet PR nor increasing enough to meet PD, taking the minimum sum of diameters as a reference at the time of study.

In the case of MM, exemplary parameters for assessing the extent of disease burden include the number of cloned plasma cells (e.g., >10% in bone marrow biopsies or from any number in other tissue biopsies; plasmacytoma), the presence of monoclonal proteins (accessory proteins) in serum or urine, evidence of damage to the final organ associated with plasma cell disorders (e.g., hypercalcemia (corrected calcium >2.75 mmol/l), renal insufficiency due to myeloma; anemia (hemoglobin <10 g/dl), and/or bone lesions (lytic lesions or osteoporosis with compression fracture)).

In the case of DLBCL, exemplary parameters for assessing disease burden include cell morphology (e.g., centromeric, immune and metaplastic cells), gene expression, miRNA expression, and protein expression (e.g., expression of BCL2, BCL6, MUM1, LMO2, MYC, and p 21).

In some aspects, the response rate in a subject, such as a subject with CLL, is based on the response criteria of the international chronic lymphocytic seminar (IWCLL) (Hallek, et al, blood 2008, jun 15;111 (12): 5446-5456). In some aspects, these criteria are described as Complete Remission (CR), which in some aspects requires the absence of peripheral blood cloned lymphocytes, the absence of lymphadenopathy, the absence of hepatomegaly or splenomegaly, the absence of constitutional symptoms and satisfactory blood count by immunophenotyping, complete remission with incomplete bone marrow recovery (CRi), which in some aspects is described as CR above, but without normal blood count, partial Remission (PR), which in some aspects is described as a decrease in lymphocyte count of 50% or more, a decrease in lymphadenopathy of 50% or a decrease in liver or spleen of 50% or more, while peripheral blood count is improved, progressive Disease (PD), which in some aspects is described as an increase in lymphocyte count of 50% or more (up to >5x10⁹/L), an increase in lymphadenopathy of 50% or more, an increase in liver or spleen size of 50% or more, richter conversion or new cytopenia due to CLL, and stable disease, which in some aspects is described as a disorder that does not meet the criteria of CR, cri, PR or PD.

In some embodiments, the subject exhibits CR OR if the subject's lymph node size is less than OR about 20mm, size is less than OR about 10mm, OR size is less than OR about 10mm within 1 month of administration of a dose of cells.

In some embodiments, no exponential cloning of CLL is detected in the subject's bone marrow (or in greater than 50%, 60%, 70%, 80%, 90% or more of the subject's bone marrow treated according to the method). In some embodiments, exponential cloning of CLL is assessed by IgH depth sequencing. In some embodiments, the exponential clone is not detected at or about or at least at or about 1, 2, 3, 4,5, 6, 12, 18, or 24 months after administration of the cell.

In some embodiments, the subject exhibits a morphological disorder if the number of blast cells in the bone marrow is greater than or equal to 5% (e.g., as monitored by light microscopy), such as greater than or equal to 10% blast cells in the bone marrow, greater than or equal to 20% blast cells in the bone marrow, greater than or equal to 30% blast cells in the bone marrow, greater than or equal to 40% blast cells in the bone marrow, or greater than or equal to 50% blast cells in the bone marrow. In some embodiments, the subject exhibits complete or clinical remission if the number of blast cells in the bone marrow is less than 5%.

In some embodiments, the subject may exhibit complete remission, but there is a small fraction of residual leukemia cells that are morphologically undetectable (by optical microscopy). A subject is said to exhibit Minimal Residual Disease (MRD) if the subject exhibits less than 5% of the blast cells in the bone marrow and exhibits a molecularly detectable B-cell malignancy. In some embodiments, a molecularly detectable B cell malignancy can be evaluated using any of a variety of molecular techniques that allow for sensitive detection of small numbers of cells. In some aspects, these techniques include PCR assays that can determine fusion transcripts resulting from unique Ig/T cell receptor gene rearrangements or chromosomal translocations. In some embodiments, flow cytometry can be used to identify B-cell malignancy cells based on leukemia cell-specific immunophenotyping. In some embodiments, molecular detection of B cell malignancy cells can detect as low as 1 leukemia cell in 100,000 normal cells. In some embodiments, if at least or more than 1 leukemia cell is detected in 100,000 cells (such as by PCR or flow cytometry), the subject exhibits a molecularly detectable MRD. In some embodiments, the disease burden of the subject is molecular detectable or MRD^-, such that in some cases leukemia cells cannot be detected in the subject using PCR or flow cytometry techniques.

In the case of leukemia, the extent of disease burden can be determined by evaluating leukemia cells remaining in blood or bone marrow. In some embodiments, the subject exhibits a morphological disorder if the blast cells in the bone marrow are greater than or equal to 5%, e.g., as detected by light microscopy. In some embodiments, the subject exhibits complete or clinical remission if the number of blast cells in the bone marrow is less than 5%.

In some embodiments, for leukemia, the subject may exhibit complete remission, but there is a small fraction of morphologically undetectable (by optical microscopy) residual leukemia cells. A subject is said to exhibit Minimal Residual Disease (MRD) if the subject exhibits less than 5% of the blast cells in the bone marrow and exhibits a molecularly detectable B cell malignancy. In some embodiments, a molecularly detectable B cell malignancy can be evaluated using any of a variety of molecular techniques that allow for sensitive detection of small numbers of cells. In some aspects, these techniques include PCR assays that can determine fusion transcripts resulting from unique Ig/T cell receptor gene rearrangements or chromosomal translocations. In some embodiments, flow cytometry can be used to identify B-cell malignancy cells based on leukemia cell-specific immunophenotyping. In some embodiments, molecular detection of B cell malignancy cells can detect as low as 1 leukemia cell in 100,000 normal cells. In some embodiments, if at least or more than 1 leukemia cell is detected in 100,000 cells (such as by PCR or flow cytometry), the subject exhibits a molecularly detectable MRD. In some embodiments, the disease burden of the subject is molecular detectable or MRD^-, such that in some cases leukemia cells cannot be detected in the subject using PCR or flow cytometry techniques.

In some embodiments, the methods and/or administration of a cell therapy, such as a T cell therapy (e.g., a T cell expressing a TCR or CAR), and/or a DGK inhibitor reduce the disease burden as compared to the disease burden at a time immediately prior to administration of the immunotherapy (e.g., T cell therapy) and/or DGK inhibitor.

In some aspects, administration of an immunotherapy (e.g., T cell therapy) and/or a DGK inhibitor may inhibit an increase in disease burden, and this may be evidenced by no change in disease burden.

In some embodiments, the method reduces the burden of the disease or condition (e.g., the number of tumor cells, tumor size, duration of patient survival or event-free survival) to a greater extent and/or for a longer period of time than the burden reduction of the disease or condition observed using alternative therapies, such as therapies in which the subject receives immunotherapy (e.g., T cell therapy alone, in the absence of administration of a DGK inhibitor). In some embodiments, the disease burden is reduced to a greater extent or for a longer period of time following administration of a combination therapy of an immunotherapy (e.g., T cell therapy) and a DGK inhibitor than the reduction in disease burden achieved by administration of each agent alone (e.g., administration of a DGK inhibitor to a subject that does not receive an immunotherapy (e.g., T cell therapy) or administration of an immunotherapy (e.g., T cell therapy) to a subject that does not receive a DGK inhibitor).

In some embodiments, the burden of a disease or condition in a subject is detected, evaluated, or measured. In some aspects, the disease burden can be detected by detecting the total number of disease or disease-related cells (e.g., tumor cells) in the subject or in an organ, tissue, or body fluid (e.g., blood or serum) of the subject. In some embodiments, the disease burden (e.g., tumor burden) is determined by measuring the number or extent of metastases. In some aspects, the survival, survival over time, degree of survival, presence of event-free or symptom-free survival, or duration of time or recurrence-free survival of the subject is assessed. In some embodiments, any symptom of a disease or condition is assessed. In some embodiments, measurement criteria for the burden of a disease or condition are specified. In some embodiments, exemplary parameters for determining include specific clinical outcomes indicative of improvement or amelioration of a disease or condition (e.g., a tumor). The parameters include the duration of disease control, which includes Complete Response (CR), partial Response (PR) or Stable Disease (SD) (see, e.g., response Evaluation CRITERIA IN Solid Tumors (RECIST) guidelines), objective Remission Rate (ORR), progression Free Survival (PFS), and total survival (OS). Specific thresholds for parameters can be set to determine the efficacy of the methods of combination therapy provided herein.

In some aspects, the disease burden is measured or detected prior to administration of an immunotherapy (e.g., T cell therapy), after administration of an immunotherapy (e.g., T cell therapy), but prior to administration of a DGK inhibitor, and/or after administration of both therapies (e.g., T cell therapy and DGK inhibitor). In the case of multiple administrations of one or more steps of the combination therapy, the disease burden in some embodiments may be measured before or after, or at times between, any of the administration steps, doses, and/or administration periods.

In some embodiments, the methods provided herein reduce the burden, or at least at or about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent, as compared to just prior to administration of the immunotherapy (e.g., T cell therapy) and DGK inhibitor. In some embodiments, the disease burden, tumor size, tumor volume, tumor mass, and/or tumor burden or volume (bulk) is reduced by at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90% or more after administration of the immunotherapy (e.g., T cell therapy) and DGK inhibitor compared to immediately before administration of the immunotherapy (e.g., T cell therapy) and/or DGK inhibitor.

In some embodiments, reducing disease burden by the method includes inducing complete remission in morphology, e.g., as assessed 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more than 6 months after administration (e.g., initiation) of the combination therapy.

In some aspects, the determination of minimal residual disease (e.g., as measured by multiparameter flow cytometry) is negative, or the level of minimal residual disease is less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05%.

In some embodiments, the method improves event-free survival or overall survival of the subject as compared to other methods. For example, in some embodiments, at 6 months after treatment by the methods of combination therapy provided herein, the event-free survival rate or probability of a subject treated by the methods is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some aspects, the overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the subject treated with the method exhibits event-free survival, relapse-free survival, or survival for at least 6 months or at least 1,2,3, 4, 5,6,7, 8, 9, or 10 years. In some embodiments, the time to progression is improved, such as greater than or about 6 months or at least 1,2,3, 4, 5,6,7, 8, 9, or 10 years of time to progression.

In some embodiments, the probability of recurrence is reduced after treatment by the method as compared to other methods. For example, in some embodiments, at 6 months after the method of using the combination therapy, the probability of recurrence is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%.

In some cases, the pharmacokinetics of the administered cells (e.g., adoptive transfer cells) are determined to assess availability (e.g., bioavailability of the administered cells). Methods for determining the pharmacokinetics of adoptive transfer cells may include withdrawing peripheral blood from a subject to whom the engineered cells have been administered, and determining the number or ratio of engineered cells in the peripheral blood. Methods for selecting and/or isolating cells can include the use of Chimeric Antigen Receptor (CAR) specific antibodies (e.g., brentjens et al, sci. Transl. Med.2013Mar;5 (177): 177ra 38) protein L (Zheng et al, J. Transl. Med.2012Feb; 10:29), epitope tags (such as Strep-Tag sequences introduced directly into specific sites of the CAR, thereby directly evaluating the CAR using binding reagents for Strep-Tag) (Liu et al (2016) Nature Biotechnology,34:430; international patent application publication No. WO 2015095895) and monoclonal antibodies that specifically bind to CAR polypeptides (see International patent application publication No. WO 2014190273). In some cases, exogenous marker genes can be used in conjunction with engineered cell therapies to allow detection or selection of cells and, in some cases, also promote cell suicide. In some cases, truncated epidermal growth factor receptor (EGFRt) can be co-expressed with a transgene of interest (e.g., CAR) in the transduced cells (see, e.g., U.S. patent No. 8,802,374). EGFRt may contain cetuximab by antibodyOr other therapeutic anti-EGFR antibodies or binding molecules, which can be used to recognize or select cells that have been engineered with an EGFRt construct and another recombinant receptor, such as a recombinant antigen receptor (CAR), and/or eliminate or isolate cells of the expressed body. See U.S. Pat. No. 8,802,374 and Liu et al, nature Biotech.2016april;34 (4): 430-434).

In some embodiments, the number of car+ T cells in a biological sample (e.g., blood) obtained from a patient can be determined over a period of time after administration of a cell therapy, e.g., determining the pharmacokinetics of the cells. In some embodiments, the number of car+ T cells (optionally car+cd8+ T cells and/or car+cd4+ T cells) detected in the blood of a subject or a majority of subjects treated by the method is greater than 1 cell per μl, greater than 5 cells per μl, or greater than 10 cells per μl.

Toxicity and adverse results

In embodiments of the provided methods, toxicity or other adverse consequences to a subject are monitored, including treatment-related consequences, e.g., cytokine Release Syndrome (CRS) or Neurotoxicity (NT) in a subject administered a provided combination therapy comprising a cell therapy (e.g., T cell therapy) and a DGK inhibitor. In some embodiments, the provided methods are performed to reduce the risk of toxic consequences or symptoms, toxicity promoting features, factors, or characteristics, such as indicating severe Cytokine Release Syndrome (CRS) or severe neurotoxicity or symptoms or outcomes associated therewith.

In some embodiments, such as compared to certain other cell therapies, the provided methods do not result in, or reduce the high incidence or likelihood of, toxicity or toxic outcome (severe Neurotoxicity (NT) or severe Cytokine Release Syndrome (CRS)). In some embodiments, the method does not result in or increase the risk of severe NT (sNT), severe CRS (sccrs), macrophage activation syndrome, oncolytic syndrome, fever at or about 38 degrees celsius for 3 days or more, and plasma levels of CRP at or about 20 mg/dL. In some embodiments, greater than or greater than about 30%, 35%, 40%, 50%, 55%, 60% or more of the subjects treated according to the provided methods do not exhibit any level of CRS or any level of neurotoxicity. In some embodiments, no more than 50% of subjects treated (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of subjects treated) have a Cytokine Release Syndrome (CRS) higher than grade 2 and/or a neurotoxicity higher than grade 2. In some embodiments, at least 50% of subjects treated according to the methods (e.g., at least 60%, at least 70%, at least 80%, at least 90% or more of the subjects treated) do not exhibit severe toxicity results (e.g., severe CRS or severe neurotoxicity), such as do not exhibit grade 3 or higher neurotoxicity and/or do not exhibit severe CRS, or do not exhibit such results for a period of time following the treatment (such as within one week, two weeks, or one month of administration of cells).

In some aspects, the subject is monitored and/or the method reduces the risk of indicating Cytokine Release Syndrome (CRS) or severe CRS (sccrs) or a toxic outcome associated therewith. CRS (e.g., sCRS) may occur in some cases after adoptive T cell therapy and administration of other biologic products to a subject. See Davila et al, SCI TRANSL MED, 224 a25 (2014), brentjens et al, sci.Transl.Med.5,177ra38 (2013), grupp et al, N.Engl.J.Med.368,1509-1518 (2013), and Kochenderfer et al, blood 119,2709-2720 (2012), xu et al, CANCER LETTERS 343 (2014) 172-78.

Typically, CRS is caused by excessive systemic immune responses mediated by, for example, T cells, B cells, NK cells, monocytes and/or macrophages. These cells can release a large number of inflammatory mediators, such as cytokines and chemokines. Cytokines may elicit an acute inflammatory response and/or induce endothelial organ damage, which may lead to microvascular leakage, heart failure, or death. Serious life threatening CRS can lead to lung infiltration and lung injury, renal failure or disseminated intravascular coagulation. Other serious, life threatening toxicities may include cardiac toxicity, respiratory distress, nervous system toxicity, and/or liver failure. CRS may be treated using anti-inflammatory therapies, such as anti-IL-6 therapies (e.g., anti-IL-6 antibodies, e.g., tolizumab), or antibiotics or other agents as described.

The results, signs, and symptoms of CRS are known and include those described herein. In some embodiments, a particular outcome, sign, and symptom and/or amount or extent thereof may be specified when the particular dosing regimen or administration achieves or does not achieve a given outcome, sign, or symptom associated with CRS.

In the case of CAR-expressing cells, CRS typically occurs 6-20 days after infusion of CAR-expressing cells. See Xu et al CANCER LETTERS 343 (2014) 172-78. In some cases, CRS occurs less than 6 days or more than 20 days after CAR T cell infusion. The incidence and time of CRS may be related to baseline cytokines or tumor burden at the time of infusion. Typically, CRS involves elevated serum levels of Interferon (IFN) - γ, tumor Necrosis Factor (TNF) - α, and/or Interleukin (IL) -2. Other cytokines that may be rapidly induced in CRS are IL-1β, IL-6, IL-8, and IL-10.

Exemplary outcomes associated with CRS include fever, stiffness, chills, hypotension, dyspnea, acute Respiratory Distress Syndrome (ARDS), encephalopathy, ALT/AST elevation, renal failure, cardiac conditions, hypoxia, neurological disorders, and death. Neurological complications include delirium, epileptic-like activities, confusion, difficulty finding words, aphasia, and/or becoming conscious chaos. Other CRS-related consequences include fatigue, nausea, headache, seizures, tachycardia, myalgia, rash, acute vascular leak syndrome, liver function damage, and renal failure. In some aspects, CRS is associated with an increase in one or more factors (serum ferritin, d-dimer, aminotransferase, lactate dehydrogenase, and triglycerides), or with hypofibrinogenemia or hepatosplenomegaly.

In some embodiments, the CRS-related results include one or more of sustained fever, e.g., fever at a particular temperature, e.g., greater than or about 38 degrees celsius for two or more days, e.g., three or more days, or at least 3 consecutive days, e.g., four or more days, fever at or about 38 degrees celsius, elevation of cytokines (such as a maximum fold change, e.g., at least 75-fold, or a maximum fold change of at least one of the cytokines) (e.g., at least 250-fold, as measured by at least one venous vasoactive agent) compared to pretreatment levels of at least two cytokines (e.g., interferon gamma (ifnγ), GM-CSF, IL-6, IL-10, flt-3L, fractal chemokines (FRACKTALKINE), and IL-5, and/or tumor necrosis factor alpha (tnfα)), and/or at least one clinical signs of toxicity such as hypotension (e.g., as measured by at least one venous vasoactive agent), hypoxia (e.g., at₂) levels below or about 90% in a neurological condition, or conditions, including a change in the neurological condition, or conditions.

Exemplary CRS-related results include elevated or high serum levels of one or more factors, including cytokines and chemokines and other CRS-related factors. Exemplary results further include an increase in synthesis or secretion of one or more of the factors. The synthesis or secretion may be performed by T cells or cells that interact with T cells, such as innate immune cells or B cells.

In some embodiments, CRS-related serum factors or CRS-related results include inflammatory cytokines and/or chemokines including interferon gamma (IFN-gamma), TNF-a, IL-1 beta, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Ra, granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage Inflammatory Protein (MIP) -1, tumor necrosis factor alpha (TNF alpha), IL-6, and IL-10, IL-1 beta, IL-8, IL-2, MIP-1, flt-3L, fractal chemokines, and/or IL-5. In some embodiments, the factor or result comprises C-reactive protein (CRP). In addition to being an easily measurable risk factor for early CRS, CRP is also a marker for cell expansion. In some embodiments, subjects with high CRP levels (such as ≡15 mg/dL) are measured to suffer from CRS. In some embodiments, subjects with high CRP levels are measured not to suffer from CRS. In some embodiments, the measurement of CRS includes measuring CRP and another factor indicative of CRS.

In some embodiments, one or more inflammatory cytokines or chemokines are monitored before, during, or after T cell therapy treatment and/or DGK inhibitor treatment. In some aspects, the one or more cytokines or chemokines include IFN-gamma, TNF-alpha, IL-2, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Ralpha, granulocyte macrophage colony-stimulating factor (GM-CSF), or Macrophage Inflammatory Protein (MIP). In some embodiments, IFN-gamma, TNF-alpha and IL-6 are monitored.

CRS criteria associated with CRS pathogenesis have been established to predict which patients are more likely to be at risk of developing sCRS (see Davilla et al Science translational media.2014; 6 (224): 224ra 25). Including fever, hypoxia, hypotension, altered nerves, elevated serum levels of inflammatory cytokines, such as seven cytokine groups (ifnγ, IL-5, IL-6, IL-10, flt-3L, fractal chemokines and GM-CSF), the elevation caused by treatment can be correlated well with both pretreatment tumor burden and sCRS symptoms. Other guidelines for diagnosis and management of CRS are known (see, e.g., lee et al, blood.2014;124 (2): 188-95). In some embodiments, the criteria reflecting CRS ratings are those detailed in table 2 below.

In some embodiments, if following administration, a subject is considered to develop "severe CRS" ("sCRS") in response to or subsequent to administration of a cell therapy or a cellular dose thereof, the subject exhibits (1) fever of at least 38 degrees Celsius for at least 3 days, (2) an increase in cytokines, including any of (a) at least 75-fold change in at least one of the 7 cytokine groups as compared to the level immediately following administration of interferon gamma (IFNgamma), GM-CSF, IL-6, IL-10, flt-3L, fractal chemokine, and IL-5, and/or (b) at least 250-fold change in at least one of the 7 cytokine groups as compared to the level immediately following administration of interferon gamma (IFNgamma), GM-CSF, IL-6, IL-10, flt-3L, fractal chemokine, and IL-5, and (c) at least one clinical toxic sign, such as hypotension (requiring at least one venous vasoactive pressor hypoxia (₂%) or a < neurological condition or a < status, including a change in one or more of the neurological conditions and/or a change in the chaotic state. In some embodiments, the heavy CRS includes 3 or higher order CRS, such as those listed in table 2.

In some embodiments, the results associated with heavy CRS or 3-grade CRS or higher (such as 4-base or higher) include one or more of sustained fever, e.g., fever at a particular temperature, e.g., above or about 38 degrees celsius, for two or more days, e.g., three or more days, e.g., four or more days or at least three consecutive days; an elevation of cytokines compared to pretreatment levels of at least two cytokines (e.g., at least two of interferon gamma (ifnγ), GM-CSF, IL-6, IL-10, flt-3L, fractal chemokines, and IL-5, and/or tumor necrosis factor alpha (tnfα)), e.g., a maximum fold change of at least or about 75-fold, or at least at or about 250-fold, e.g., at least one of the cytokines, and/or at least one clinical toxic sign such as hypotension (e.g., as measured by at least one vasoactive pressor), hypoxia (e.g., less than at or about 90% plasma oxygen (PO₂) levels), and/or one or more neurological disorders (including mental state changes, consciousness chaos, and epilepsy). In some embodiments, the heavy CRS includes CRS that are required to be managed or cared for in an Intensive Care Unit (ICU).

In some embodiments, the CRS such as severe CRS encompasses a combination of (1) sustained fever (fever at least 38 degrees celsius for at least 3 days) and (2) serum levels of CRP at or about 20 mg/dL. In some embodiments, the CRS encompasses hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation. In some embodiments, the dose of vasopressor is increased in a second or subsequent administration.

In some embodiments, severe CRS or grade 3 CRS encompasses elevated glutamate pyruvate transaminase, elevated aspartate aminotransferase, chills, febrile neutrophil depletion, headache, left ventricular dysfunction, encephalopathy, hydrocephalus, and/or tumors.

Methods of measuring or detecting various results may be specified.

In some aspects, the toxicity outcome of a therapy (such as a cell therapy) is or is associated with or indicative of neurotoxicity or severe neurotoxicity. In some embodiments, symptoms associated with clinical risk of neurotoxicity include confusion, delirium, expressive aphasia, confusion, myoclonus, somnolence, altered state of consciousness, tics, epileptic-like activity, epilepsy (optionally as demonstrated by electroencephalogram [ EEG ]), elevated beta amyloid levels (aβ), elevated glutamate levels, and elevated oxygen free radical levels. In some embodiments, neurotoxicity is graded based on severity (e.g., using a grade 1-5 (see, e.g., guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6,657-666 (December 2010); national institute of cancer—general toxicity Standard version 4.03 (NCI-CTCAE v 4.03).

In some cases, the neurological symptom may be an early symptom of sCRS. In some embodiments, the neurological symptoms begin to manifest 5 to 7 days after the infusion of the cell therapy. In some embodiments, the duration of the nervous system change may range from 3 to 19 days. In some cases, recovery from nervous system changes occurs after the disappearance of other symptoms sCRS. In some embodiments, treatment with anti-IL-6 and/or steroid(s) does not accelerate the resolution time or extent of the nervous system change.

In some embodiments, if following administration, a subject is considered to develop "severe neurotoxicity" in response to or secondary to administration of a cell therapy or a cellular dose thereof, the subject exhibits symptoms from limited self-care (e.g., bathing, dressing, eating, toilet, taking), 1) symptoms of peripheral motor neuropathy, including inflammation or degeneration of peripheral motor nerves, 2) symptoms of peripheral sensory neuropathy, including inflammation or degeneration of peripheral sensory nerves, sensory consciousness chaos, such as sensory distortion leading to abnormal and unpleasant sensations, neuralgia, such as intense pain along a nerve or group of nerves, and/or paresthesias, such as dysesthesias of sensory neurons, leading to tingling, numbness, pressure, cold and hot abnormal skin sensations without irritation. In some embodiments, severe neurotoxicity comprises a grade 3 or higher neurotoxicity, such as set forth in table 3. In some embodiments, severe neurotoxicity is considered prolonged grade 3 if grade 3 neurotoxicity symptoms last for 10 days or more.

In some embodiments, the method reduces symptoms associated with CRS or neurotoxicity as compared to other methods. In some aspects, the provided methods reduce symptoms, outcomes, or factors associated with CRS (including symptoms, outcomes, or factors associated with severe CRS or 3-or higher CRS) as compared to other methods. For example, a subject treated according to the present methods may lack symptoms, results, or factors that are detectable and/or have a reduction in CRS (e.g., severe CRS or CRS of grade 3 or higher, such as any of those described in table 2, for example). In some embodiments, a subject treated according to the present methods may have reduced symptoms of neurotoxicity (such as limb weakness or numbness, memory, vision and/or intellectual loss, uncontrollable compulsive and/or compulsive behavior, delusions, headache, cognitive and behavioral problems, including loss of motor control, cognitive deterioration, and autonomic nervous system dysfunction and sexual dysfunction) as compared to subjects treated by other methods. In some embodiments, a subject treated according to the present methods may have reduced symptoms associated with peripheral motor neuropathy, peripheral sensory neuropathy, sensory awareness chaos, neuralgia, or paresthesia.

In some embodiments, the methods reduce the consequences associated with neurotoxicity (including damage to the nervous system and/or brain, such as neuronal death). In some aspects, the methods reduce the level of factors associated with neurotoxicity, such as beta amyloid (aβ), glutamate, and oxygen radicals.

In some embodiments, the toxicity result is Dose Limiting Toxicity (DLT). In some embodiments, the toxicity results in the absence of dose limiting toxicity. In some embodiments, dose Limiting Toxicity (DLT) is defined as any grade 3 or higher toxicity as described or assessed by any of the known or published guidelines for assessing specific toxicity, such as any of the above descriptions and including the National Cancer Institute (NCI) adverse event common terminology standard (CTCAE) version 4.0.

In some embodiments, the provided embodiments result in a low rate or risk of toxicity, e.g., CRS or neurotoxicity or severe CRS or neurotoxicity, e.g., CRS of grade 3 or higher or neurotoxicity, such as observed with a dose of T cells administered according to the provided combination therapies and/or the provided articles or compositions. In some cases, this allows for the administration of the cell therapy on an outpatient basis. In some embodiments, administration of the cell therapy (e.g., a dose of T cells (e.g., tcr+ or car+ T cells)) according to the provided methods and/or provided articles or compositions is performed on an outpatient basis or does not require hospitalization of the subject, such as requiring overnight hospitalization.

In some aspects, subjects to which the cell therapy (e.g., a dose of T cells (e.g., tcr+ or car+ T cells)) is administered according to the provided methods and/or provided articles or compositions include subjects treated on an outpatient basis, unless or until the subject exhibits a toxic sign or symptom (such as neurotoxicity or CRS), no intervention for treating any toxicity is administered prior to or concurrently with administration of the cell dose.

In some embodiments, if a subject (including an outpatient therapy-based subject) to whom the cell therapy (e.g., a dose of T cells (e.g., tcr+ or car+ T cells)) exhibits fever, the subject is administered or instructed to receive or administer an antipyretic therapy. In some embodiments, fever in a subject is characterized by a body temperature of the subject that is (or is measured as) at or above a certain threshold temperature or level. In some aspects, the threshold temperature is a temperature associated with at least low heat, at least moderate heat, and/or at least high heat. In some embodiments, the threshold temperature is a particular temperature or range. For example, the threshold temperature may be at or about or at least at or about 38, 39, 40, 41, or 42 degrees celsius, and/or may be in a range of or about 38 degrees celsius to at or about 39 degrees celsius, in a range of or about 39 degrees celsius to at or about 40 degrees celsius, in a range of or about 40 degrees celsius to at or about 41 degrees celsius, or in a range of or about 41 degrees celsius to at or about 42 degrees celsius.

In some embodiments, the treatment intended for antipyresis comprises treatment with an antipyretic. Antipyretics may include any agent, composition or ingredient that is antipyretic, such as one of a number of drugs known to have antipyretic effects, such as NSAIDs (such as ibuprofen, naproxen, ketotifen and nimesulide), salicylates such as aspirin, choline salicylate, magnesium salicylate and sodium salicylate, paracetamol, acetaminophen, analgin, nalbumetone (Nabumetone), benazedone (Phenaxone), antipyrine, antipyretics. In some embodiments, the antipyretic is acetaminophen. In some embodiments, acetaminophen may be administered orally or intravenously at a dose of 12.5mg/kg up to every four hours. In some embodiments, it is or includes ibuprofen or aspirin.

In some embodiments, if the fever is sustained fever, an alternative treatment for treating toxicity is administered to the subject. For a subject at an outpatient treatment, if the subject has and/or is determined to have or will have sustained fever, the subject is instructed to return to the hospital. In some embodiments, the subject has and/or is determined or is considered to have sustained fever if he or she exhibits a temperature at or above the relevant threshold temperature, and the fever or body temperature of the subject does not decrease, or does not decrease by more than a specific amount (e.g., more than 1 ℃, and typically does not fluctuate about, or does not fluctuate more than about 0.5 ℃, 0.4 ℃, 0.3 ℃, or 0.2 ℃) after a specific treatment, such as a treatment intended for antipyretic, such as with an antipyretic, e.g., an NSAID or a salicylic acid, e.g., ibuprofen, acetaminophen, or aspirin. For example, a subject is considered to have sustained fever if he or she exhibits or is determined to exhibit fever (which does not decrease or decrease more than at or about 0.5 ℃, 0.4 ℃, 0.3 ℃, or 0.2 ℃, or does not decrease by at or about 1%, 2%, 3%, 4%, or 5%) at or about 38 or 39 ℃ even after treatment with an antipyretic, such as acetaminophen. In some embodiments, the dose of the antipyretic is a dose that is generally effective in reducing fever or a particular type of fever, such as fever associated with a bacterial or viral infection (e.g., a local or systemic infection), such as in a subject.

In some embodiments, the subject has and/or is determined or considered to have sustained fever if he or she exhibits fever at or above the relevant threshold temperature, and wherein the subject's fever or body temperature does not fluctuate by about, or by more than about 1 ℃, and typically does not fluctuate by about, or by more than about 0.5 ℃, 0.4 ℃,0.3 ℃, or 0.2 ℃. Such absence of fluctuations above or in a certain amount is typically measured over a given period of time (such as over a period of 24 hours, 12 hours, 8 hours, 6 hours, 3 hours, or 1 hour, which may be measured from the first sign of heat generation or the first temperature above a specified threshold). For example, in some embodiments, a subject is considered or determined to exhibit sustained fever if he or she exhibits fever at least at or about or at or about 38 or 39 degrees celsius (which does not fluctuate more than at or about 0.5 ℃, 0.4 ℃,0.3 ℃, or 0.2 ℃ over a period of 6 hours, over a period of 8 hours, or over a period of 12 hours, or over a period of 24 hours).

In some embodiments, the fever is sustained fever, in some aspects, the subject is treated at a time when the subject has been determined to have sustained fever, such as within one, two, three, four, five, six or less hours of the first time of the determination or after a primary treatment (such as a cell therapy, such as a dose of T cells, e.g., tcr+ or car+ T cells) that has the potential to induce toxicity.

In some embodiments, one or more interventions or agents for treating toxicity (such as toxicity targeted therapies) are administered at or immediately after a time when the subject is determined or confirmed (such as first determined or confirmed) to exhibit sustained fever (e.g., as measured according to any of the preceding embodiments). In some embodiments, the one or more toxicity targeted therapies are administered within the identified or determined period of time (such as within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours thereof).

Articles of manufacture and kits

Also provided are articles of manufacture containing dgkα and/or dgkζ inhibitors and components (e.g., engineered cells, and/or compositions thereof) for T cell therapy. The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed of various materials, such as glass or plastic. The container in some embodiments contains a composition that is itself or in combination with another composition that is effective for treating, preventing, and/or diagnosing the condition. In some embodiments, the container has a sterile access end. Exemplary containers include intravenous solution bags, vials (including those having a stopper penetrable by an injection needle), or bottles or vials for oral administration. The label or package insert may indicate that the composition is used to treat a disease or condition.

The article of manufacture may comprise (a) a first container and a composition contained therein, wherein the composition comprises an engineered cell for the T cell therapy (e.g., any of the compositions described in section I-a), and (B) a second container and a composition contained therein, wherein the composition comprises a second agent, such as a dgkα and/or dgkζ inhibitor (e.g., any of the inhibitors described in section I-B). The article of manufacture may further comprise a third container and a composition contained therein, wherein the composition comprises a checkpoint inhibitor (e.g., any one as described in section I-C). The article of manufacture may further comprise a pharmaceutical instruction indicating that the composition is useful for treating a particular condition. Or alternatively, or in addition, the article of manufacture may further comprise another or the same container containing a pharmaceutically acceptable buffer. It may further comprise other materials such as other buffers, diluents, filters, needles and/or syringes.

Definition of the definition

Unless otherwise indicated, all technical and scientific terms or terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter names pertains. In some cases, terms with commonly understood meanings as defined herein are used for clarity and/or for ready reference, and inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is commonly understood in the art.

As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". It is to be understood that the aspects and variations described herein include "consisting of" and/or "consisting essentially of" aspects and variations.

Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as a inflexible limitation on the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as having individual values within the range. For example, when a range of values is provided, it is to be understood that each intervening value, between the upper and lower limit of that range and any stated or intervening value in that stated range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

As used herein, the term "about" refers to a range of common errors for the respective values that are readily known in the art. References herein to "about" a value or parameter include (and describe) embodiments that relate to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein, a "subject" is a human. As used herein, the terms "subject" and "patient" are used interchangeably to refer to a human unless specifically indicated otherwise.

As used herein, the term "treatment" (and grammatical variations thereof, such as "treatment" or "treatment") refers to the complete or partial amelioration of a disease or condition or disorder or symptoms, adverse reactions, or outcomes, or phenotypes associated therewith. Desirable therapeutic effects include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. These terms are not meant to completely cure the disease or completely eliminate any symptoms or effect on all symptoms or outcomes.

As used herein, "delay of disease progression" means delay, impediment, slowing, delaying, stabilizing, inhibiting, and/or postnatal disease (such as cancer) progression. The length of this delay may vary depending on the disease and/or the medical history of the individual being treated. It is apparent that a sufficient or significant delay may actually cover prophylaxis, i.e. the individual does not develop a disease. For example, advanced cancers (such as metastasis) may be delayed.

As used herein, "preventing" includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay the progression of a disease or slow the progression of a disease.

As used herein, "inhibiting" a function or activity refers to reducing the function or activity as compared to the same condition other than the condition or parameter of interest (or alternatively, as compared to another condition). For example, the cells reduce the growth rate of a tumor as compared to the growth rate of a tumor in the absence of cells that inhibit tumor growth.

An "effective amount" (in the case of administration) of an agent (e.g., a pharmaceutical formulation, cell or composition) refers to an amount that is effective at the necessary dose/amount and time period to achieve the desired result (such as a therapeutic or prophylactic result).

A "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation or an engineered cell) refers to an amount that is effective at the necessary dose and time period to achieve the desired therapeutic result, such as a pharmacokinetic or pharmacodynamic effect for treating a disease, condition, or disorder, and/or treatment. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex and weight of the subject, the immunomodulatory polypeptide or engineered cell to which it is administered. In some embodiments, provided methods involve administering an immunomodulatory polypeptide, engineered cell, or composition in an effective amount (e.g., a therapeutically effective amount).

"Prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the requisite dosage and time period. Typically, but not necessarily, since the prophylactic dose is administered to the subject prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The term "pharmaceutical formulation" refers to a formulation that is in such a form as to allow the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the formulation is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical formulation that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, the expression nucleotide or amino acid position "corresponding to" a nucleotide or amino acid position in a published sequence (such as listed in the sequence listing) refers to a nucleotide or amino acid position that is identified when aligned with the published sequence to maximize identification using a standard alignment algorithm (such as the GAP algorithm). By aligning the sequences, the corresponding residues can be identified, for example, using conserved and identical amino acid residues as a guide. Generally, to identify the corresponding position, the amino acid sequences are aligned for highest order matching (see, e.g., computational Molecular Biology, lesk, a.m., code, oxford University Press, new york, 1988;Biocomputing:Informatics and Genome Projects,Smith,D.W, code, ACADEMIC PRESS, new york, 1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M, and Griffin, h.g., code ,Humana Press,New.Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987; and sequences ANALYSIS PRIMER, gribskov, M and Devereux, j., eds., M Stockton Press, new york, 1991; carrello et al (1988) SIAM J APPLIED MATH 48:1073).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and are introduced into the genome of a host cell. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. These vectors are referred to herein as "expression vectors". Among these vectors are viral vectors such as retroviruses, e.g., gamma retroviruses and lentiviral vectors.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" which include primary transformed cells and their derived progeny, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny selected or selected for the same function or biological activity in the initially transformed cell.

As used herein, a description of a cell or cell population being "positive" for a particular marker refers to the detectable presence on or in a cell of the particular marker (typically a surface marker). When referring to a surface marker, the term refers to the presence of surface expression detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detected by flow cytometry at a level that is substantially higher than the level of staining detected by the same method with an isotype-matched control under otherwise identical conditions, and/or at a level that is substantially similar to the level of staining of cells that are positive for the known marker, and/or at a level that is substantially higher than the level of staining of cells that are negative for the known marker.

As used herein, a description of a cell or cell population being "negative" for a particular marker refers to the absence of a substantially detectable presence on or in the cell of the particular marker (typically a surface marker). When referring to a surface marker, the term refers to the absence of surface expression detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is undetectable by flow cytometry at a level that is substantially higher than the level of staining detected by the same method under otherwise identical conditions with a isotype-matched control, and/or at a level that is substantially lower than the level of staining of cells that are positive for the known marker, and/or at a level that is substantially similar to the level of staining of cells that are negative for the known marker.

Amino acid substitutions may include substitution of one amino acid in the polypeptide with another amino acid. The substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions. Amino acid substitutions can be introduced into a binding molecule of interest (e.g., an antibody) and products of a desired activity screened (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

Amino acids can generally be grouped according to the following common side chain properties:

(1) Hydrophobic norleucine Met, ala, val, leu, ile;

(2) Neutral hydrophilic Cys, ser, thr, asn, gln;

(3) Acid, asp, glu;

(4) Basicity His, lys, arg;

(5) Residues affecting chain orientation, gly, pro;

(6) Aromatic Trp, tyr, phe.

In some embodiments, conservative substitutions may involve swapping one member of the classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions may involve swapping one member of these classes for another class.

As used herein, with respect to amino acid sequences (reference amino acid sequences), the terms "percent (%) amino acid sequence identity" and "percent identity" when used are defined as the percentage of amino acid residues in a candidate sequence (e.g., a subject antibody or fragment) that are identical to amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the greatest percent sequence identity, and do not consider any conservative substitutions as part of the sequence identity. Sequence alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Suitable parameters for aligning sequences can be determined, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

As used herein, a composition refers to any mixture of two or more products, substances, or compounds (including cells). It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

"DGK alpha and/or DGK zeta inhibitor" refers to "an inhibitor of DGK alpha and/or DGK zeta enzyme activity," both referring to an inhibitor of human DGK alpha and/or human DGK zeta, such as DGK zeta having the amino acid sequence shown as SEQ ID NO:190 or the amino acid sequence shown as SEQ ID NO:190 (without naturally occurring amino acids in DGK alpha (e.g., his tail or certain N-terminal amino acids)), and having the amino acid sequence shown as SEQ ID NO:191 or the amino acid sequence shown as SEQ ID NO:191 (without naturally occurring amino acids in DGK zeta (e.g., his tail or certain N-terminal amino acids)).

Target proteins (e.g., DGK, PD-1, PD-L1, and CTLA 4) as used herein refer to human target proteins unless explicitly indicated otherwise or the context clearly indicates otherwise. For example, "mouse DGK" refers to a mouse form of DGK, as it is specifically indicated.

As used herein, the phrase "compound and/or pharmaceutically acceptable salt thereof" refers to at least one compound, at least one salt of the compound, or a combination thereof. For example, the compounds of formula (I) and/or pharmaceutically acceptable salts thereof include a compound of formula (I), two compounds of formula (I), a pharmaceutically acceptable salt of a compound of formula (I) and one or more compounds of formula (I), and a pharmaceutically acceptable salt of two or more compounds of formula (I).

Unless otherwise indicated, any atom that does not satisfy a valence state is considered to have enough hydrogen atoms to satisfy the valence state.

Throughout the specification, one skilled in the art can select groups and substituents thereof to provide stable moieties and compounds.

According to convention in the art, structural formulae herein are usedTo describe bonds as points of attachment of moieties or substituents to the core or host structure.

As used herein, the terms "halo" and "halogen" refer to F, cl, br, and I.

The term "cyano" refers to the group-CN.

The term "amino" refers to the group-NH₂.

The term "oxo" refers to the group = O.

As used herein, the term "alkyl" refers to both branched and straight chain saturated aliphatic hydrocarbon groups containing, for example, 1 to 12 carbon atoms, 1 to 6 carbon atoms, and 1 to 4 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl and tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylpentyl, 2-ethylbutyl, 3-methylpentyl and 4-methylpentyl. When a number appears after the symbol "C" in the subscript format, the subscript more specifically defines the number of carbon atoms that a particular group may contain. For example, "C_1–4 alkyl" represents a straight or branched alkyl group having one to four carbon atoms.

As used herein, the term "fluoroalkyl" is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups substituted with one or more fluorine atoms. For example, "C_1–4 fluoroalkyl" is intended to include C₁、C₂、C₃ and C₄ alkyl groups substituted with one or more fluorine atoms. Representative examples of fluoroalkyl groups include, but are not limited to, -CF₃ and-CH₂CF₃.

The term "cyanoalkyl" includes both branched and straight chain saturated alkyl groups substituted with one or more cyano groups. For example, "cyanoalkyl" includes-CH₂CN、-CH₂CH₂ CN and C_1–4 cyanoalkyl.

The term "aminoalkyl" includes both branched and straight-chain saturated alkyl groups substituted with one or more amino groups. For example, "aminoalkyl" includes-CH₂NH₂、-CH₂CH₂NH₂ and C_1–4 aminoalkyl.

The term "hydroxyalkyl" includes both branched and straight chain saturated alkyl groups substituted with one or more hydroxyl groups. For example, "hydroxyalkyl" includes-CH₂OH、-CH₂CH₂ OH and C_1–4 hydroxyalkyl.

The term "alkenyl" refers to a straight or branched hydrocarbon group containing 2 to 12 carbon atoms and at least one carbon-carbon double bond. Exemplary such groups include vinyl or allyl. For example, "C_2-6 alkenyl" means straight and branched alkenyl groups containing 2 to 6 carbon atoms.

The term "alkynyl" refers to a straight or branched hydrocarbon group containing 2 to 12 carbon atoms and at least one carbon-carbon triple bond. Exemplary such groups include ethynyl. For example, "C_2-6 alkynyl" means straight and branched alkynyl groups containing 2 to 6 carbon atoms.

As used herein, the term "cycloalkyl" refers to a molecule derived from a non-aromatic mono-or polycyclic hydrocarbon by removal of one hydrogen atom from a saturated ring carbon atom. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. When a number appears after the symbol "C" in the subscript format, the subscript more specifically defines the number of carbon atoms that a particular cycloalkyl group may contain. For example, "C_3–6 cycloalkyl" represents a cycloalkyl group containing 3 to 6 carbon atoms.

As used herein, the term "alkoxy" refers to an alkyl group attached to the parent molecular moiety through an oxygen atom, e.g., a methoxy group (-OCH₃). For example, "C_1–3 alkoxy" means an alkoxy group containing 1 to 3 carbon atoms.

The terms "fluoroalkoxy" and "-O (fluoroalkyl)" denote fluoroalkyl groups as defined above linked through an oxygen linker (-O-). For example, "C_1–4 fluoroalkoxy" is intended to include C₁、C₂、C₃ and C₄ fluoroalkoxy groups.

The term "alkylene" refers to a saturated carbon chain having two points of attachment to the core or host structure. The alkylene group has the structure- (CH₂)_n -, examples of alkylene chains include-CH₂CH₂–、–CH₂CH₂CH₂ -and- (CH₂)_2-4 -.

The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The compound (e.g., a compound of formula (I) or formula (II)) can form a pharmaceutically acceptable salt for use in the methods described herein. Unless otherwise indicated, reference to a compound is understood to include reference to one or more pharmaceutically acceptable salts thereof. The term "salt" means an acid-and/or base-addition pharmaceutically acceptable salt formed with inorganic and/or organic acids and bases. Furthermore, the term "salt" may include zwitterionic (inner salts), for example, when the compounds of formula (I) contain a basic moiety such as an amine or pyridine or imidazole ring and an acidic moiety such as a carboxylic acid. Preferred pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are such as, for example, acceptable metal and amine salts, wherein the cation has no significant effect on the toxicity or biological activity of the salt. However, other salts may be useful, for example, in isolation or purification steps that may be used in the preparation, and are therefore encompassed herein. Salts of the compounds (e.g., compounds of formula (I)) may be formed, for example, by reacting the compounds (e.g., compounds of formula (I)) with an amount of an acid or base (such as an equivalent amount) in a medium such as a salt precipitating medium or by lyophilization in an aqueous medium.

Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, e.g., trifluoroacetic acid, adipates, alginates, ascorbates, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphoric acid, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, gluconate, citrate, camphoric acid, polygluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, caproate, hydrochloride (formed with hydrochloric acid), hydrobromide (formed with hydrogen bromide), hydroiodic acid, maleate (formed with maleic acid), 2-hydroxyethanesulfonate, lactate, methanesulfonate (formed with methanesulfonic acid), 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectate, persulfate, 3-phenylpropionate, phosphate, bitrates, pivalate, propionate, salicylate, succinate, sulfate (such as those formed with sulfuric acid), sulfonate (such as those herein), sulfonate (such as those mentioned herein), thiosulfonate (such as those mentioned herein), toluene (tosylates), toluene (such as those mentioned herein), and the like.

Exemplary base addition salts include ammonium salts, alkali metal salts (such as sodium, lithium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts, barium salts, zinc salts and aluminum salts), salts with organic bases (e.g., organic amines such as trialkylamines, such as triethylamine, procaine, dibenzylamine, N-benzyl- β -phenethylamine, 1-amphetamine, N' -dibenzylethylenediamine-diamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine or similar pharmaceutically acceptable amines), and salts with amino acids (such as arginine, lysine, etc.). Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others. Preferred salts include monohydrochloride, bisulfate, methanesulfonate, phosphate, or nitrate.

The compound (e.g., a compound of formula (I)) may be provided as an amorphous solid or a crystalline solid. Lyophilization may be used to provide a solid of a compound (e.g., a compound of formula (I)).

It is further understood that solvates (e.g., hydrates) of compounds (e.g., compounds of formula (I)) may also be used in the methods described herein. The term "solvate" means a physical association of a compound (e.g., a compound of formula (I)) with one or more solvent molecules (organic or inorganic). Such physical bonding includes hydrogen bonding. In some cases, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be able to separate. "solvate" encompasses both solution phases and separable solvates. Exemplary solvate solutes include hydrates, ethanolates, methanolates, isopropanolates, acetonitrile solvates and ethyl acetate solvates. Solvation methods are known in the art.

Various forms of prodrugs are well known in the art and are described in the following:

a) THE PRACTICE of MEDICINAL CHEMISTRY, camille G.Wermuth et al, ch 31, (academic Press, 1996);

b) Design of Prodrugs, h.bundegaard, inc. (Elsevier, 1985);

c) A Textbook of Drug DESIGN AND Development, P.Krogsgaard-Larson and H.

Bundgaard, code Ch 5, pgs 113-191 (Harwood Academic Publishers, 1991), and

D) Hydrolysis in Drug and Prodrug Metabolism, bernard Testa and Joachim M.Mayer,

(Wiley-VCH,2003)。

Furthermore, the compound (e.g., a compound of formula (I)) may be isolated and purified after its preparation to obtain a composition containing equal to or greater than 99% by weight of the compound (e.g., a compound of formula (I)) ("substantially pure") that is then used or formulated as described herein. Such "substantially pure" compounds (e.g., compounds of formula (I)) are also encompassed herein.

"Stable compound" and "stable structure" are meant to indicate that the compound is sufficiently robust to survive isolation to a useful purity from the reaction mixture and formulation into an effective therapeutic agent. The compounds used herein are intended to include stable compounds.

The compounds of the present invention are intended to include all isotopes of atoms present in the compounds of the present invention. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and not limitation, isotopes of hydrogen include deuterium (D) and tritium (T). Isotopes of carbon include¹³ C and¹⁴ C. Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by techniques similar to those described herein using an appropriate isotopically-labeled reagent in place of an otherwise-used non-labeled reagent.

Illustrative embodiments

The provided embodiments include:

1. a method of treatment, the method comprising administering a combination therapy to a subject suffering from cancer, comprising:

(a) T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered on day 1 of the combination therapy, and

(B) Dgkα and/or dgkζ inhibitors, wherein administration of the inhibitors begins on any of days 1-8 (including days 1 and 8) of the combination therapy, and the inhibitors are administered once daily, or between about 21 and 42 consecutive days (including days 21 and 42).

2. The method of embodiment 1, wherein administration of the inhibitor begins after administration of the T cell therapy.

3. A method of treatment comprising administering to a subject suffering from cancer a dgkα and/or dgkζ inhibitor, wherein the inhibitor is administered as part of a T cell therapy comprising the inhibitor and live engineered T cells that comprise recombinant receptors for antigens expressed by cancer cells, wherein:

Prior to beginning administration of the inhibitor, the subject has previously administered the T cell therapy, wherein the T cell therapy is administered on day 1 of the combination therapy;

The administration of the inhibitor is started on any of days 1-8 (including days 1 and 8) of the combination therapy, and

The inhibitor is administered once daily, or between about 21 and 42 consecutive days (including 21 and 42 days).

4. A method of treatment, the method comprising administering a combination therapy to a subject suffering from cancer, comprising:

(a) T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered on day 1 of the combination therapy, and

(B) Dgkα and/or dgkζ inhibitors, wherein administration of the inhibitors begins on any of days 1-8 (including days 1 and 8) of the combination therapy, and the inhibitors are continuously administered at least until the number of engineered T cells of the T cell therapy in the subject peaks after administration of the T cell therapy.

5. The method of embodiment 4, wherein administration of the inhibitor begins after administration of the T cell therapy.

6. A method of treatment comprising administering to a subject suffering from cancer a dgkα and/or dgkζ inhibitor, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells that express a recombinant receptor for an antigen expressed by cancer cells, wherein:

Prior to beginning administration of the inhibitor, the subject has previously administered the T cell therapy, wherein the T cell therapy is administered on day 1 of the combination therapy;

The administration of the inhibitor is started on any of days 1-8 (including days 1 and 8) of the combination therapy, and

The administration of the inhibitor is continued at least until the number of engineered T cells of the T cell therapy in the subject peaks after administration of the T cell therapy.

7. The method of embodiment 4, wherein administration of the inhibitor begins on day 1 of combination therapy prior to administration of the T cell therapy.

8. A method of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein:

The T cell therapy is administered on day 1 of the combination therapy;

The administration of the inhibitor is started on day 1 of the combination therapy prior to the administration of the T cell therapy, and

The administration of the inhibitor is continued at least until the number of engineered T cells of the T cell therapy in the subject peaks after administration of the T cell therapy.

9. The method of any one of embodiments 4-8, wherein the inhibitor is administered daily, every two days, or every three days.

10. The method of any one of embodiments 4-9, wherein the inhibitor is administered daily.

11. The method of any one of embodiments 4-10, wherein the inhibitor is administered once per day of administration of the inhibitor.

12. The method of any one of embodiments 4-11, wherein the inhibitor is administered until the number of engineered T cells of T cell therapy in the subject peaks after administration of the T cell therapy.

13. The method of any one of embodiments 4-12, wherein the inhibitor is administered at or over a period of time between about 21 and 42 days (including 21 and 42 days).

14. A method of treatment, the method comprising administering a combination therapy to a subject suffering from cancer, comprising:

(a) T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered on day 1 of the combination therapy, and

(B) A DGK alpha and/or DGK zeta inhibitor, wherein administration of the inhibitor starts on any of days 1-8 (including days 1 and 8) of the combination therapy, and the inhibitor is administered at a therapeutically effective dose at or for a period of time between about 21 and 42 days.

15. The method of embodiment 14, wherein administration of the inhibitor begins after administration of the T cell therapy.

16. A method of treatment comprising administering to a subject suffering from cancer a dgkα and/or dgkζ inhibitor, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells that express a recombinant receptor for an antigen expressed by cancer cells, wherein:

Prior to beginning administration of the inhibitor, the subject has previously administered the T cell therapy, wherein the T cell therapy is administered on day 1 of the combination therapy;

Administration of the inhibitor begins on any of days 1-8 (including days 1 and 8) of the combination therapy, and

The inhibitor is administered at a therapeutically effective dose at or over a period of between about 21 and 42 days.

17. The method of embodiment 14, wherein administration of the inhibitor begins on day 1 of combination therapy prior to administration of the T cell therapy.

18. A method of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein:

The T cell therapy is administered on day 1 of the combination therapy;

The administration of the inhibitor is started on day 1 of the combination therapy prior to the administration of the T cell therapy, and

The inhibitor is administered at a therapeutically effective dose at or over a period of between about 21 and 42 days.

19. The method of any one of embodiments 14-18, wherein the therapeutically effective dose provides a therapeutically effective inhibitor dose for a period of time of between about 21 and 42 days.

20. The method of any one of embodiments 14-19, wherein the inhibitor is administered every 2 days or 3 days.

21. The method of any one of embodiments 1-20, wherein administration of the inhibitor begins on any one of days 1-6 (including days 1 and 6) of the combination therapy.

22. The method of any one of embodiments 1-21, wherein administration of the inhibitor begins on any one of days 1-4 (including days 1 and 4) of the combination therapy.

23. The method of any one of embodiments 1-22, wherein administration of the inhibitor begins on day 1 or day 2 of the combination therapy.

24. The method of any one of embodiments 1-23, wherein administration of the inhibitor begins on day 1 of the combination therapy.

25. The method of any one of embodiments 1 and 21-24, wherein administration of the inhibitor begins on day 1 of combination therapy prior to administration of the T cell therapy.

26. A method of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein:

The T cell therapy is administered on day 1 of the combination therapy;

The administration of the inhibitor is started on day 1 of the combination therapy prior to the administration of the T cell therapy, and

The inhibitor is administered once daily or over a period of time between about 21 and 42 consecutive days, including 21 and 42 days.

27. The method of any one of embodiments 1-26, wherein the inhibitor is administered at or over a period of between about 21 and 35 days (including 21 and 35 days).

28. The method of any one of embodiments 1-27, wherein the inhibitor is administered at or over a period of time between about 21 and 28 days (including 21 and 28 days).

29. The method of any one of embodiments 1-28, wherein the inhibitor is administered at or over a period of time between about 28 and 42 days (including 28 and 42 days).

30. The method of any one of embodiments 1-27 and 29, wherein the inhibitor is administered at or over a period of time between about 28 and 35 days (including 28 and 35 days).

31. The method of any one of embodiments 1-30, wherein the inhibitor is administered over a period of time of at or about 28 days.

32. A method of treatment, the method comprising administering a combination therapy to a subject suffering from cancer, comprising:

(a) T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered on day 1 of the combination therapy, and

(B) A dgkα and/or dgkζ inhibitor, wherein administration of the inhibitor begins on day 1 of the combination therapy, and the inhibitor is administered once daily on days 1-28 (including days 1 and 28) of the combination therapy.

33. The method of embodiment 32, wherein administration of the inhibitor begins after administration of the T cell therapy.

34. A method of treatment comprising administering to a subject suffering from cancer a dgkα and/or dgkζ inhibitor, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells that express a recombinant receptor for an antigen expressed by cancer cells, wherein:

Prior to beginning administration of the inhibitor, the subject has previously administered the T cell therapy, wherein the T cell therapy is administered on day 1 of the combination therapy;

administration of the inhibitor begins on day 1 of the combination therapy, and

The inhibitor is administered once daily on days 1-28 (including days 1 and 28) of the combination therapy.

35. The method of embodiment 32, wherein administration of the inhibitor is initiated prior to administration of the T cell therapy.

36. A method of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing recombinant receptors for antigens expressed by cancer cells, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a dgkα and/or dgkζ inhibitor, wherein:

The T cell therapy is administered on day 1 of the combination therapy;

The administration of the inhibitor is started on day 1 of the combination therapy prior to the administration of the T cell therapy, and

The inhibitor is administered once daily on days 1-28 (including days 1 and 28) of the combination therapy.

37. The method of any one of embodiments 1-36, wherein administration of the inhibitor begins within 12 hours or about 12 hours of administration of the T cell therapy.

38. The method of any one of embodiments 1-37, wherein administration of the inhibitor begins within 6 hours or about 6 hours of administration of the T cell therapy.

39. The method of any one of embodiments 1-38, wherein administration of the inhibitor begins within 4 hours or about 4 hours of administration of the T cell therapy.

40. The method of any one of embodiments 1-39, wherein administration of the inhibitor begins within 2 hours or about 2 hours of administration of the T cell therapy.

41. The method of any one of embodiments 1-40, wherein administration of the inhibitor begins within 1 hour or about 1 hour of administration of the T cell therapy.

42. The method of any one of embodiments 1,4, 9-14, 19-24, 27-32, and 37-41, wherein administration of the inhibitor is initiated on day 1 of the combination therapy concurrently with administration of the T cell therapy.

43. The method of any one of embodiments 1,4, 9-14, 19-24, 27-32, and 37-42, wherein administration of the inhibitor begins during administration of the T cell therapy on day 1 of the combination therapy.

44. The method of any one of embodiments 1-43, wherein prior to administration of the T cell therapy, the subject has been pretreated with a lymphocyte removal therapy comprising administration of fludarabine and/or cyclophosphamide.

45. The method of embodiment 44, wherein the method further comprises administering a lymphocyte removal therapy to the subject.

46. The method of embodiment 44 or embodiment 45, wherein the lymphocyte removal therapy comprises administration of:

Cyclophosphamide at or between about 200 and 400mg/m² (including 200 and 400mg/m²), daily administration for or between about 2 and 4 days (including 2 and 4 days), and/or

Fludarabine at or between about 20 and 40mg/m² (including 20 and 40mg/m²) is administered daily for or between about 2 and 4 days (including 2 and 4 days).

47. The method of any one of embodiments 44-46, wherein the lymphocyte removal therapy comprises administering cyclophosphamide at or about 300mg/m² and fludarabine at or about 30mg/m² for or for about 3 days per day.

48. The method of any one of embodiments 44-47, wherein the T cell therapy is administered at or between about 2 and 7 days (including days 2 and 7) after administration of the lymphocyte removal therapy.

49. The method of any one of embodiments 1-48, wherein the inhibitor is a dgkα inhibitor other than a significant inhibitor of dgkζ.

50. The method of any one of embodiments 1-48, wherein the inhibitor is a dgkζ inhibitor other than a significant inhibitor of dgkα.

51. The method of any one of embodiments 1-48, wherein the inhibitor is a dgkα and dgkζ inhibitor.

52. The method of any one of embodiments 1-51, wherein the inhibitor is not a significant inhibitor of other DGKs.

53. The method of any one of embodiments 1-52, wherein the inhibitor is a compound of formula (I):

Or a pharmaceutically acceptable salt thereof, wherein:

R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, C_3–4 cycloalkyl substituted with 0 to 4R_1a, C_1–3 alkoxy substituted with 0 to 4R_1a, -NR_aR_a、–S(O)_nR_e or-P (O) R_eR_e;

Each R_1a is independently F, cl, -CN, -OH, -OCH₃, or-NR_aR_a;

Each R_a is independently H or C_1–3 alkyl;

Each R_e is independently C_3–4 cycloalkyl or C_1–3 alkyl substituted with 0 to 4R_1a;

R₂ is H, C_1–3 alkyl substituted with 0 to 4R_2a, or C_3–4 cycloalkyl substituted with 0 to 4R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), C_3–4 cycloalkyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₃ is H, F, cl, br, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_3–4 cycloalkyl, C_3–4 fluorocycloalkyl or-NO₂;

R₄ is –CH₂R_4a、–CH₂CH₂R_4a、–CH₂CHR_4aR_4d、–CHR_4aR_4b or-CR_4aR_4bR_4c;

R_4a and R_4b are independently:

(i) C_1–6 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy, -NR_aR_a、–S(O)₂R_e, or-NR_aS(O)₂R_e;

(ii) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH)_1–3O(C_1–3 alkyl), C_1–3 Fluoroalkoxy 、–O(CH)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl, -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–6 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or

(Iii) C_1–4 alkyl substituted with one cyclic group selected from the group consisting of C_3–6 cycloalkyl, heterocyclyl, aryl and heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from the group consisting of F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–6 cycloalkyl;

or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f;

each R_f is independently F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–6 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

R_4d is-OCH₃;

Each R_c is independently H or C_1–2 alkyl;

R_d is phenyl substituted with 0 to 1 substituents selected from F, cl, -CN, -CH₃, and-OCH₃;

Each R₅ is independently-CN, C_1–6 alkyl substituted with 0 to 4R_g, C_2–4 alkenyl substituted with 0 to 4R_g, C_2–4 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 4R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 4R_g, - (CH₂)_1–2 (heterocyclyl substituted with 0 to 4R_g)), a compound of formula (i), - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

Each R_g is independently F, cl, -CN, -OH, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -O (CH₂)_1–2O(C_1–2 alkyl), or-NR_cR_c;

m is 0, 1,2 or 3, and

N is 0, 1 or 2.

54. The method of embodiment 53, wherein the inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:

R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, cyclopropyl substituted with 0 to 3R_1a, C_1–3 alkoxy substituted with 0 to 3R_1a, -NR_aR_a、–S(O)_nCH₃ or-P (O) (CH₃)₂;

Each R_1a is independently F, cl or-CN;

Each R_a is independently H or C_1–3 alkyl;

R₂ is H or C_1–2 alkyl substituted with 0 to 2R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), cyclopropyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₃ is H, F, cl, br, -CN, C_1–2 alkyl, -CF₃, cyclopropyl or-NO₂;

R_4a and R_4b are independently:

(i) C_1–4 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy and-NR_aR_a;

(ii) C_3–6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, -CH₂OH、–(CH₂)_1–2O(C_1–2 alkyl), 0 to 4, C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CH)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy, -O (CH)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl, -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl), -O (CH₂)_1–2(C_3–4 cycloalkyl), -O (CH₂)_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or

(Iii) C_1–3 alkyl substituted with one cyclic group selected from the group consisting of C_3–6 cycloalkyl, heterocyclyl, phenyl and heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from the group consisting of F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl) and C_3–4 cycloalkyl;

or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f;

each R_f is independently F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–4 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

And each R₅ is independently-CN, C_1–5 alkyl substituted with 0 to 4R_g, C_2–3 alkenyl substituted with 0 to 4R_g, c_2–3 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 3R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 3R_g, - (CH₂)_1–2 (heterocyclyl substituted with 0 to 4R_g)), a compound of formula (i), - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl).

55. The method of embodiment 54, wherein the inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, having the structure:

Wherein:

R₁ is-CN;

R₂ is-CH₃;

R₃ is H, F or-CN;

r₄ is:

56. the method of embodiment 53, wherein the inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, having the structure:

57. The compound of any one of embodiments 1-52, wherein the inhibitor is a compound of formula (II):

Or a salt thereof, wherein:

R₁ is H, F, cl, br, -CN, C_1–3 alkyl substituted with 0 to 4R_1a, C_3–4 cycloalkyl substituted with 0 to 4R_1a, C_1–3 alkoxy substituted with 0 to 4R_1a, -NR_aR_a、–S(O)_nR_e or-P (O) R_eR_e;

Each R_1a is independently F, cl, -CN, -OH, -OCH₃, or-NR_aR_a;

Each R_a is independently H or C_1–3 alkyl;

Each R_e is independently C_3–4 cycloalkyl or C_1–3 alkyl optionally substituted with 0 to 4R_1a;

R₂ is H, C_1–3 alkyl substituted with 0 to 4R_2a, or C_3–4 cycloalkyl substituted with 0 to 4R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), C_3–4 cycloalkyl, C_3–4 alkenyl, or C_3–4 alkynyl;

R₄ is –CH₂R_4a、–CH₂CH₂R_4a、–CH₂CHR_4aR_4d、–CHR_4aR_4b or-CR_4aR_4bR_4c;

R_4a and R_4b are independently:

(i) C_1–6 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy, -NR_aR_a、–S(O)₂R_e, or-NR_aS(O)₂R_e;

(ii) C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, phenyl or 5-to 10-membered heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, through 0 to 4, C_1–2 bromoalkyl, C_1–2 cyanoalkyl, C_1–4 hydroxyalkyl, - (CH₂)_1–2O(C_1–3 alkyl), C_1–4 alkoxy, C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy, -O (C_1–4 hydroxyalkyl), a, -O (CR_xR_x)_1-3O(C_1-3 alkyl), C_1–3 fluoroalkoxy 、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–CH₂NR_aNR_a、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -CR_xR_x)_0-2NR_aC(O)O(C_1-4 alkyl, -P (O) (C_1–3 alkyl)₂、–S(O)₂(C_1–3 alkyl, - (CR_xR_x)_1-2(C_3-4 cycloalkyl), - (CR_xR_x)_1–2 (morpholinyl), - (CR_xR_x)_1–2 (difluoromethyl), - (CR_xR_x)_1–2 (dimethylmorpholinyl)) and the like, - (CR_xR_x)_1–2 (oxaazabicyclo [2.2.1] heptane), (CR_xR_x)_1–2 (oxaazaspiro [3.3] heptane), - (CR_xR_x)_1–2 (methylpiperazinonyl), - (CR_xR_x)_1–2 (acetylpiperazinyl)) and the like, - (CR_xR_x)_1–2 (piperidinyl), - (CR_xR_x)_1–2 (difluoropiperidinyl), - (CR_xR_x)_1–2 (methoxypiperidinyl), - (CR_xR_x)_1–2 (hydroxypiperidinyl)), and the like, -O (CR_xR_x)_0–2(C_3–6 cycloalkyl), -O (CR_xR_x)_0–2 (methylcyclopropyl), -O (CR_xR_x)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CR_xR_x)_0–2 (oxetanyl)), -O (CR_xR_x)_0–2 (methylazetidinyl)), -O (CR_xR_x)_0–2 (tetrahydropyranyl) -O (CR_xR_x)_1–2 (morpholinyl)), -O (CR_xR_x)_0–2 (thiazolyl), Cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, dioxolanyl, pyrrolidone group and R_d, or

(Iii) C_1–4 alkyl substituted with one cyclic group selected from C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, mono-or bicyclic aryl, or 5-to 10-membered heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), and C_3–6 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl or 3-to 6-membered heterocyclyl, each substituted with 0 to 3R_f;

each R_f is independently F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–6 alkyl, C_1–3 fluoroalkyl, C_1–3 alkoxy, C_1–3 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–6 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

R_4d is-OCH₃;

Each R_c is independently H or C_1–2 alkyl;

r_d is phenyl substituted with 0 or 1 substituent selected from F, cl, -CN, -CH₃, and-OCH₃;

Each R₅ is independently-CN, C_1–6 alkyl substituted with 0 to 4R_g, C_2–4 alkenyl substituted with 0 to 4R_g, C_2–4 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, Phenyl substituted with 0 to 4R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 4R_g, - (CH₂)_1–2 (4-to 10-membered heterocyclyl substituted with 0 to 4R_g), and, - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

Each R_g is independently F, cl, -CN, -OH, C_1–3 alkoxy, C_1–3 fluoroalkoxy, -O (CH₂)_1–2O(C_1–2 alkyl), or-NR_cR_c;

m is 0, 1,2 or 3, and

N is 0, 1 or 2.

58. The method of embodiment 57, wherein the inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein:

R₁ is H, F, cl, br, -CN, -OH, C_1–3 alkyl substituted with 0 to 4R_1a, cyclopropyl substituted with 0 to 3R_1a, C_1–3 alkoxy substituted with 0 to 3R_1a, -NR_aR_a、–S(O)_nCH₃, or-P (O) (CH₃)₂;

R₂ is H or C_1–2 alkyl substituted with 0 to 2R_2a;

Each R_2a is independently F, cl, -CN, -OH, -O (C_1–2 alkyl), cyclopropyl, C_3–4 alkenyl, or C_3–4 alkynyl, R_4a and R_4b are independently:

(i) -CN or C_1–4 alkyl substituted with 0 to 4 substituents independently selected from F, cl, -CN, -OH, -OCH₃、–SCH₃、C_1–3 fluoroalkoxy and-NR_aR_a;

(ii) C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, phenyl or 5-to 10-membered heteroaryl, each independently selected from F, cl, br, -CN, -OH, C_1–6 alkyl, C_1–3 fluoroalkyl, through 0 to 4, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1-2 hydroxyalkyl, -CH₂NR_aR_a、–(CH₂)_1–2O(C_1–2 alkyl, - (CH₂)_1–2NR_xC(O)O(C_1-2 alkyl), C_1–4 alkoxy, -O (C_1–4 hydroxyalkyl), -O (CR_xR_x)_1–2O(C_1–2 alkyl), C_1–3 fluoroalkoxy, C_1–3 cyanoalkoxy 、–O(CH₂)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl, -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), -P (O) (C_1–2 alkyl)₂、–S(O)₂(C_1–3 alkyl), - (CH₂)_1–2(C_3–4 cycloalkyl), -CR_xR_x (morpholinyl), -CR_xR_x (difluoromethyl), -CR_xR_x (dimethylmorpholinyl), -CR_xR_x (oxaazabicyclo [2.2.1] heptanyl), -CR_xR_x (oxaazaspiro [3.3] heptanyl), -CR_xR_x (methylpiperazinonyl), -CR_xR_x (acetylpiperazinyl), and-, -CR_xR_x (piperidinyl), -CR_xR_x (difluoropiperidinyl), -CR_xR_x (methoxypiperidinyl), -CR_xR_x (hydroxypiperidinyl), -O (CH₂)_0–2(C_3–4 cycloalkyl), -O (CH₂)_0–2 (methylcyclopropyl), -O (CH₂)_0–2 ((ethoxycarbonyl) cyclopropyl), -O (CH₂)_0–2 (oxetanyl)), -O (CH₂)_0–2 (methylazetidinyl), -O (CH₂)_1–2 (morpholinyl), -O (CH₂)_0–2 (tetrahydropyranyl), -O (CH₂)_0–2 (thiazolyl)), Cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (t-butoxycarbonyl) azetidinyl, dioxolanyl, pyrrolidone group, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d, or

(Iii) C_1–3 alkyl substituted with one cyclic group selected from C_3–6 cycloalkyl, 4-to 10-membered heterocyclyl, mono-or bicyclic aryl, or 5-to 10-membered heteroaryl, said cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy 、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3 alkyl), -NR_aC(O)(C_1–3 alkyl), -NR_aC(O)O(C_1–4 alkyl), and C_3–4 cycloalkyl;

Or R_4a and R_4b together with the carbon atom to which they are attached form a C_3–6 cycloalkyl group or a 3-to 6-membered heterocyclyl group, each substituted with 0 to 3R_f;

Each R_f is independently F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, -OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c, or a cyclic group selected from C_3–6 cycloalkyl, 3-to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, cl, br, -OH, -CN, C_1–4 alkyl, C_1–2 fluoroalkyl, C_1–3 alkoxy, C_1–2 fluoroalkoxy, and-NR_cR_c;

R_4c is C_1–4 alkyl or C_3–6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, cl, -OH, C_1–2 alkoxy, C_1–2 fluoroalkoxy and-CN;

Each R₅ is independently-CN, C_1–5 alkyl substituted with 0 to 4R_g, C_2–3 alkenyl substituted with 0 to 4R_g, c_2–3 alkynyl substituted with 0 to 4R_g, C_3–4 cycloalkyl substituted with 0 to 4R_g, phenyl substituted with 0 to 3R_g, oxadiazolyl substituted with 0 to 3R_g, pyridinyl substituted with 0 to 3R_g, - (CH₂)_1–2 (4-to 10-membered heterocyclyl substituted with 0 to 4R_g), and, - (CH₂)_1–2NR_cC(O)(C_1–4 alkyl), - (CH₂)_1–2NR_cC(O)O(C_1–4 alkyl), - (CH₂)_1–2NR_cS(O)₂(C_1–4 alkyl), -C (O) (C_1–4 alkyl), -C (O) OH, -C (O) O (C_1–4 alkyl), -C (O) O (C_3–4 cycloalkyl), -C (O) NR_aR_a or-C (O) NR_a(C_3–4 cycloalkyl);

Each R_x is independently H or-CH₃, and

M is 1, 2 or 3.

59. The method of embodiment 58, wherein the inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, having the structure:

R₁ is-CN;

R₂ is-CH₃;

r_5a is-CH₃ or-CH₂CH₃, and

R_5c is-CH₃、–CH₂CH₃ or-CH₂CH₂CH₃.

60. The method of embodiment 57, wherein the inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, having the structure:

61. The method of embodiment 57, wherein the inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, having the structure:

62. The method of any one of embodiments 1-13 and 21-61, wherein the inhibitor is administered in a therapeutically effective amount.

63. The method of embodiment 62, wherein a therapeutically effective dose provides an inhibitor dose that is effective for a period of time during which the inhibitor is administered.

64. The method of any one of embodiments 1-63, wherein the inhibitor is administered in an amount of between or about 0.25 and 250mg (including 0.25 and 250 mg) per day.

65. The method of any one of embodiments 1-64, wherein the inhibitor is administered in an amount of between or about 0.5 and 100mg (including 0.5 and 100 mg) per day.

66. The method of any one of embodiments 1-65, wherein the inhibitor is administered orally.

67. The method of any one of embodiments 1-66, wherein the recombinant receptor is an engineered T cell receptor (eTCR).

68. The method of any one of embodiments 1-66, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR).

69. The method of embodiment 68, wherein the antigen is CD19 and the CAR is an anti-CD 19 CAR.

70. The method of embodiment 68 or embodiment 69, wherein the CAR is(Li Jimai, lisocabtagene maraleucel), TECARTUS^TM (buckyolol, brexucabtagene autoleucel), KYMRIAH^TM (s Li Fuming, tisagenlecleucel), or YESCARTA^TM (alzerland, axicabtagene ciloleucel).

71. The method of any one of embodiments 68-70, wherein the T cell therapy is(Li Jimai, lisocabtagene maraleucel), TECARTUS^TM (buckyolol, brexucabtagene autoleucel), KYMRIAH^TM (s Li Fuming, tisagenlecleucel) or YESCARTA^TM (alzerland, axicabtagene ciloleucel).

72. The method of embodiment 68, wherein the antigen is BCMA and the CAR is anti-BCMACAR.

73. The method of embodiment 68 or embodiment 72, wherein the CAR is(Ai Dika barren, idecabtagene vicleucel) or CARVYKTI^TM (sidaky olanexide, ciltacabtagene autoleucel).

74. The method of any one of embodiments 68, 72 and 73, wherein the T cell therapy is(Ai Dika Bajin, idecabtagene vicleucel) or CARVYKTI^TM (Sida-based Orthoxel, ciltacabtagene autoleucel).

75. The method of any one of embodiments 1-74, wherein the cancer is a solid tumor.

76. The method of any one of embodiments 1-74, wherein the cancer is a hematological (liquid) tumor.

77. The method of any one of embodiments 1-76, wherein the cancer is a B cell malignancy.

78. The method of any one of embodiments 1-74, 76 and 77, wherein the cancer is leukemia.

79. The method of any one of embodiments 1-74, 76 and 77, wherein the cancer is lymphoma.

80. The method of any one of embodiments 1-74, 76 and 77, wherein the cancer is myeloma.

81. The method of embodiment 80, wherein the myeloma is multiple myeloma.

82. The method of any one of embodiments 1-81, wherein the cancer is relapsed or refractory.

83. The method of any one of embodiments 1-82, wherein the T cell therapy comprises between or about 0.1x10⁶ and 1,000x10⁶ (including 0.1x10⁶ and 1,000x10⁶) total weight group receptor expressing T cells.

84. The method of any one of embodiments 1-83, wherein the T cell therapy comprises between or about 10x10⁶ and 1,000x10⁶ (including 10x10⁶ and 1,000x10⁶) total number of total recombinant receptor expressing T cells.

85. The method of any one of embodiments 1-84, wherein the T cell therapy comprises between or about 10x10⁶ and 500x10⁶ (including 10x10⁶ and 500x10⁶) total number of total recombinant receptor-expressing T cells.

86. The method of any one of embodiments 83-85, wherein the recombinant receptor-expressing T cell is a live recombinant receptor-expressing T cell.

87. The method of any one of embodiments 1-86, wherein the T cell therapy is administered intravenously.

88. The method of any one of embodiments 1-87, wherein the T cells of the T cell therapy are autologous to the subject.

89. The method of any one of embodiments 1-87, wherein the T cells of the T cell therapy are allogeneic to the subject.

90. The method of any one of embodiments 1-89, wherein the T cells of the T cell therapy are human T cells.

Examples

[1] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLE 1 Synthesis of DGK inhibitors

DGKi Compound 1

4- ((2 R,5 s) -4- (bis (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -6-bromo-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridine-3-carbonitrile

DGKi Compound 2

1- (Bis (4-fluorophenyl) methyl) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl) piperazine-2-carboxylic acid methyl ester

DGKi Compound 3

(R) -4- (4- (bis (4-fluorophenyl) methyl) -3-methylpiperazin-1-yl) -6-bromo-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridine-3-carbonitrile

DGKi Compound 4

(R) -8- (4- (bis (4-fluorophenyl) methyl) -3-methylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2, 7-dicarboxylic acid carbonitrile

DGKi Compound 5

8- [ (2S, 5R) -4- [ (4-fluorophenyl) (phenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

DGKi Compounds 6 and 7

8- [ (2S, 5R) -4- [ (4-fluorophenyl) (phenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

DGKi Compound 8

4- [ (2 S,5 r) -4- [ (4-chlorophenyl) (4-fluorophenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -6-methoxy-1-methyl-1, 2-dihydro-1, 5-naphthyridin-2-one

DGKi Compound 9

8- [ (2S, 5R) -4- { [2- (difluoromethyl) -4-fluorophenyl ] methyl } -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

DGKi Compound 10

8- [ (2S, 5R) -4- [ (4-fluorophenyl) (4-methylphenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

DGKi Compound 11

8- [ (2S, 5R) -4- [1- (2, 6-difluorophenyl) ethyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

DGKi Compounds 12 to 14

8- ((2S, 5R) -4- (1- (2, 4-difluorophenyl) propyl) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

Intermediate 1

6-Cyano-3- (N-methylacetamido) pyridine-2-carboxylic acid ethyl ester

To a stirred pale yellow solution of 3- (N-methylacetamido) -1- (l 1-oxo radical) -1l 4-pyridine-2-carboxylic acid ethyl ester (50 g,210 mmol) in DCM (500 mL) was added cyanotrimethylsilane (39.4 mL, 254 mmol) at room temperature. The reaction mixture was stirred for 10min and the mixture was cooled to-10 ℃. Then benzoyl chloride (34.1 mL, 254 mmol) was added over 15min through a 50mL addition funnel, followed by TEA (41.0 mL, 254 mmol) over 20min through a 50mL addition funnel. During the TEA addition, an exothermic reaction was observed. The reaction mixture became a cloudy mixture (TEA salt) which was stirred at the same temperature for 2.5h. The reaction was quenched with 10% NaHCO₃ solution (500 mL) and extracted with DCM (3X 300 mL). The combined organic layers were washed with brine (2×250 mL) then dried over Na₂SO₄ and concentrated to give a yellow crude material. By using crude EA/petroleum ether as eluentRedieSep normal phase silica gel column. The product was isolated by 65-70% EA/petroleum ether and the fractions were concentrated to give ethyl 6-cyano-3- (N-methylacetamido) pyridine-2-carboxylate (43 g,83% yield) as a light brown liquid, LCMS: M/z=248.0 (M+H), rt 1.255min, LC-MS method: column-KINETEX-XB-C18 (75X 3mM-2.6 μm), mobile phase A:10mM ammonium formate/water: acetonitrile (98:2), mobile phase B:10mM ammonium formate/water: acetonitrile (2:98), gradient: over 4 min, 20-100% B, flow rate 1.0mL/min, then hold at 100% B for 0.6 min, flow rate 1.5mL/min, then gradient: over 0.1 min, 100-20% B, flow rate 1.5mL/min.

Intermediate 2

8-Hydroxy-5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

KHMDS (4.80 mL,4.37 mmol) was added to a stirred solution of ethyl 6-cyano-3- (N-methylacetamido) pyridine-2-carboxylate (0.9 g,3.64 mmol) in tetrahydrofuran (10 mL) at-78℃over 10 min. The reaction mixture was stirred for 15min. The reaction mixture was slowly warmed to room temperature over 30min and then stirred for another 90min. The reaction mixture was cooled to 0 ℃. The reaction was quenched with saturated sodium bicarbonate solution (70 mL). The mixture was diluted with ethyl acetate (2 x100 mL). The aqueous layer was collected and acidified with 1.5N HCl to adjust pH to 3.0. The mixture was stirred for 15min to form a solid block, which was filtered through a buchner funnel to obtain 550mg of 8-hydroxy-5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile as a brown solid, 75% yield. LCMS: M/z=202.0 (m+h), rt 0.361min, lc-MS method: column-KINETEX-XB-C18 (75X 3mM-2.6 μm), mobile phase a:10mM ammonium formate/water: acetonitrile (98:2), mobile phase B:10mM ammonium formate/water: acetonitrile (2:98), gradient: 4min,20-100% B, flow rate 1.0mL/min followed by 100% B for 0.6 min, flow rate 1.5mL/min, followed by gradient: 0.1 min, 100-20% B, flow rate 1.5mL/min.

Intermediate 3

8-Chloro-5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

To a stirred solution of 8-hydroxy-5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile (0.55 g,2.73 mmol) in acetonitrile (10 mL) was added POCl₃ (1.53 mL,16.4 mmol). The reaction mixture was heated to 85 ℃ over 5min and stirred for 16h. The reaction mixture was concentrated under reduced pressure to obtain a crude material. The reaction mixture was cooled to 0 ℃. The reaction was quenched with saturated sodium bicarbonate solution (50 mL). The reaction was diluted with DCM (3X 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure to give 8-chloro-5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile (0.25 g,29.1% yield) as a brown solid. LCMS: M/z=220.2 (m+h), rt 1.528min, lc-MS method: column-KINETEX-XB-C18 (75X 3mM-2.6 μm), mobile phase a:10mM ammonium formate/water: acetonitrile (98:2), mobile phase B:10mM ammonium formate/water: acetonitrile (2:98), gradient: 4 min, 20-100% B, flow rate 1.0mL/min, then 100% B0.6 min, flow rate 1.5mL/min, then gradient: 0.1 min, 100-20% B, flow rate 1.5mL/min.

Intermediate 4

Stereochemistry A

(Cyanomethyl) trimethyl phosphonium iodide

(Cyanomethyl) trimethyl phosphonium iodide was prepared according to the general procedure described in Zarago, F., et al, J.org. chem.2001,66, 2518-2521. In a 1L round bottom flask, trimethylphosphine in toluene (100 mL,100 mmol) was diluted with THF (50.0 mL) and toluene (50.0 mL) and then cooled in an ice bath. The reaction mixture was vigorously stirred while iodoacetonitrile (7 ml,16.7g,68.3 mmol) was added dropwise to give a tan precipitate. The cooling bath was removed and the reaction mixture was stirred at room temperature overnight. The flask was placed in ultrasound to break up any agglomerated solids. The reaction mixture was stirred for an additional 4 hours. The solid was collected by filtration and dried under vacuum to give (cyanomethyl) trimethyl phosphonium iodide (16.6 g,68.3mmol,68.3% yield).¹H NMR(400MHz,DMSO-d₆ ) δ4.03 (d, j=16.4hz, 2 h), 2.05 (d, j=15.4hz, 9 h).

Intermediate 5

Stereochemistry same chirality (hemochiral)

8- ((2S, 5R) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile, TFA

To a solution of 6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl triflate (65 g,195 mmol) and tert-butyl (2R, 5S) -2, 5-dimethylpiperazine-1-carboxylate (43.9 g,205 mmol) in acetonitrile (1.3L) was added DIPEA (0.102L, 585 mmol). The solution was stirred at 80 ℃ for 6 hours. The solvent was removed and the crude residue chromatographed on silica gel (product Rf 0.4 in 100% ethyl acetate). The product (2R, 5S) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl) -2, 5-dimethylpiperazine-1-carboxylic acid tert-butyl ester (75 g,189mmol,97% yield. LCMS: M/z=398.2 (M+H); rt 2.7min. Method: column-Kinetex XB-C18 (75X 3mM-2.6 μm), flow rate 1mL/min, gradient time 4min;20% solvent B to 100% solvent B; monitoring at 254nm (solvent A:98% water: 2% acetonitrile; 10mM ammonium formate; solvent B:2% water: 98% acetonitrile; 10mM ammonium formate).

To a solution of (2 r,5 s) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl) -2, 5-dimethylpiperazine-1-carboxylic acid tert-butyl ester (30 g,75 mmol) in ethyl acetate (1000 mL) was added HCl (4M in dioxane) (189 mL,755 mmol) and the temperature was allowed to reach room temperature while stirring for 6h. LC/MS analysis showed the product mass to be 90% at 0.60RT, while the amide byproduct mass was 4% at 0.44RT (consistent with nitrile hydrolysis). The reaction mixture was diluted with methyl tert-butyl ether (MTBE, 2000 mL), stirred for 15min, and the HCl salt of the product was filtered and washed with MTBE (100 mL). HCl salt was dissolved in water (300 ml) and the pH was adjusted to 8 using 10% aqueous sodium bicarbonate. The organic fraction was extracted with DCM (5X 250 ml). The combined organic layers were washed with water (2×300 mL), dried over sodium sulfate and concentrated to give 8- ((2 s,5 r) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile (20 g,65.2mmol,86% yield). LCMS: M/z= 298.2 (m+h); rt 0.5min. The method comprises the steps of carrying out column-Kinetex XB-C18 (75X 3mM-2.6 μm), flowing at 1mL/min, gradient time of 4min, 20% of solvent B to 100% of solvent B, monitoring at 254nm (solvent A:98% of water: 2% of acetonitrile, 10mM of ammonium formate, solvent B:2% of water: 98% of acetonitrile, 10mM of ammonium formate) .¹H NMR(400MHz,CDCl₃)δ7.79(d,J＝8.8Hz,1H),7.70(d,J＝12,3.2Hz,1H),6.29(s,1H),3.80(dd,J＝8.8Hz,1H)3.70(m,1H),3.65(s,3H),3.29(m,2H),2.80(m,2H),1.19(d,J＝6Hz,3H),1.15(d,J＝6Hz,3H).¹³C NMR(75MHz,Chloroform-d)δ161.9,155.0,138.5,137.0,128.2,125.0,122.2,117.2,111.3,56.5,51.9,50.0,49.5,29.0,18.8,15.4.

Intermediate 6

8-Chloro-5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

A2-dram flask containing 8-hydroxy-5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile (192 mg,0.780 mmol) was charged with magnetic stirring bar and acetonitrile (3.1 mL). Subsequently, DIEA (0.272 mL,1.560 mmol) was added to the suspension. The reaction mixture was stirred for 1-2 minutes until the reaction mixture became a uniform yellow solution. To the reaction mixture was added phosphorus oxychloride (0.131 ml,1.404 mmol). The vial was capped under nitrogen with a vent for an oil bubbler. The reaction mixture was stirred at room temperature for 1.5 hours, and then benzyl triethyl ammonium chloride (200 mg,0.878 mmol) was added to the reaction mixture. The vials were capped under nitrogen atmosphere and immersed in an oil bath (65 ℃) and heated for 1 hour. The reaction mixture was cooled and the reaction volatiles were removed in vacuo using a rotary evaporator. The reaction residue was dissolved in ethyl acetate, poured into a beaker containing ice (10 mL), and then transferred to a separatory funnel. The aqueous phase was extracted with ethyl acetate. The organic extracts were combined and washed sequentially with 1.5M K₂HPO₄, saturated aqueous sodium bicarbonate and brine. The organic extract was dried over magnesium sulfate, filtered and the solvent removed in vacuo to give 204mg of a brown crystalline solid. LCMS: waters Acquity UPLC BEH C column, 2.1X105 mm,1.7 μm particles, mobile phase A:100% water containing 0.05% trifluoroacetic acid, mobile phase B:100% acetonitrile containing 0.05% trifluoroacetic acid, temperature: 40 ℃, gradient: 1.5 min, 2-98% B followed by 98% B for 0.5 min, flow rate: 0.8mL/min, UV detection at 220nm. Retention time = 1.01min, [ m+h ] observed adduct, [ 265.0 (weak ionization) observed mass.¹ H NMR (CHLOROFORM-d) δ8.03 (d, j=8.8 hz, 1H), 7.89-7.97 (m, 1H), 3.82 (s, 3H).

Intermediate 7

8- ((2S, 5R) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile, TFA

To a solution of 6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl triflate (65 g,195 mmol) and tert-butyl (2R, 5S) -2, 5-dimethylpiperazine-1-carboxylate (43.9 g,205 mmol) in acetonitrile (1.3L) was added DIPEA (0.102L, 585 mmol). The solution was stirred at 80 ℃ for 6 hours. The solvent was removed and the crude residue was chromatographed on silica gel (product Rf 0.4 in 100% ethyl acetate). The product (2R, 5S) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl) -2, 5-dimethylpiperazine-1-carboxylic acid tert-butyl ester (75 g,189mmol,97% yield). LCMS: M/z=398.2 (m+h); rt 2.7min. The method comprises the steps of carrying out column-Kinetex XB-C18 (75X 3mM-2.6 μm), flowing at 1mL/min, gradient time of 4min, 20% of solvent B to 100% of solvent B, monitoring at 254nm (solvent A:98% of water: 2% of acetonitrile, 10mM of ammonium formate, solvent B:2% of water: 98% of acetonitrile, and 10mM of ammonium formate.

To a solution of (2 r,5 s) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl) -2, 5-dimethylpiperazine-1-carboxylic acid tert-butyl ester (30 g,75 mmol) in ethyl acetate (1000 mL) was added HCl (4M in dioxane) (189 mL,755 mmol) and the temperature was allowed to reach room temperature while stirring for 6h. LC/MS analysis showed that the product mass was 90% at 0.60RT and the amidation by-product mass was 4% at 0.44RT (consistent with nitrile hydrolysis). The reaction mixture was diluted with methyl tert-butyl ether (MTBE, 2000 mL), stirred for 15min, and the HCl salt of the product was filtered and washed with MTBE (100 mL). HCl salt was dissolved in water (300 ml) and the pH was adjusted to 8 using 10% aqueous sodium bicarbonate. The organic fraction was extracted with DCM (5X 250 mL). The combined organic layers were washed with water (2×300 mL), dried over sodium sulfate and concentrated to give 8- ((2 s,5 r) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile (20 g,65.2mmol,86% yield). LCMS: M/z= 298.2 (m+h); rt 0.5min. The method comprises the steps of column-Kinetex XB-C18 (75X 3mM-2.6 μm), flow rate of 1mL/min, gradient time of 4min, 20% solvent B to 100% solvent B, monitoring at 254nm (solvent A:98% water: 2% acetonitrile; 10mM ammonium formate; solvent B:2% water: 98% acetonitrile; 10mM ammonium formate: .¹H NMR(400MHz,CDCl₃)δ7.79(d,J＝8.8Hz,1H),7.70(d,J＝12,3.2Hz,1H),6.29(s,1H),3.80(dd,J＝8.8Hz,1H)3.70(m,1H),3.65(s,3H),3.29(m,2H),2.80(m,2H),1.19(d,J＝6Hz,3H),1.15(d,J＝6Hz,3H).¹³C NMR(75MHz, chloroform-d). Delta. 161.9,155.0,138.5,137.0,128.2,125.0,122.2,117.2,111.3,56.5,51.9,50.0,49.5,29.0,18.8,15.4. Stereochemistry, i.e., homochiral.

Method for synthesizing DGKi Compound 1

4- ((2 R,5 s) -4- (bis (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -6-bromo-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridine-3-carbonitrile

To a stirred solution of 6-bromo-3-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl trifluoromethane sulfonate (80 mg,0.194 mmol) in acetonitrile (5 mL) was added DIPEA (0.102 mL, 0.552 mmol) and HCl salt of (2 s,5 r) -1- (bis (4-fluorophenyl) methyl) -2, 5-dimethylpiperazine (75 mg,0.214 mmol). The reaction mixture was stirred at 85 ℃ overnight. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (15 mL). The organic layer was washed with brine, dried over Na₂SO₄ and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography using 24g flash chromatography eluting with 50-80% etoac/petroleum ether. The fractions were concentrated under reduced pressure to give 4- ((2 r,5 s) -4- (bis (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -6-bromo-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridine-3-carbonitrile (95 mg,85% yield); LCMS: M/z= 578.2 (m+h); rt 3.916min.

Method for synthesizing DGKi Compound 2

1- (Bis (4-fluorophenyl) methyl) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl) piperazine-2-carboxylic acid methyl ester

To a stirred solution of 8-chloro-5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile (22.90 mg,0.104 mmol) in DMA (1 mL) and tert-butanol (4 mL) was added the TFA salt of 1- (bis (4-fluorophenyl) methyl) piperazine-2-carboxylate (40 mg,0.087 mmol) and cesium carbonate (85 mg,0.261 mmol) under a nitrogen atmosphere followed by chloro (2-dichlorophenylphosphino-2 ',6' -diisopropyloxy-1, 1' -biphenyl) [2- (2 ' -amino-1, 1' -biphenyl) ] palladium (II) (3.37 mg, 4.34. Mu. Mol). The reaction vessel was immersed in an oil bath at 70 ℃. The bath temperature was raised to 90 ℃ over 2min and the reaction mixture was stirred for 16h. The reaction mixture was filtered through a pad of celite and concentrated in high vacuum to give a brown gum. The crude material was purified by preparative HPLC under the conditions of column Sunfire C18,19X150mM,5 μm particles, mobile phase A10 mM ammonium acetate pH 4.5 containing acetic acid, mobile phase B acetonitrile, gradient 30-100% B over 15 minutes followed by 100% B for 5 minutes, flow rate 17mL/min. The product containing fractions were combined and evaporated by centrifugation to give methyl 1- (bis (4-fluorophenyl) methyl) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl) piperazine-2-carboxylate (3.5 mg,6.23 μmol,7.17% yield). LCMS, M/z=530.2 (m+h), rt 2.20min. LC-MS method column-X Bridge BEH XP C18 (50×2.1mM 2.5 μm; flow rate 1.1mL/min; gradient time 3min; temperature: 50 ℃ C., 0% solvent B to 100% solvent B; monitoring at 220nm (solvent A:95% water: 5% acetonitrile; 10mM ammonium acetate; solvent B:5% water: 95% acetonitrile; 10mM ammonium acetate) ).¹H NMR(400MHz,DMSO-d₆)δppm 8.16(d,J＝8.8Hz,1H),8.08(d,J＝9.0Hz,1H),7.57(dd,J＝8.8,5.6Hz,2H),7.42-7.28(m,2H),7.22-7.08(m,4H),6.14(s,1H),5.17(s,1H),4.78(d,J＝12.2Hz,1H),3.64(d,J＝12.0Hz,1H),3.59(s,3H),3.54(s,3H),3.45-3.35(m,2H),3.15(dd,J＝12.5,3.9Hz,1H),3.04(td,J＝11.7,2.9Hz,1H),2.71-2.63(m,1H).

Method for synthesizing DGKi Compound 3

(R) -4- (4- (bis (4-fluorophenyl) methyl) -3-methylpiperazin-1-yl) -6-bromo-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridine-3-carbonitrile

To a stirred solution of 6-bromo-3-cyano-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridin-4-yl trifluoromethane sulfonate (100 mg,0.243 mmol) in acetonitrile (8 mL) was added DIPEA (0.127 mL, 0.428 mmol) and HCl salt of (R) -1- (bis (4-fluorophenyl) methyl) -2-methylpiperazine (82 mg,0.243 mmol). The reaction mixture was heated to 85 ℃ over 5min and stirred for 1h. The reaction mixture was concentrated under high vacuum to give a brown gum. By using the crude compound12G silica gel column purification, 60-67% ethyl acetate/petroleum ether to obtain (R) -4- (4- (bis (4-fluorophenyl) methyl) -3-methylpiperazin-1-yl) -6-bromo-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridine-3-carbonitrile (90 mg,42.7% yield), LCMS: M/z= 566.0 (M+2H), rt 2.23min as a brown gum. LC-MS method column-AQUITY UPLC BEH C18 (3.0X10 mM) 1.7 μm, mobile phase A buffer: acetonitrile (95:5), mobile phase B buffer: acetonitrile (5:95), buffer: 10mM ammonium acetate, gradient: 20-100% B over 2.0 min, followed by 100% B for 0.2 min, flow rate 0.7mL/min.

Method for synthesizing DGKi Compound 4

(R) -8- (4- (bis (4-fluorophenyl) methyl) -3-methylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2, 7-dicarboxylic acid carbonitrile

To a stirred solution of (R) -4- (4- (bis (4-fluorophenyl) methyl) -3-methylpiperazin-1-yl) -6-bromo-1-methyl-2-oxo-1, 2-dihydro-1, 5-naphthyridine-3-carbonitrile (90 mg, 0.1599 mmol) in NMP (5 mL) under nitrogen was added zinc (2.085 mg,0.032 mmol) and zinc cyanide (37.4 mg,0.319 mmol). Nitrogen purging was continued for 3min and dppf (5.30 mg, 9.57. Mu. Mol) and Pd₂(dba)₃ (14.6 mg,0.016 mmol) were added. The reaction mixture was heated to 80 ℃ over 5min and stirred for 4h. The reaction mixture was filtered through a pad of celite and concentrated under high vacuum to give a brown gum. The crude material was purified via preparative HPLC. HPLC method of column-SUNFIRE C (150 mM x19mM ID,5 μm), mobile phase A10 mM ammonium acetate/water, mobile phase B acetonitrile, gradient 40-60% B over 3.0 min, 17mL/min flow rate, followed by 60-100% B17 min flow rate, 17mL/min flow rate. The fractions containing the product were combined and concentrated under high vacuum. The sample was then diluted with (etoh\h₂ O, 1:3) and lyophilized overnight to give (R) -8- (4- (bis (4-fluorophenyl) methyl) -3-methylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2, 7-carbonitrile (50 mg,61.4% yield) as a pale yellow solid. LCMS, M/z= 511.2 (m+h), rt 3.520min. LC-MS method column-KINETEX-XB-C18 (75X 3 mM-2.6. Mu.), mobile phase A10 mM ammonium formate/water: acetonitrile (98:2), mobile phase B10 mM ammonium formate/water: acetonitrile (2:98), gradient 20-100% B over 4 min, flow 1.0mL/min, then 100% B for 0.6 min, flow 1.5mL/min, then gradient 100-20% B over 0.1 min, flow rate 1.5mL/min.¹H NMR(400MHz,DMSO-d₆)δppm 8.26(d,J＝8.8Hz,1H),8.15(d,J＝9.0Hz,1H),7.56(dd,J＝11.9,8.7Hz,2H),7.57(dd,J＝11.7,8.8Hz,2H),7.16(t,J＝8.9Hz,4H),4.90(s,1H),4.10(d,J＝13.0Hz,1H),4.01(d,J＝12.5Hz,1H),3.86(dd,J＝12.2,2.9Hz,1H),3.66-3.55(m,1H),3.53(s,3H),3.08-2.97(m,1H),2.97-2.90(m,1H),2.90(s,1H),1.03(d,J＝6.6Hz,3H).

Method for synthesizing DGKi compound 5

8- [ (2S, 5R) -4- [ (4-fluorophenyl) (phenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

In a 2-dram sealed reaction vessel, 8- ((2 s,5 r) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile, TFA (41.1 mg, 100. Mu. Mol), (4-fluorophenyl) (phenyl) methanol (28.3 mg, 140. Mu. Mol) and (cyanomethyl) trimethyl phosphonium iodide (48.6 mg, 200. Mu. Mol) were mixed in acetonitrile (200. Mu.l). Hunig's base (75. Mu.L, 429. Mu. Mol) was added and the reaction mixture was heated at 110℃for 2 hours. The reaction mixture was directly injected into a 12g silica gel column and eluted with 20-100% ethyl acetate/hexanes to give example 182 as a diastereomeric mixture. Analytical LC/MS conditions were injected volume vol=3 μl, initial% B0, final% B100, gradient time 2 min, flow rate 1mL/min, wavelength 220nm, solvent versus acetonitrile/water/TFA, solvent a10% acetonitrile, 90% water/0.05% TFA, solvent B10% water, 90% acetonitrile/0.05% TFA, column Acquity BEH C18.x50mm1.7 μm, furnace temperature=40 ℃. LC\MS results, retention time 1.4 min, observed quality 482.5 (M⁺).

The crude material was further purified via preparative LC/MS under conditions of column XBIdge C18,200mM x19mM,5 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, gradient: held at 47% B0 min for 20min 47-87% B followed by 100% B4 min, flow rate: 20mL/min, column temperature: 25 ℃. Fraction collection triggered by MS signal, fractions containing product were combined and dried via centrifugal evaporation to obtain 14.4mg of the title compound (30% yield). The molecular weight 481.575 was calculated. LC\MS Condition QC-ACN-TFA-XB observed MS ion 482.2, retention time 1.6 min .¹H NMR(500MHz,DMSO-d₆)δ8.18-8.10(m,1H),8.06(d,J＝8.8Hz,1H),7.68-7.48(m,4H),7.39-7.26(m,2H),7.25-7.08(m,3H),6.00(s,1H),4.67(br s,1H),4.59(br d,J＝6.7Hz,1H),3.76-3.62(m,1H),3.55(br d,J＝12.8Hz,1H),3.15-3.04(m,1H),2.90-2.81(m,1H),2.36(br dd,J＝17.4,11.9Hz,1H),1.35-1.28(m,3H),1.24(s,1H),1.07(br t,J＝5.6Hz,3H).

Method for synthesizing DGKi Compounds 6 and 7

8- [ (2S, 5R) -4- [ (4-fluorophenyl) (phenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

Example 5 was separated into the individual diastereomers using chiral solid phase chromatography, column CHIRALPAK OJ-H,21X250mm, 5 microns, mobile phase 90% CO₂/10% methanol, flow conditions 45mL/min, detector wavelength 225nm, sample details 500. Mu.L, 15mg dissolved in 1mL methanol/acetonitrile.

The first eluting diastereomer, example 6 (66.4 mg), was isolated in 20.2% yield. The final purity was determined using analytical LC/MS. Conditions 1 were column Waters XBiridge C18,2.1mM x50mM,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, temperature 50 ℃, fraction 0% B to 100% B over 3min followed by 100% B for 0.50min, flow rate 1mL/min, detection: MS and UV (220 nm). The sample was sampled at 1 results of 100.0% purity, 482.1% observed mass, and 2.49min retention time. Conditions were applied 2 columns of Waters XBridge C18,2.1mm x50mm,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 0.1% trifluoroacetic acid, mobile phase B95:5 acetonitrile in water with 0.1% trifluoroacetic acid, temperature 50 ℃, gradient from 0% B to 100% B over 3min, followed by 100% B for 0.50min, flow rate 1mL/min, detection: MS and UV (220 nm). The result of the sample introduction 2 is that the purity is 100.0%, the observed quality is 482.11, and the retention time is 1.75min.

The second eluting diastereomer, example 7 (71.7 mg), was isolated in 21.9% yield. Analytical LC/MS was used to determine the final purity. Conditions 1 were column Waters XBRID C18,2.1mM x50mM,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, temperature 50 ℃, gradient 0% B to 100% B over 3min followed by 100% B for 0.50min, flow rate 1mL/min, detection: MS and UV (220 nm). The sample was sampled at 1 results of 100.0% purity, 482.11% observed mass, 2.51min retention time. Conditions were applied 2 columns of Waters XBridge C18,2.1mm x50mm,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 0.1% trifluoroacetic acid, mobile phase B95:5 acetonitrile in water with 0.1% trifluoroacetic acid, temperature 50 ℃, gradient from 0% B to 00% B over 3min, followed by 100% B for 0.50min, flow rate 1mL/min, detection: MS and UV (220 nm). The result of the sample introduction 2 is that the purity is 100.0%, the observed quality is 482.1, and the retention time is 1.76min.

Method for synthesizing DGKi Compound 8

4- [ (2 S,5 r) -4- [ (4-chlorophenyl) (4-fluorophenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -6-methoxy-1-methyl-1, 2-dihydro-1, 5-naphthyridin-2-one

4- ((2S, 5R) -2, 5-dimethylpiperazin-1-yl) -6-methoxy-1-methyl-1, 5-naphthyridin-2 (1H) -one (50 mg,0.165 mmol) and 1- (bromo (4-chlorophenyl) methyl) -4-fluorobenzene (49.5 mg,0.165 mmol) were mixed with diisopropylethylamine (0.173 mL,0.992 mmol) in acetonitrile (3 mL) and the reaction mixture was heated at 55deg.C overnight. LC/MS indicated that the reaction was complete. The crude material was purified by preparative LC/MS under the conditions of column XBridge C18,200mM x19mM,5 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, gradient: 42% B0 min over 25 min 42-82% B followed by 100% B5 min, flow rate: 20mL/min, column temperature: 25 ℃. Fraction collection was triggered by MS signal. The fractions containing the product were combined and dried via centrifugal evaporation. The molecular weight 521.03 was calculated. LC\MS conditions QC-ACN-AA-XB observed MS ion 521.1, retention time 2.77 min.

Method for synthesizing DGKi Compound 9

8- [ (2S, 5R) -4- { [2- (difluoromethyl) -4-fluorophenyl ] methyl } -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

To a solution of 8- ((2 s,5 r) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile (30 mg,0.081 mmol) in DMF (2 mL) was added 2- (difluoromethyl) -4-fluorobenzaldehyde (16.86 mg,0.097 mmol). The solution was stirred at room temperature for 1 hour. Sodium cyanoborohydride (15.22 mg,0.242 mmol) was added and the reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated completion of the reaction. The crude material was purified by preparative LC/MS under the conditions of column XBridge C18,200mM x19mM,5 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, gradient: hold at 31% B0 min over 25 min, 31-71% B followed by 100% B5 min, flow rate: 20mL/min, column temperature: 25 ℃. Fraction collection was triggered by MS and UV signals. The fractions containing the product were combined and dried via centrifugal evaporation. The yield of the product was 13.0mg and the estimated purity by LCMS analysis was 100%. The final purity was determined using analytical LC/MS. Conditions 1 were column Waters XBridge C18,2.1mm x50mm,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 0.1% trifluoroacetic acid, mobile phase B95:5 acetonitrile in water with 0.1% trifluoroacetic acid, temperature 50 ℃, gradient 3min0% B to 100% B followed by 100% B for 0.5min, flow rate 1mL/min, detection: MS and UV (220 nm). The sample was sampled at 1 results of 100.0% purity, 456.08% observed mass, and 1.39min retention time. Inject 2 conditions were column 10mM ammonium acetate in water, 5:95 acetonitrile in mobile phase A, 10mM ammonium acetate in water, 50℃in mobile phase B, 3min0% B to 100% B in gradient followed by 100% B for 0.50min, flow rate 1mL/min, detection: MS and UV (220 nm). The sample introduction was carried out with a purity of 100.0%, an observed mass of 456.07 and a retention time of 2.22min. After 3min% B, it was then kept at 100% B0.50min, flow rate 1mL/min, MS and UV (220 nm) detection. The sample introduction was carried out with a purity of 100.0%, an observed mass of 456.07 and a retention time of 2.22min.

Method for synthesizing DGKi Compound 10

8- [ (2S, 5R) -4- [ (4-fluorophenyl) (4-methylphenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

To a mixture of 8- ((2 s,5 r) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile, TFA (68.6 mg,60% wt.,0.1 mmol), (cyanomethyl) trimethylonium iodide (48.6 mg,0.200 mmol) and (4-fluorophenyl) (p-tolyl) methanol (26.0 mg,0.120 mmol) in acetonitrile (0.3 mL) was added Hunig's base (0.105 mL,0.600 mmol). The reaction mixture was stirred at 110℃for 2 hours, then (cyanomethyl) trimethylonium iodide (48.6 mg,0.200 mmol), (4-fluorophenyl) (p-tolyl) methanol (26.0 mg,0.120 mmol) and Hunig's base (0.058 mL,0.300 mmol) were added in two portions. The reaction mixture was stirred at 110 ℃ for an additional 2 hours. The crude reaction mixture was directly injected onto 12g Si-REDISEP RF, and used for flash chromatography by 20-100% ethyl acetate/hexane. The products containing the fractions were combined and dried by vacuum. The resulting material was further purified via preparative LC/MS under conditions of column XBIdge C18,200mm x19mm,5 μm particles, mobile phase A5:95 acetonitrile water 0.1% trifluoroacetic acid, mobile phase B95:5 acetonitrile water 0.1% trifluoroacetic acid, gradient 20-60% B over 25 min at 20% B0 min, followed by 100% B5 min at 20mL/min at a flow rate of 25℃column temperature. Fraction collection was triggered by MS and UV model. The fractions containing the product were combined and dried via centrifugal evaporation. The yield of the diastereomeric product TFA salt was 47.1mg.

The diastereoisomeric product was resolved into two diastereoisomers using SFC-Chiral chromatography, with the following conditions: column: chiral AD,30x250mm,5 micron particles, mobile phase: 80% CO₂/20% IPA w/0.1% DEA, flow rate: 100mL/min, column temperature: 25 ℃. The title compound was collected as the second elution peak, >91% de. The molecular weight 495.602 was calculated. The final purity was determined using analytical LC/MS. Conditions 1 were column Waters XBiridge C18,2.1mM x50mM,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, temperature 50 ℃, gradient 0% B to 100% B over 3min followed by 100% B0.5min, flow rate 1mL/min, detection: MS and UV (220 nm). The sample was sampled 1 for 97.6% purity, 496.26% observed mass and 2.52min retention time. Conditions were applied 2 columns of Waters XBridge C18,2.1mm x50mm,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 0.1% trifluoroacetic acid, mobile phase B95:5 acetonitrile in water with 0.1% trifluoroacetic acid, temperature 50 ℃, gradient from 0% B to 100% B over 3min, then 100% B for 0.50min, flow rate 1mL/min, detection: MS and UV (220 nm). The result of the sample introduction 2 is that the purity is 98.2%, the observed quality is 496.28, and the retention time is 1.73min.

Method for synthesizing DGKi Compound 11

8- [ (2S, 5R) -4- [1- (2, 6-difluorophenyl) ethyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

To a mixture of 2- (1-bromoethyl) -1, 3-difluorobenzene (15.12 mg,0.065 mmol) and 5-methyl-6-oxo-8- (piperazin-1-yl) -5, 6-dihydro-1, 5-naphthyridine-2, 7-carbonitrile, TFA (34.0 mg,60% wt.,0.05 mmol) in acetonitrile (0.3 mL) was added Hunig's base (0.052 mL,0.300 mmol). The mixture was stirred at 55 ℃ for 2 hours. LCMS indicated complete conversion to product. The crude material was purified by preparative LC/MS under the conditions of column XBridge C18,200mM x19mM,5 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, gradient: hold at 37% B0 min over 20 min, 37-77% B, followed by 100% B5 min, flow rate: 20mL/min, column temperature: 25 ℃. Fraction collection was triggered by MS and UV signals. The fractions containing the product were combined and dried via centrifugal evaporation. The yield of the product was 12.1mg. The molecular weight 437.495 was calculated. The final purity was determined using analytical LC/MS. Conditions 1 were column Waters XBiridge C18,2.1mM x50mM,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, temperature 50 ℃, gradient 0% B to 100% B over 3min followed by 100% B0.50min, flow rate 1mL/min, detection: MS and UV (220 nm). The sample was sampled at1 results of 100.0% purity, 438.14% observed mass, 2.36min retention time. Conditions were applied 2 columns of Waters XBridge C18,2.1mm x50mm,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 0.1% trifluoroacetic acid, mobile phase B95:5 acetonitrile in water with 0.1% trifluoroacetic acid, temperature 50 ℃, gradient from 0% B to 100% B over 3min, followed by 100% B for 0.50min, flow rate 1mL/min, detection: MS and UV (220 nm). The result of the sample introduction 2 is that the purity is 100.0%, the observed quality is 438.14, and the retention time is 1.2min.

Method for synthesizing DGKi compounds 12-14

8- ((2S, 5R) -4- (1- (2, 4-difluorophenyl) propyl) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

To a mixture of 8- ((2S, 5R) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile (29.7 mg,0.1 mmol) and 1- (1-bromopropyl) -2, 4-difluorobenzene (25.9 mg,0.110 mmol) in acetonitrile (0.3 mL) was added Hunig's base (87. Mu.L, 0.500 mmol). The mixture was stirred on a hot plate at 55 ℃ for 16 hours. The crude material was purified by preparative LC/MS under the conditions of column XBridge C18,200mm x19mm,5- μm particles, mobile phase a 5:95 acetonitrile water 0.1% trifluoroacetic acid, mobile phase B95:5 acetonitrile water 0.1% trifluoroacetic acid, gradient 3-43% B over 25 minutes at 3% B0 min, followed by 100% B5 minutes at 20mL/min, column temperature 25 ℃. Fraction collection was triggered by MS and UV signals. The product-containing fractions were combined and dried via centrifugal evaporation. Stereochemistry: diastereomeric mixtures.

The diastereoisomeric mixture of compound 12 synthesized DGKi was further separated by using SFC-Chiral chromatography to resolve the two homochiral diastereoisomers, provided that the column was Chiral OD,30x250mm.5 micron particles, mobile phase 15% IPA/85% CO₂ w/0.1% DEA, flow rate 100mL/min, detector wavelength 220nm.

DGKi Compound 13 (isomer 1) was collected as the first elution peak at 95% de. Stereochemistry, i.e., homochiral.

DGKi Compound 14 (isomer 2) was collected as the second elution peak at 95% de. Stereochemistry, i.e., homochiral.

Method for synthesizing DGKi compound 15

8- (4- (Bis (4-fluorophenyl) methyl) piperazin-1-yl) -5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

DMF was bubbled with nitrogen for 1 hour. In a 1-dram bottle were added zinc (0.95 mg,0.015 mmol), palladium (I) bromo (tri-tert-butylphosphine) dimer (9.96 mg,0.013 mmol) and 4- (4- (bis (4-fluorophenyl) methyl) piperazin-1-yl) -6-bromo-1-methyl-3-nitro-1, 5-naphthyridin-2 (1H) -one (21.38 mg,0.037 mmol). Bubbling DMF (0.3 mL) was added and the mixture capped under nitrogen and immersed in a 50 ℃ oil bath for 15 minutes. Zinc dicyano (2.86 mg,0.024 mmol) was added. The reaction mixture was capped under nitrogen and immersed in a 50 ℃ oil bath for 3 hours. LC/MS analysis indicated completion of the reaction. The crude material was purified by preparative LC/MS under the conditions of column XBridge C18,19x200mM,5 μm particles, mobile phase a:5:95 acetonitrile: water containing 10mM ammonium acetate, mobile phase B:95:5 acetonitrile: water containing 10mM ammonium acetate, gradient 50-90% B over 15 minutes followed by 100% B for 5 minutes, flow rate 20 mL/min. The fractions containing the product were combined and dried via centrifugal evaporation. The title compound (11.4 mg) was isolated in 59.7% yield.

Or alternatively synthesized by mixing a solution of 8-chloro-5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile (750 mg,2.83 mmol) in DMF (6 mL) with 1- (bis (4-fluorophenyl) methyl) piperazine (899 mg,3.12 mmol)) followed by the addition of Hunig's base (0.990 mL,5.67 mmol). The reaction mixture was stirred at room temperature overnight. LC/MS analysis indicated completion of the reaction. The crude material was filtered and purified by preparative HPLC using aqueous acetonitrile containing ammonium acetate as buffer to yield 1.02g of a yellow solid. Two analytical LC/MS feeds were used to determine final purity. Conditions 1 were column Waters Acquity UPLC BEH C, 2.1X105 mM,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, temperature 50 ℃, gradient 0-100% B over 3min, followed by 100% B for 0.75 min, flow rate 1.0 mL/min, UV at 220nm. Conditions of sample introduction 2 were column Waters Acquity UPLC BEH C, 2.1x50mm,1.7μm particles, mobile phase A5:95 acetonitrile water 0.1% trifluoroacetic acid, mobile phase B95:5 acetonitrile water 0.1% trifluoroacetic acid, temperature 50 ℃, gradient 0-100% B over 3 minutes followed by 100% B for 0.75 minutes, flow rate 1.0 mL/min, UV detection at 220nm. The sample was sampled 1 for 100% purity, 517.0% observed mass and 2.4 minutes retention time. The results of the sample introduction were 98.4% purity, 517.0% observed mass and 1.7 min retention time.¹ H NMR (500 MHz, chloroform -d)δ7.88(d,J＝8.7Hz,1H),7.76(d,J＝8.9Hz,1H),7.40(dd,J＝8.5,5.5Hz,4H),7.02(t,J＝8.7Hz,4H),4.34(s,1H),3.68(s,3H),3.62-3.55(m,4H),2.64(br s,4H).¹³C NMR(126MHz, chloroform -d)δ163.0,161.0,155.4,147.0,138.0,137.7,137.7,135.9,132.4,129.5,129.2,129.2,126.0,123.1,116.5,115.8,115.6,74.3,51.6,51.2,29.7.)

Method for synthesizing DGKi Compound 16

8- [ (2S, 5R) -4- [ bis (4-methylphenyl) methyl ] -2, 5-dimethylpiperazin-1-yl ] -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile

To a mixture of (cyanomethyl) trimethylphosphine iodide (46.2 mg,0.19 mmol), di-p-tolylmethanol (23.46 mg,0.108 mmol) and 8- ((2 s,5 r) -2, 5-dimethylpiperazin-1-yl) -5-methyl-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile, TFA (72.4 mg,54% wt,0.095 mmol) in acetonitrile (0.3 mL) was added Hunig's base (0.10 mL,0.57 mmol). The reaction mixture was stirred at 110 ℃ for 2 hours. The crude material was purified by preparative LC/MS under the conditions of column XB ridge C18,200mM x19mM,5 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, gradient: 55% B0 min, 55-95% B over 20 min, followed by 100% B4 min, flow rate: 20mL/min, column temperature: 25 ℃. Fraction collection was triggered by MS and UV signals. The product-containing fractions were combined and dried via centrifugal evaporation. The yield of the product was 23.4mg. The molecular weight 491.639 was calculated. Analytical LC/MS was used to determine the final purity. Conditions 1 were column Waters XBRID C18,2.1mM x50mM,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 10mM ammonium acetate, mobile phase B95:5 acetonitrile in water with 10mM ammonium acetate, temperature 50 ℃, gradient 0% B to 100% B over 3min followed by 100% B0.75 min, flow rate 1mL/min, detection: MS and UV (220 nm). The sample was sampled at 1 results of 100.0% purity, 492.21% observed mass, 2.77min retention time. Conditions were applied 2 columns of Waters XBridge C18,2.1mm x50mm,1.7 μm particles, mobile phase A5:95 acetonitrile in water with 0.1% trifluoroacetic acid, mobile phase B95:5 acetonitrile in water with 0.1% trifluoroacetic acid, temperature 50 ℃, gradient from 0% B to 100% B over 3min, followed by 100% B for 0.75min, flow rate 1mL/min, detection: MS and UV (220 nm). Sample introduction 2 results, purity 100.0%, observed quality 492.2%, retention time :1.71min.¹H NMR(400MHz,DMSO-d₆)δppm 8.15(d,J＝8.5Hz,1H),8.04-8.09(m,1H),7.81(s,4H),7.57-7.63(m,2H),7.12-7.19(m,2H),6.00(s,1H),4.82(s,1H),4.52-4.63(m,1H),3.64-3.76(m,1H),3.51-3.58(m,4H),2.99-3.10(m,1H),2.86(br d,J＝8.5Hz,1H),2.28-2.37(m,1H),1.31(d,J＝6.5Hz,3H),1.07(d,J＝6.5Hz,3H).¹³C NMR(100.66MHz,DMSO-d₆)δppm 162.4,160.9,159.9,153.5,148.0,138.7,138.6,135.0,132.6,129.3(d,J＝8.0Hz),128.8(d,J＝10.0Hz),124.0,122.8,118.6,117.5,115.6,115.4,109.8,104.8,69.0,51.8,49.4,48.9,47.2,28.6,13.4,7.4.

DGKi Compounds 17 and 18

4- ((2 S,5 r) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) propyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile

To a stirred solution of 4- ((2 s,5 r) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, TFA (0.12 g,0.27 mmol) in acetonitrile (10 mL) was added DIPEA (0.14 mL,0.82 mmol), 1- (1-chloropropyl) -4- (trifluoromethyl) benzene (0.12 g,0.55 mmol) and sodium iodide (0.04 g,0.27 mmol). The reaction mixture was heated at 85 ℃ for 16h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure to obtain a crude product which was purified by preparative HPLC [ HPLC method: column: sunfire C18,150X19mM ID,5 μm; mobile phase A:10mM ammonium acetate/water; mobile phase B: acetonitrile; gradient: over 18 minutes, 0-100% B followed by 100% B for 5 minutes; flow rate: 17mL/min ]. The fractions were concentrated under reduced pressure and lyophilized from EtOH/H₂ O (1:5) to give compounds 17 and 18.

Compound 17 (10 mg,7% yield), LCMS: M/z=513.3 (M+H); rt 2.52min, (LCMS method: column: XB ridge BEH XP C18 (50X 2.1) mM,2.5 μm mobile phase A:95% water: 5% acetonitrile, 10mM ammonium formate, mobile phase B:5% water: 95% acetonitrile, 10mM ammonium formate, flow rate: 1.1mL/min, temperature: 50 ℃ C., time) (min):0-4;％B:0-100;¹H NMR(400MHz,DMSO-d₆)δ8.24(d,J＝6.6Hz,1H),7.98(d,J＝9.0Hz,1H),7.73(d,J＝8.1Hz,2H),7.56(d,J＝7.1Hz,2H),5.83-5.48(m,1H),4.98-4.86(m,1H),3.64(br.s.,1H),3.43(s,3H),3.08(d,J＝9.8Hz,1H),2.93-2.82(m,2H),2.42-2.26(m,1H),2.13-2.08(m,1H),1.98-1.82(m,3H),1.66-1.54(m,1H),1.44-1.31(m,1H),0.98-0.91(br.s.,3H),0.69-0.53(m,6H).

Compound 18 (3 mg,2% yield), LCMS: M/z=513.3 (M+H); rt 2.54min, (LCMS method: column: XB ridge BEH XP C18 (50X 2.1) mM,2.5 μm mobile phase A:95% water: 5% acetonitrile, 10mM ammonium formate, mobile phase B:5% water: 95% acetonitrile, 10mM ammonium formate, flow rate: 1.1mL/min, temperature: 50 ℃ C., time) (min):0-4;％B:0-100;¹H NMR(400MHz,DMSO-d₆)δ8.28-8.19(m,1H),8.01-7.95(m,1H),7.72(d,J＝7.8Hz,2H),7.58(d,J＝8.6Hz,2H),6.06-5.28(m,1H),5.08-4.76(m,1H),3.64-3.50(m,2H),3.43(s,3H),3.16-3.08(m,1H),2.25-2.14(m,2H),2.00-1.83(m,3H),1.57-1.53(m,3H),1.03-0.89(m,3H),0.65-0.54(m,6H).

DGKi Compounds 19 and 20

4- ((2 S,5 r) -5-ethyl-2-methyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile

To a stirred solution of 4- ((2 s,5 r) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, TFA (70 mg,0.22 mmol) in acetonitrile (2 mL) was added DIPEA (0.12 mL,0.67 mmol), 1- (1-chloroethyl) -4- (trifluoromethyl) benzene (93 mg,0.45 mmol), sodium iodide (33.6 mg,0.22 mmol) and heated at 85 ℃ for 16h at room temperature. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (100 mL). The organic layer was washed with brine, dried over Na₂SO₄ and concentrated under reduced pressure to obtain the crude product which was purified by preparative HPLC [ HPLC method: column: sunfire C18 (150mmx19.2mm ID,5 μm), mobile phase a=10 mM ammonium acetate/water, mobile phase b=acetonitrile, flow rate: 19mL/min ], fractions were concentrated under reduced pressure, diluted with EtOH/H₂ O (1:5) and lyophilized to obtain compounds 19 and 20.

Compound 19 (9 mg,8% yield), LCMS: M/z=485.1 (M+H); rt 2.34min, (LCMS method: column: XB ridge BEH XP C18 (50×2.1 mM), 2.5 μm, mobile phase A:95% water: 5% acetonitrile, 10mM ammonium acetate, mobile phase B:5% water: 95% acetonitrile, 10mM ammonium acetate, flow rate: 1.1mL/min, temperature: 50 ℃ C., time) (min):0-3;％B:0-100.¹H NMR(400MHz,DMSO-d₆)δppm 8.32-8.17(m,1H),8.05-7.94(m,1H),7.76-7.66(m,2H),7.66-7.55(m,2H),6.11-5.42(m,1H),5.10-4.79(m,1H),3.78-3.59(m,2H),3.44(s,3H),3.17-3.05(m,1H),2.64-2.55(m,1H),2.26-2.09(m,1H),1.65-1.34(m,3H),1.31-1.16(m,5H),1.01(br t,J＝7.1Hz,3H).

Compound 20 (9 mg,8% yield), LCMS: M/z=485.1 (M+H); rt 2.29min, (LCMS method: column: XB ridge BEH XP C18 (50×2.1 mM), 2.5 μm, mobile phase A:95% water: 5% acetonitrile, 10mM ammonium acetate, mobile phase B:5% water: 95% acetonitrile, 10mM ammonium acetate, flow rate: 1.1mL/min, temperature: 50 ℃ C., time) (min):0-3;％B:0-100％).¹H NMR(400MHz,DMSO-d₆)δppm 8.24(br d,J＝8.6Hz,1H),7.99(d,J＝9.0Hz,1H),7.73(d,J＝8.3Hz,2H),7.61(br d,J＝8.3Hz,2H),5.87-5.63(m,1H),5.10-4.79(m,1H),3.90-3.80(m,1H),3.44(s,3H),3.46-3.15(m,1H),2.89-2.73(m,2H),2.41-2.34(m,1H),1.63-1.34(m,5H),1.29(br d,J＝6.1Hz,3H),0.79-0.64(m,3H).

DGKi Compounds 21 and 22

4- ((2 S,5 r) -5-ethyl-4- ((4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile

To a stirred solution of 4- ((2 s,5 r) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, TFA (0.5 g,1.17 mmol) in acetonitrile (10 mL) was added DIPEA (1.02 mL,5.86 mmol) followed by 2- (bromo (4-fluorophenyl) methyl) -5- (trifluoromethyl) pyridine (0.78 mg,2.35 mmol). The reaction mixture was heated at 80 ℃ for 3h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure to obtain a crude product, which was purified by preparative HPLC (HPLC method: column: INERTSIL ODS21.2X250mm,5 μm; mobile phase A:0.1% TFA/water; mobile phase B: acetonitrile; gradient: 30-80% B over 14 min followed by 100% B5 min; flow rate: 17 mL/min), the fractions were concentrated under reduced pressure and lyophilized from (EtOH/H₂ O, 1:5) to obtain compound 21 and compound 22.

Compound 21:140mg,21% yield, LCMS: M/z= 566.2 (M+H); rt 3.26min; (LCMS method: column-Kinetex XB-C18 (75X 3mM-2.6 μm), mobile phase A:98% water: 2% acetonitrile, 10mM ammonium formate, mobile phase B:2% water: 98% acetonitrile, 10mM ammonium formate, flow rate: 1.0mL/min, temperature: 50 ℃ C., time (min):0-4;％B:0-100％).¹H NMR(400MHz,DMSO-d₆)δppm 8.83(br s,1H),8.19-8.31(m,2H),7.95-8.12(m,2H),7.53-7.63(m,2H),7.12-7.26(m,2H),5.41-6.26(m,1H),4.79-5.20(m,2H),3.60-3.74(m,1H),3.44(s,3H),2.73-2.87(m,1H),2.22-2.42(m,2H),1.40-1.68(m,5H),0.53-0.71(m,3H).

Compound 22:155mg,23% yield, LCMS: M/z= 566.2 (M+H); rt 3.25min; (LCMS method: column-Kinetex XB-C18 (75X 3mM-2.6 μm), mobile phase A:98% water: 2% acetonitrile, 10mM ammonium formate, mobile phase B:2% water: 98% acetonitrile, 10mM ammonium formate, flow rate: 1.0mL/min, temperature: 50 ℃ C., time (min):0-4;％B:0-100％).¹H NMR(400MHz,DMSO-d₆)δppm 8.92(s,1H),8.17-8.27(m,2H),7.90-8.02(m,2H),7.60-7.67(m,2H),7.14-7.22(m,2H),5.52-6.07(m,1H),4.87-5.08(m,2H),3.39-3.71(m,4H),2.69-2.78(m,1H),2.37-2.45(m,1H),1.37-1.69(m,5H),0.58-0.77(m,3H).

DGKi Compounds 23 to 24

(4- ((2 S,5 r) -4- ((4-chlorophenyl) (pyridin-2-yl) methyl) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile

To a stirred solution of 4- ((2 s,5 r) -5-ethyl-2-methylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (100 mg,0.32 mmol) in acetonitrile (5 mL) was added DIPEA (0.3 mL,1.60 mmol) followed by 2- (bromo (4-chlorophenyl) methyl) pyridine (181 mg,0.64 mmol). The reaction mixture was heated at 80 ℃ for 3h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure to obtain a crude product, which was purified by preparative HPLC (HPLC method: column: cellulose-5 (250 x 20 id) 5 μm, mobile phase a:0.1% dea/IPA, mobile phase B:0.1% dea/ACN, gradient: 90% B, then held at 100% B for 5 min, flow rate: 18 mL/min), the fractions were concentrated under reduced pressure and lyophilized from (EtOH/H₂ O, 1:5) to obtain compound 23 and compound 24.

Compound 23:24mg,14% yield, LCMS: M/z=514.2 (M+H); rt 2.94min; (LCMS method: column-Kinetex XB-C18 (75X 3mM-2.6 μm), mobile phase A:98% water: 2% acetonitrile, 10mM ammonium formate, mobile phase B:2% water: 98% acetonitrile, 10mM ammonium formate, flow rate: 1.0mL/min, temperature: 50 ℃ C., time (min):0-4;％B:0-100％).¹H NMR(400MHz,DMSO-d₆):δppm 8.52(d,J＝4.5Hz,1H),8.23(d,J＝9.0Hz,1H),7.96-8.02(m,1H),7.75-7.81(m,1H),7.59-7.68(m,3H),7.39(d,J＝8.5Hz,2H),7.22-7.29(m,1H),5.54-5.95(m,1H),4.81-5.07(m,2H),3.39-3.68(m,5H),2.69-2.76(m,1H),2.35-2.44(m,1H),1.37-1.67(m,5H),0.58-0.67(m,3H).

Compound 24:22mg,13% yield, LCMS: M/z=514.2 (M+H); rt 2.94min; LCMS method: column-Kinetex XB-C18 (75X 3mM-2.6 μm), mobile phase A:98% water: 2% acetonitrile, 10mM ammonium formate, mobile phase B:2% water: 98% acetonitrile, 10mM ammonium formate, flow rate: 1.0mL/min, temperature: 50 ℃ C., time (min):0-4;％B:0-100％).¹H NMR(400MHz,DMSO-d₆):δppm 8.41-8.45(m,1H),8.23(d,J＝9.0Hz,1H),7.96-8.02(m,1H),7.78-7.85(m,2H),7.53-7.61(m,2H),7.40(d,J＝8.5Hz,2H),7.20-7.26(m,1H),5.52-5.97(m,1H),4.87-5.04(m,1H),4.78-4.86(m,1H),3.37-3.71(m,4H),2.72-2.78(m,1H),2.54-2.63(m,1H),2.35-2.46(m,1H),1.40-1.64(m,5H),0.58-0.70(m,3H).

DGKi Compounds 25 and 26

4- ((2 S,5 r) -4- ((3-cyclopropyl-1, 2, 4-oxadiazol-5-yl) (4-fluorophenyl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile

To a stirred solution of 2- ((2R, 5S) -4- (6-cyano-1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidin-4-yl) -2, 5-diethylpiperazin-1-yl) -2- (4-fluorophenyl) acetic acid (0.045 g,0.09 mmol), N-hydroxycyclopropane formamidine (9.4 mg,0.09 mmol) in DMF (2 mL) was added BOP (0.01 g,0.23 mmol) and triethylamine (0.04 mL,0.23 mmol) at room temperature. After 2 hours, the reaction mixture was heated at 110 ℃ for 3 hours. The reaction mixture was cooled to room temperature and evaporated under reduced pressure to obtain the crude product, which was purified via preparative HPLC. Chiral separation method, column DAD-1-Cellulose-2 (250X 4.6 mm), 5 μm. Mobile phase, 0.1% DEA/acetonitrile, flow rate 2.0 mL/min.

Compound 25 (1.9 mg,6% yield) LCMS M/z 543.3 (M+H), rt 2.21min, LCMS method column XBRID BEH XP C18 (50×2.1) mM,2.5 μm, mobile phase A95% water 5% acetonitrile, 10mM ammonium acetate, mobile phase B5% water 95% acetonitrile, 10mM ammonium acetate, flow rate 1.1mL/min, temperature 50 ℃ and time (min):0-3;％B:0-100％).¹H NMR(400MHz,DMSO-d₆)δppm 8.29-8.16(m,1H),8.06-7.92(m,1H),7.75-7.58(m,2H),7.26(m,2H),6.01-5.32(m,1H),5.28(br s,1H),5.00-4.79(m,1H),3.66-3.56(m,1H),3.43(s,3H),2.65-2.57(m,1H),2.44-2.34(m,2H),2.18-2.00(m,1H),1.95-1.74(m,2H),1.68-1.34(m,2H),1.15-1.02(m,2H),0.93-0.83(m,2H),0.81-0.62(m,6H).

Compound 26 (1.0 mg,3% yield) LCMS M/z 543.3 (M+H) rt 2.20min LCMS method column XBridge BEH XP C18 (50X 2.1) mM,2.5 μm mobile phase A95% water 5% acetonitrile 10mM ammonium acetate mobile phase B5% water 95% acetonitrile 10mM ammonium acetate flow rate 1.1mL/min temperature 50 ℃ time (min):0-3;％B:0-100％).¹H NMR(400MHz,DMSO-d₆)δppm 8.23(d,J＝8.8Hz,1H),8.06-7.91(m,1H),7.62(dd,J＝6.2,7.5Hz,2H),7.26(t,J＝8.8Hz,2H),5.92-5.31(m,1H),5.29(s,1H),4.96-4.78(m,1H),3.60-3.50(m,1H),3.43(s,3H),3.25-3.10(m,1H),2.97-2.75(m,2H),2.27-1.65(m,3H),1.49-1.24(m,2H),1.11-0.97(m,2H),0.94-0.75(m,5H),0.74-0.50(m,3H).

DGKi Compounds 27 and 28

4- ((2 S,5 r) -4- ((4-fluorophenyl) (5- (trifluoromethyl) pyridin-2-yl) methyl) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile

To a stirred solution of 4- ((2 s,5 r) -2, 5-dimethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile (1 g,3.35 mmol) in acetonitrile (10 mL) was added DIPEA (5.9 mL,33.5 mmol) followed by 2- (bromo (4-fluorophenyl) methyl) -5- (trifluoromethyl) pyridine (2.24 g,6.70 mmol). The reaction mixture was heated at 80 ℃ for 4h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure to obtain a crude product, which was purified by preparative HPLC (HPLC method: column: sunfire C18,150x19mm ID,5 μm; mobile phase A:0.1% TFA/water; mobile phase B: acetonitrile: meOH (1:1); gradient: 50-100% B over 20min followed by 100% B5 min; flow rate: 19 mL/min), the fractions were concentrated under reduced pressure and lyophilized from (EtOH/H₂ O, 1:5) to obtain compound 27 and compound 28.

Compound 27:110mg,6% yield, LCMS: M/z= 552.2 (M+H); rt 3.09min; (LCMS method: column-Kinetex XB-C18 (75X 3mM-2.6 μm), mobile phase A:98% water: 2% acetonitrile, 10mM ammonium formate, mobile phase B:2% water: 98% acetonitrile, 10mM ammonium formate, flow rate: 1.0mL/min, temperature: 50 ℃ C., time (min):0-4;％B:20-100％).¹H NMR(400MHz,DMSO-d₆)δppm 8.83(s,1H),8.22(d,J＝9.0Hz,2H),8.11-7.95(m,2H),7.71-7.58(m,2H),7.25-7.13(m,2H),5.76-5.44(m,1H),5.13-4.67(m,2H),3.86-3.49(m,1H),3.44(s,3H),3.19-3.08(m,1H),2.84(dd,J＝3.8,12.3Hz,1H),2.38-2.26(m,1H),1.67-1.39(m,3H),1.11-0.86(m,3H).

Compound 28:145mg,8% yield, LCMS: M/z= 552.2 (M+H); rt 3.09min; (LCMS method: column-Kinetex XB-C18 (75X 3mM-2.6 μm), mobile phase A:98% water: 2% acetonitrile, 10mM ammonium formate, mobile phase B:2% water: 98% acetonitrile, 10mM ammonium formate, flow rate: 1.0mL/min, temperature: 50 ℃, time (min):0-4;％B:0-100％).¹H NMR(400MHz,DMSO-d₆)δppm 8.91(s,1H),8.27-8.16(m,2H),7.99(d,J＝9.0Hz,2H),7.69-7.57(m,2H),7.23-7.13(m,2H),5.77-5.41(m,1H),5.09-4.62(m,2H),3.90-3.65(m,1H),3.44(s,3H),3.14-3.02(m,1H),2.80-2.74(m,1H),1.61-1.40(m,3H),1.10-0.93(m,3H)[1H masked by solvent peaks ].

DGKi Compounds 29 and 30

4- ((2 S,5 r) -4- (1- (4- (cyclopropylmethoxy) -2-fluorophenyl) propyl) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile

To a stirred solution of 4- ((2 s,5 r) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, HCl (200 mg,0.55 mmol) in acetonitrile (5 mL) was added DIPEA (0.3 mL,1.65 mmol), sodium iodide (83 mg,0.55 mmol) and 1- (1-chloropropyl) -4- (cyclopropylmethoxy) -2-fluorobenzene (268 mg,1.1 mmol). The reaction mixture was heated at 80 ℃ for 16h. The reaction mixture was cooled to room temperature. Another batch of 1- (1-chloropropyl) -4- (cyclopropylmethoxy) -2-fluorobenzene (268 mg,1.102 mmol) was added and heating continued for an additional 16h. The reaction mixture was cooled, the solvent removed under reduced pressure and the residue dissolved in ethyl acetate (10×20 mL). The organic layer was washed with brine, dried over Na₂SO₄, concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC. HPLC method of EXRS (20X 250mM,5 μm), mobile phase A-10mM ammonium acetate/water R, mobile phase A-B acetonitrile, flow rate 20mL/min.

Fraction 1 was concentrated under reduced pressure and the product was diluted with (EtOH/H₂ O, 1:5) and lyophilized to give compound 29 (35 mg,11.6% yield); LCMS: m/z 533.4[ M+H ]⁺, rt 1.57min; (LCMS method: column: KINETIX XB C (75X 3mM,2.6 μm); mobile phase A:10mM ammonium acetate/water (pH 3.3), mobile phase B: acetonitrile) .¹HNMR(DMSO-d₆,400MHz)δ(ppm)8.23(d,J＝9.0Hz,1H),7.97(d,J＝9.0Hz,1H),7.33(m,1H),6.62-6.92(m,2H),5.29-6.06(m,1H),4.70-5.05(m,1H),3.82(m,3H),3.43(s,3H),2.99-3.10(m,1H),2.80-2.87(m,1H),2.63-2.78(m,1H),2.33(s,1H),1.74-2.11(m,3H),1.51-1.66(m,1H),1.17-1.46(m,3H),0.84-1.01(m,3H),0.61-0.78(m,6H),0.53-0.61(m,2H),0.29-0.35(m,2H).

Fraction 2 was concentrated under reduced pressure and the product was diluted with (EtOH/H₂ O, 1:5) and lyophilized to give compound 30 (37 mg,12.35% yield); LCMS: m/z 533.4[ M+H ]⁺, rt 2.72min; (LCMS method: column: KINETIX XB C (75 x3mM,2.6 μm); mobile phase A:10mM ammonium acetate/water (pH 3.3), mobile phase B: acetonitrile) .¹HNMR(DMSO-d₆,400MHz):δ(ppm)8.13-8.35(m,1H),7.98(m,1H),7.38(m,1H),6.61-6.89(m,2H),5.18-6.15(m,1H),4.66-5.13(m,1H),3.63-3.90(m,3H),3.43(s,3H),3.25(m,1H),3.00-3.15(m,1H),2.63-2.70(m,1H),2.26-2.38(m,1H),1.81(m,3H),1.35-1.61(m,2H),1.15-1.26(m,2H),0.88-1.00(m,3H),0.61-0.71(m,6H),0.51-0.59(m,2H),0.32(m,2H).

DGKi Compounds 31 and 32

4- ((2 S,5 r) -2, 5-diethyl-4- (1- (4- (trifluoromethyl) phenyl) butyl) piperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile

To a stirred solution of 4- ((2 s,5 r) -2, 5-diethylpiperazin-1-yl) -1-methyl-2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile, HCl (0.4 g,1.1 mmol) in acetonitrile (10 mL) was added DIPEA (0.6 mL,3.31 mmol) followed by 1- (1-chlorobutyl) -4-trifluoromethyl) benzene (0.783 g,3.31 mmol) and sodium iodide (0.165 g,1.102 mmol). The reaction mixture was heated at 85 ℃ for 16h. The reaction mixture was filtered through a celite pad, washed with ethyl acetate and the filtrate was concentrated under reduced pressure to give the crude compound which was purified by preparative HPLC [ HPLC method: column YMC ExRS (250 mM x21.2mM,5 μm) mobile phase a=10 mM ammonium acetate/water pH 4.5. Mobile phase B = acetonitrile gradient 80% B over 2 minutes followed by 100% B for 16 minutes; flow rate: 19 mL/min) to obtain compounds 31 and 32.

Compound 31 (10 mg,1.7% yield), LCMS: M/z= 527.4 (M+H); rt 2.626min; [ LCMS method: column: xbridge BEH XP C18 (50×2.1 mM), 2.5 μm; mobile phase A:95% water: 5% acetonitrile; 10mM NH₄ OAC; mobile phase B:5% water: 95% acetonitrile; 10mM NH₄ OAC; flow rate: 1.1mL/min; temperature: 50 ℃ C. Time) (min)].¹H NMR(400MHz,DMSO-d₆)δ8.30-8.16(m,1H),7.98(d,J＝9.0Hz,1H),7.72(d,J＝8.3Hz,2H),7.56(br d,J＝7.8Hz,2H),5.86-5.44(m,1H),5.01-4.77(m,1H),3.730-3.718(m,1H),3.46(s,3H),3.43-3.35(m,1H)3.13-3.01(m,1H),2.93-2.75(m,2H),2.38-2.26(m,1H),2.17-1.74(m,3H),1.63-1.22(m,3H),1.01-0.86(m,4H),0.84-0.75(m,3H),0.73-0.54(m,3H).

Compound 32 (7.2 mg,1.23% yield), LCMS: M/z= 527.3 (M+H); rt 2.654min; [ LCMS method: column: XBRID BEH XP C18 (50×2.1) mM,2.5 μm; mobile phase A:95% water: 5% acetonitrile; 10mM NH₄ OAC; mobile phase B:5% water: 95% acetonitrile; 10mM NH4OAC; flow rate: 1.1mL/min; temperature: 50 ℃ C; time) (min)].¹H NMR(400MHz,DMSO-d₆)δ＝8.29-8.15(m,1H),7.96-8.02(m,1H),7.70(d,J＝8.1Hz,2H),7.58(br d,J＝8.1Hz,2H),6.09-5.22(m,1H),5.13-4.66(m,1H),3.68-3.52(m,2H),3.43(s,3H),3.28-3.04(m,2H),2.60-2.53(m,1H),2.25-2.12(m,1H),2.04-1.68(m,3H),1.60-1.29(m,3H),1.05-0.74(m,7H),0.59(t,J＝7.5Hz,3H).

DGKi Compounds 33 and 34

1-Methyl-4- ((2 s,5 r) -2-methyl-5-propyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) -2-oxo-1, 2-dihydropyrido [3,2-d ] pyrimidine-6-carbonitrile

To a solution of 6-chloro-1-methyl-4- ((2 s,5 r) -2-methyl-5-propyl-4- (1- (4- (trifluoromethyl) phenyl) ethyl) piperazin-1-yl) pyrido [3,2-d ] pyrimidin-2 (1H) -one (0.1 g,0.19 mmol) in DMF (2 mL) was added zinc cyanide (0.046 g,0.39 mmol), zinc (0.7 mg,9.8 μmol) and triethylamine (0.1 mL,0.59 mmol) under an argon atmosphere followed by dichloro [9, 9-dimethyl-4, 5-bis (diphenylphosphino) xanthen ] palladium (II) (0.015 g,0.02 mmol). The reaction mixture was heated at 90 ℃ overnight. The reaction mixture was diluted with EtOAc (50 mL) and passed throughThe pad was filtered and washed with additional ethyl acetate (2×50 mL). The filtrate was washed with water (50 mL), brine, dried over Na₂SO₄ and concentrated under reduced pressure to give a crude product, which was purified by preparative HPLC (HPLC method: column: YMC EXRS (250X 19mM,5 μm); mobile phase A:10mM ammonium acetate/water pH 4.5; mobile phase B: acetonitrile flow rate: 20 mL/min) to give compound 33 and compound 34.

Compound 33 (13 mg,14% yield). LCMS: m/z=499.3 [ M+H ]⁺; rt 2.376min (LCMS method: column: XB ridge BEH XP C18 (50×2.1mM,2.5 μm), mobile phase A:95% water: 5% acetonitrile, 10mM NH₄ OAc, mobile phase B:5% water:95% acetonitrile, 10mM NH₄ OAc, flow rate: 1.1mL/min, temperature) :50℃).¹H NMR(400MHz,DMSO-d₆)δ(ppm)＝8.22(br d,J＝8.8Hz,1H),7.98(d,J＝8.8Hz,1H),7.70-7.72(m,2H),7.59-7.61(m,2H),5.84-5.59(m,1H),5.10-4.67(m,1H),3.91-3.75(m,1H),3.38-3.43(m,4H),2.86-2.70(m,2H),2.47-2.36(m,1H),1.63-1.51(m,1H),1.47-1.18(m,8H),0.9-0.99(m,1H),0.75-0.59(m,3H).

Compound 34 (13 mg,13% yield), LCMS: m/z=499.3 [ M+H ]⁺, rt 2.436min, (LCMS method: column: XBRID BEH XP C18 (50×2.1mM,2.5 μm), mobile phase A:95% water: 5% acetonitrile, 10mM NH₄ OAc, mobile phase B:5% water: 95% acetonitrile, 10mM NH₄ OAc, flow rate: 1.1mL/min, temperature :50℃).¹H NMR(400MHz,DMSO-d₆)δ(ppm)＝8.25(br d,J＝2.4Hz,1H),8.06-7.92(m,1H),7.77-7.65(m,2H),7.65-7.54(m,2H),6.09-5.44(m,1H),5.04-4.68(m,1H),3.81-3.59(m,2H),3.44(s,3H),3.28-3.13(m,1H),2.52-2.61(m,1H),2.24-2.05(m,1H),1.72-1.48(m,2H),1.47-1.15(m,8H),0.98-0.75(m,3H).

Biological assays

The pharmacological properties of the compounds described herein may be demonstrated by a number of bioassays.

1. In vitro DGK inhibitor assay

The dgkα and dgkζ reactions were performed using extruded liposomes (dgkα and dgkζ LIPGLO assays) or detergent/lipid micelle substrates (dgkα and dgkζ assays). The reaction was performed in 50mM MOPS pH 7.5, 100mM NaCl, 10mM MgCl₂、1μM CaCl₂ and 1mM DTT (assay buffer). The reaction using the detergent/lipid micelle substrate also contained 50mM octyl B-D-glucopyranoside. The lipid substrate concentrations used for the detergent/lipid micelle substrate reaction were 11mM PS and 1mM DAG. The lipid substrate concentrations used for the extrusion liposome reaction were 2mM PS, 0.25mM DAG and 2.75mM PC. The reaction was performed in 150. Mu.M ATP. The enzyme concentrations of DGK alpha and DGK zeta were 5nM.

Compound inhibition studies were performed by transferring 50nL drops of each test compound (maximum concentration 10mM, 11 spots per compound, 3-fold dilution series) dissolved in DMSO into wells of a white 1536 well plate (Corning 3725). An enzyme/substrate solution with a final reaction concentration of 2x was prepared by mixing 2.5ml of 4x enzyme solution (20 nM dgkα or dgkζ (prepared as described below) in assay buffer) and 2.5ml of 4x liposome or 4x detergent/lipid micelle solution (composition described below) and incubated at room temperature for 10 minutes. Next, 1. Mu.L of 2 Xenzyme/substrate solution was added to the wells containing the test compound, and 1. Mu.L of 300uM ATP was added to start the reaction. The reaction was allowed to proceed for 1 hour, after which 2. Mu.L of Glo Reagent (Promega V9101) was added and incubated for 40 minutes. Subsequently, 4. Mu.L of kinase assay reagent was added and incubated for 30 minutes. Fluorescence was recorded using an EnVision microplate reader. Percent inhibition was calculated by ATP conversion generated by the no enzyme control reaction (100% inhibition) and the vector only reaction (0% inhibition). The 11 concentrations of compounds were evaluated to determine IC₅₀.

Preparation of 4x detergent/lipid micelles

Detergent/lipid micelles were prepared by mixing 15g phosphatidylserine (Avanti 840035P) and 1g diacylglycerol (800811O) in a 2L round bottom flask and dissolving in 150mL chloroform. Chloroform was removed by rotary evaporation under high vacuum. The resulting colorless gum was resuspended in 400mL 50mM MOPS pH 7.5, 100mM NaCl, 20mM NaF, 10mM MgCl₂、1μM CaCl₂, 1mM DTT and 200mM octyl glucoside by thorough mixing. The detergent/lipid micelle solution was split into 5mL aliquots and stored at-80 ℃.

Preparation of 4x liposomes

The lipid composition for the 4x liposome solution was 5mol% dag (Avanti 800811O), 40mol% ps (Avanti 840035P) and 55mol% pc (Avanti 850457), with a total lipid concentration of 15.2mg/mL. PC, DAG and PS were dissolved in chloroform, mixed and dried in vacuo to a thin film. The lipids were hydrated to 20mM at 50mM MOPS pH 7.5, 100mM NaCl, 5mM MgCl₂ and freeze-thawed five times. The lipid suspension was extruded 11 times through a 100nm polycarbonate filter. Dynamic light scattering was performed to confirm liposome size (50-60 nm radius). Liposome formulations can be stored up to four weeks at 4 ℃.

Baculovirus expression of human DGK alpha and DGK zeta

Human DGK-alpha-TVMV-His-pFBgate and human DGK-zeta-transcriptional variant-2-TVMV-His-pFBgate baculovirus samples were generated using the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer's protocol. The DNA used to express DGK-alpha and DGK-zeta have SEQ ID NOs 251 and 252, respectively. Baculovirus expansion was achieved using Sf9 cells infected at a 1:1500 virus/cell ratio and grown at 27 ℃ for 65 hours after transfection.

Scale-up expression of each protein was performed in Cellbag 50LWAVE-Bioreactor systems 20/50 from GE HEALTHCARE Bioscience. 12L 2X 10⁶ cells/mL Sf9 cells (expression system, davis, calif.) grown in ESF921 insect medium (expression system) were infected with the viral stock at a 1:200 virus/cell ratio and grown at 27℃for 66-68 hours after infection. Passing the infected cell culture throughThe harvest was centrifuged at 2000rpm 4℃for 20min in an RC12BP centrifuge. The cell pellet was stored at-70 ℃ until purification.

Purification of human DGK-alpha and DGK-zeta

The full-length human dgkα and dgkζ, each expressed containing a TVMV-cleavable C-terminal Hexa-His tag sequence (SEQ ID NOs 190 and 191, respectively) and produced as described above, were purified from Sf9 baculovirus-infected insect cell bodies (paste). Cells were lysed by nitrogen cavitation using nitrogen bullets (Parr Instruments) and the lysates were clarified by centrifugation. Three are usedThe clarified lysate was purified to 90% uniformity by continuous column chromatography steps on a Purifier Plus system. Three column chromatography steps included nickel affinity resin capture (i.e., HISTRAP FF chromatography, GE HEALTHCARE) followed by size exclusion chromatography (i.e., hiLoad 26/600Superdex200 preparation grade, GE HEALTHCARE for DGK-. Alpha., and HiPrep 26/600Sephacryl S300_HR,GE Healthcare for DGK-. Zeta.). The third step is ion exchange chromatography, the two subtypes being different in method. DGK alpha was purified using Q-Sepharose anion exchange chromatography (GE HEALTHCARE). Dgkζ was purified using SP Sepharose cation exchange chromatography (GE HEALTHCARE). The protein is delivered at a concentration of ≡2 mg/mL. The formulated buffers for both proteins were identical, 50mM Hepes, pH 7.2, 500mM NaCl, 10% v/v glycerol, 1mM TCEP and 0.5mM EDTA.

Raji CD4T cell IL2 assay

1536-Well IL-2 assays were performed in a 4. Mu.L volume using pre-activated CD 4T cells and Raji cells. Prior to assay, CD 4T cells were pre-activated by using 1.5. Mu.g/mL, 1. Mu.g/mL and 10. Mu.g/mL of α -CD3, α -CD28 and PHA, respectively. Raji cells were treated with 10,000ng/mL Staphylococcal Enterotoxin B (SEB). Serial dilutions of compounds were first transferred to 1536-well assay plates (Corning, # 3727) and then 2 μl of pre-activated CD 4T cells (final concentration 6000 cells/well) and 2 μl of SEB-treated Raji cells (2000 cells/well) were added. After 24 hours incubation in a 37℃/5% CO₂ incubator, 4. Mu.l of IL-2 detection reagent (Cisbio, #64IL2 PEC) was added to the assay plates. The assay plate was read on an Envision plate reader. To assess compound cytotoxicity, raji or CD 4T cells were incubated with serially diluted compounds. After 24 hours incubation, 4 μ L CELL TITER Glo (Promega, #g7572) was added and the assay plates were read on an Envision plate reader. The four parameter logarithmic formulA y=a+ ((B-A)/(1+ ((C/x)/(D))) was used to calculate the 50% effective concentration (IC₅₀), where A and B represent minimum and maximum activation or inhibition, respectively, C is IC₅₀, D is hill slope, and x represents compound concentration.

CellTiter-Glo CD 8T cell proliferation assay

Frozen naive human CD 8T cells were thawed in rpmi+10% fbs, incubated at 37 ℃ for 2h and counted. 384-well tissue culture plates were coated with 20 μl of anti-human CD3 at 0.1 μg/mL overnight at 4 ℃ in normal RPMI, removed from the plates, and then 20k/40 μl CD 8T cells were added to each well with 0.5 μg/mL soluble anti-human CD 28. After cell attachment, the compounds were repeatedly added to the cell plates. After 72h incubation in a 37℃incubator, 10. Mu. L CELLTITER-glo reagent (Promega catalog number G7570) was added to each well. Plates were vigorously shaken for 5min, incubated at room temperature for an additional 15min and CD 8T cell proliferation read at Envision. In the analysis, 0.1 μg/mL anti-CD 3 and 0.5 μg/mL anti-CD 28 stimulated CD 8T cell signal was used as background. 3. Mu.M of the reference compound, 8- (4- (bis (4-fluorophenyl) methyl) piperazin-1-yl) -5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile, was used to set the 100% range, and EC₅₀ was absolute 50% to normalize the data.

DGK AP1-reporter assay

Jurkat AP 1-fluorescence reporter was generated using the CIGNAL LENTI AP reporter (luc) kit from SABiosciences (CLS-011L).

Compounds were transferred from Echo LDV plates using an Echo550 instrument into individual wells of 384-well plates (white, solid bottom, opaque PE CulturPlate 6007768). The sample size was 30nL per well, and one target plate per source plate. Cell suspensions were prepared by transferring 40mL cells (2 x20 mL) into a clean 50mL conical tube. The cells were concentrated by centrifugation (1200 rpm;5min; ambient temperature). The supernatant was removed and all cells were suspended in RPMI (Gibco 11875) +10% fbs to a concentration of 1.35×10⁶ cells/ml. Cells were added manually using a multichannel pipette, and 30 μl/well of cell suspension was added to 384-well TC plates containing the compound, 4.0x10⁴ cells per well. Cell plates were incubated at 37 ℃ and 5% CO₂ for 20 min.

During incubation, an anti-CD 3 antibody (αcd3) solution was prepared by mixing 3 μl of aCD3 (1.3 mg/mL) with 10mL of medium [ final concentration = 0.4 μg/mL ]. Next, 1.5. Mu.l of aCD3 (1.3 mg/mL) was mixed with 0.5mL of the medium [ final concentration=4. Mu.g/mL ]. After 20 minutes, 10 μl of medium was added to each of column 1, a through M wells, and 10 μl of αcd3 (4 ug/mL) was added to each of column 1, rows N through P as a control. Then 10 μl αcd3 (0.4 ug/mL) was added to each well using a multichannel pipette. αcd3 stimulated +/-compound treated cells were incubated in 5% co₂ for 6 hours at 37 ℃.

During incubation, the Steady-Glo (Promega E2520) reagent was slowly thawed to ambient temperature. Next, 20. Mu.L of Steady-Glo reagent was added to each well using a multi-point Combi-dispenser. The air bubbles were removed by centrifugation (2000 rpm, ambient temperature, 10 seconds). Cells were incubated at ambient temperature for 5 min. Samples were characterized by fluorescence protocol using Envision plate reader instrument to measure Relative Light Units (RLU). Data were analyzed using the reference compound 8- (4- (bis (4-fluorophenyl) methyl) piperazin-1-yl) -5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile to normalize 100% inhibition.

5. Murine cytotoxic T lymphocyte assay

Antigen-specific cytolytic T-Cell (CTL) assays were developed to functionally evaluate the ability of dgkα and dgkζ inhibitors to enhance T cell mediated tumor killing activity. CD8+ T cells isolated from OT-1 transgenic mice recognize antigen presenting cell MC38, which presents the ovalbumin derived peptide SIINFEKL. Recognition of the cognate antigen initiates cytolytic activity of OT-1 antigen specific cd8+ T cells.

Functionalized CTL cells were generated by isolating OT-1 spleen cells from 8-12 week old mice and amplifying in the presence of SIINFEKL peptide at 1. Mu.g/mL and mIL2 at 10U/mL. After 3 days, fresh medium containing mIL 2U/ml was added. On day 5 of expansion, cd8+ T cells were isolated and ready for use. Activated CTL cells can be stored frozen for 6 months. 100 ten thousand MC38 tumor cells were pulsed with 1 μg/ML SIINFEKL-OVA peptide at 37℃for 3 hours, respectively. Cells were washed (3X) with fresh medium to remove excess peptide. Finally, CTL cells pretreated with DGK inhibitor for 1 hour were mixed with antigen-loaded MC38 tumor cells at a 1:10 ratio in 96-well U-bottom plates. The cells were then spun at 700rpm for 5min and placed in a 37 ℃ incubator overnight. After 24 hours, the supernatant was collected for analysis of IFN-gamma cytokine levels by alpha Lisa from PERKIN ELMER.

PHA proliferation assay

Phytohemagglutinin (PHA) -stimulated blasts from the frozen stock solution were incubated in RPMI medium (Gibco, thermoFisher Scientific, waltham, MA) supplemented with 10% fetal bovine serum (SIGMA ALDRICH, ST.LOUIS, MO) for 1 hour and then added to individual wells of 384-well plates (10,000 cells per well). Compounds were transferred to individual wells of 384-well plates and treated cells were maintained at 37℃in medium containing human IL2 (20 ng/mL) at 5% CO₂ for 72H, and growth was measured using the MTS reagent [3- (4, 5-dimethyl-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazole ] according to the manufacturer's instructions (Promega, madison, wis.). Percent inhibition was calculated by comparing the values between IL2 stimulated (0% inhibition) and unstimulated control (100% inhibition). The determination of the inhibition concentration (IC₅₀) was calculated based on 50% inhibition of the doubling rate between the IL2 stimulated and unstimulated treatments.

7. Human CD 8T cell IFN-gamma assay

Frozen naive human CD 8T cells were thawed in AIM-V medium, incubated at 37 ℃ for 2h and counted. 384-well tissue culture plates were coated with 20 μl of anti-human CD3 (0.05 μg/mL in PBS) overnight at 4 ℃ and after removal from the plates 40000 cells per 40 μl CD 8T cells containing 0.1 μg/mL soluble anti-human CD28 were added to each well. Immediately after cell attachment, compounds were transferred to the cell plates using an Echo liquid processor. After 20h incubation in a 37 ℃ incubator, 3 microliters of supernatant per well was transferred to a new 384 well white assay plate for cytokine measurement.

Interferon-gamma (IFN-gamma) was quantified using the AlphLISA kit (Cat#AL 217) as described in the manufacturer's manual (PERKIN ELMER). Counts from each well were converted to IFN-gamma concentration (pg/mL). Compound EC₅₀ values were determined by setting 0.05 μg/mL anti-CD 3 plus 0.1 μg/mL anti-CD 28 as baseline, and 3 μm co-stimulation of reference compound 8- (4- (bis (4-fluorophenyl) methyl) piperazin-1-yl) -5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile with anti-CD 3 plus anti-CD 28 as 100% activation.

8. Human CD 8T cell pERK assay

Frozen naive human CD 8T cells were thawed in AIM-V medium, incubated at 37 ℃ for 2h and counted. CD8 positive T cells were added to AIM-V medium in 384-well tissue culture plates at 20,000 cells per well. One compound was added per well, followed by the addition of bead-bound anti-human CD3 and anti-CD 28 mAb at a final concentration of 0.3 μg/mL. Cells were incubated at 37 ℃ for 10 min. The reaction was stopped by adding lysis buffer from ALPHALISA SUREFIRE kit (PERKIN ELMER, cat# ALSU-PERK-A). Lysates (5 μl per well) were transferred to new 384-well white assay plates for pERK activation measurement.

Compound EC₅₀ was determined by setting anti-CD 3 plus anti-CD 28 as baseline and 3 μm co-stimulation of 8- (4- (bis (4-fluorophenyl) methyl) piperazin-1-yl) -5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile with anti-CD 3 plus anti-CD 28 as 100% activation.

9. Human whole blood IFN-gamma assay

Human venous whole blood (22.5 μl per well) obtained from healthy donors was pre-treated with the compound for 1 hour at 37 ℃ in humidified 95% air/5% CO₂ incubator. Blood was stimulated with 2.5 μl of each of the anti-human CD3 and anti-CD 28 mabs at a final concentration of 1 μg/mL for 24 hours at 37 ℃. IFN-. Gamma.in the supernatant was measured using AlphLISA kit (Cat#AL 217).

Compound EC₅₀ was determined by setting anti-CD 3 plus anti-CD 28 as baseline and 3 μm co-stimulation of the reference compound 8- (4- (bis (4-fluorophenyl) methyl) piperazin-1-yl) -5-methyl-7-nitro-6-oxo-5, 6-dihydro-1, 5-naphthyridine-2-carbonitrile with anti-CD 3 plus anti-CD 28 as 100% activation.

Table a lists the in vitro DGK inhibition IC₅₀ activity values measured in dgkα and dgkζ liposome (LIPGLO) assays.

The compounds described herein have activity as inhibitors of one or both of dgkα and dgkζ enzymes and are therefore useful for treating diseases associated with inhibition of dgkα and dgkζ activity.

Example 2 Effect of Compound 17 on CAR-T cell function

T cell function of T cells engineered with chimeric antigen receptor (CAR-T cells) treated with exemplary DGKi (compound 17) was evaluated.

T cell compositions containing T cells expressing the exemplary anti-CD 19 CAR were generated from leukapheresis samples from two healthy adult donors, by a method comprising immune affinity based selection of T cells, by selecting cd4+ and cd8+ cells from the samples, respectively, resulting in each donor producing two compositions enriched in cd4+ and cd8+ cells, respectively. Cells of the cd4+ and cd8+ enriched compositions were activated with anti-CD 3/anti-CD 28 magnetic beads and lentivirally transduced with vectors encoding anti-CD 19 CAR, respectively. The anti-CD 19 CAR contains an anti-CD 19 single chain variable fragment (scFv), derived from a murine antibody (the variable region is derived from FMC 63), an immunoglobulin-derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and a CD 3-zeta intracellular signaling domain. The expression construct in the viral vector also contains a sequence encoding a surrogate surface marker of CAR expression, which is separated from the CAR coding sequence by a T2A ribosome jump sequence. The transduced cd4+ or cd8+ T cell populations are then individually cultured in the presence of a stimulating agent for cell expansion. Expanded cd4+ and cd8+ cells were formulated and cryopreserved and stored separately.

Prior to use, the cryopreserved cd4+ and cd8+ CAR engineered cell compositions were thawed and combined at approximately a 1:1CD4:cd8 ratio per donor to prepare combined cd4+ car+ and cd8+ car+ T cell compositions. The therapeutic effect of compound 17 added at various concentrations (0.1 nM,1nM,10nM,100nM or 1000 nM) during stimulation of the combined CAR-T cell composition with plate-binding CAR-specific anti-idiotype antibodies (see, e.g., WO 2018/023900) or during co-culture with cells expressing CD19 was then evaluated on day 0.

A.T cell phenotype

Approximately 20,000 cells of the combined CAR-T cell composition were stimulated with 3 μg/ml or 30 μg/ml plate-bound CAR-specific anti-idiotype antibodies in the presence of compound 17. After 48 hours of stimulation, the T cell surface was stained with antibodies to various activation and isolation markers and analyzed by flow cytometry for the percentage of cells positive for the marker being evaluated (% positive) and the Mean Fluorescence Intensity (MFI) of expression of the marker being evaluated. The mean log₂ fold change in surface marker expression of% positive cells or MFI from two healthy donors compared to expression without treatment with compound 17 was determined.

Figure 1A shows the mean of log₂ fold changes in% positive cells of various activation and differentiation markers in the donor (presence of compound 17 vs. absence) after stimulation with 3 μg/mL (left panel) or 30 μg/mL (right panel) CAR-specific anti-idiotype antibodies. Figure 1B shows the mean of log₂ fold changes in MFI of cells in donors (presence vs. absence of compound 17) after stimulation with 3 μg/mL (left panel) or 30 μg/mL (right panel) CAR-specific anti-idiotype antibody. In fig. 1A and 1B, positive values are indicated by circles. As shown in fig. 1A and 1B, CAR expressing cells showed increased CD69 and PD-1 expression and decreased CD27 and CD62L expression in the presence of compound 17, consistent with the activating effect phenotype of more cells after stimulation in the presence of compound 17. This effect on cell phenotype is dose dependent, particularly for high concentration CAR-specific anti-idiotype antibody (30 μg/mL) -stimulated cells.

B. Proliferation

T cell proliferation during stimulation in the presence of compound 17 was monitored for 7 days with a 30 μg/mL plate-conjugated anti-CAR anti-idiotype antibody using an Incucyte imaging system. Cell proliferation was monitored by analyzing the area of the cell image taken over time (% fusion) with increasing fusion as the cells proliferate.

Figure 2 shows the growth curve (percent cell fusion in wells) of cells in the presence of compound 17 or stimulated by control. Values of triplicate of cell compositions prepared from two different healthy donors are shown. The results show that the presence of compound 17 during stimulation does not affect T cell proliferation.

C. Cell lysis function

After co-culturing the combined CAR-T cell composition with various CD19 expressing lymphoma cells at a 2.5:1 effector to target cell ratio, the cytolytic function of T cells treated with compound 17 was evaluated in the presence of compound 17. Target lymphoma cells were labeled with NucLight Red (NLR) to allow for their tracking by microscopy. Cytolytic activity is assessed by measuring loss of viable target cells over a period of 4 to 7 days, as determined by red fluorescent signal (useLiving cell analysis system, essen Bioscience).

FIG. 3 shows the percentage of effect of CAR expressing cells on specific lysis in the presence of compound 17 (or control) when co-cultured with CD19 antigen expressing target cells, K562.CD19 cells (high CD19 expression; left panel), granta-519 cells (low CD19 expression; middle panel) and Raji cells (middle/high CD19 expression; right panel). Values of triplicate of cell compositions prepared from two different healthy donors are shown. The results show that the presence of compound 17 does not affect the cytolytic function during co-cultivation.

D. Cytokine production

Supernatants were collected after 48 hours of the co-culture described above of CAR expressing T cells and CD19 expressing tumor cells in the presence of compound 17. Cytokine production was measured from the supernatant by Meso Scale Discovery (MSD) analysis of the collected cell supernatant.

FIG. 4 shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNFα (right panel) after co-culture with K562.CD19 cells (circles), granta-519 cells (squares) and Raji cells (triangles) in the presence of compound 17. The results show that the presence of compound 17 during co-culture resulted in a dose-dependent increase in production of all three cytokines, with high concentrations of compound 17 resulting in the greatest effect during co-culture with Granta-519 cells (low CD19 expression), in particular IL-2 production.

E. summary

Taken together, these results show that treatment with compound 17 increases the cytokine production of CAR T cells and the number of cd4+ and cd8+ CAR expressing cells with an activating effect phenotype without affecting CAR T cell proliferation or cytolytic function. Furthermore, no effect of compound 17 on T cell viability was observed in these assays. These results are consistent with the observation that the presence of compound 17 enhances CAR-T cell activation under acute stimulation conditions.

Example 3 evaluation of chronically stimulated CAR-T cells simultaneously treated with Compound 17

Compound 17 was evaluated for its ability to prevent or reduce depletion of long-term stimulated CAR T cells.

To mimic the conditions of long term stimulation, approximately 20,000 anti-CD 19 CAR expressing T cells of the combined CAR-T cell composition produced from two healthy adult donors as described in example 2 were incubated with 30 μg/mL plate-binding CAR-specific anti-idiotype antibodies (see, e.g., WO 2018/02100) for a period of 7 days at 37 ℃. During this 7 day period, engineered CAR T cells were treated with compound 17 (0.1-1000 nM) simultaneously or vehicle controls were added on day 0. Similar to the results observed in example 2, following acute stimulation, simultaneous incubation of T cells with compound 17 during long-term stimulation increased the highly activated CAR-T cell population, in particular the population of cells highly expressing CD69 and PD-1, in the stimulated cells.

The stimulated CAR T cells were then re-challenged with CD19 expressing tumor cells or tumor spheres in the absence of compound 17 and evaluated for cytolytic activity and cytokine production.

A.2-dimensional CD19+ tumors

To assess cytolytic function, the stimulated CAR-T cell composition was co-cultured with various CD19 expressing lymphoma cells at a 2.5:1 effector to target cell ratio. Engineering tumor cells to express NucLight Red (NLR) and byThe number of tumor cells was monitored. Figure 5A shows the number of tumor cells during 2D co-culture with CAR T cells that have been stimulated for a long period of time in the presence of compound 17 simultaneous treatment, k562.Cd19 (left panel), granta-519 (middle panel) and Raji cells (right panel). Duplicate values of cell compositions prepared from two different healthy donors are shown. As shown, incubation with DGKi reduced the cytolytic activity of CAR-T cells on CD19 expressing lymphoma cells in a dose-dependent manner during long-term stimulation of T cells. In particular, treatment with high concentration of compound 17 during long term stimulation reduced the cytolytic activity of CAR T cells.

After 48 hours of co-culture of the long-term stimulated CAR-expressing T cells and CD 19-expressing tumor cells described above, the supernatant was collected. Cytokine production was measured from the supernatant by Meso Scalediscovery (MSD) analysis of the collected cell supernatant. FIG. 5B shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNFα (right panel) after 2D co-cultivation with K562.CD19 cells (circles), granta-519 cells (squares) and Raji cells (triangles) for 48 hours. Duplicate values of cell compositions prepared from two different healthy donors are shown. The results indicate that treatment with compound 17 during long-term stimulation resulted in a dose-dependent increase in production of all three cytokines.

B. three-dimensional spherical CD19+ tumor

To further evaluate the activity of the chronically stimulated cells, chronically stimulated CAR-T cells that had been treated with DGKi simultaneously were re-challenged with either a549 cd19+ tumor spheres or Granta-519 tumor spheres at an effector to target cell ratio of 1:2 for up to 14 days. Tumor spheres were engineered to express red fluorescent dye to allow tumor cell lysis to be monitored by microscopy during the assay.

Antigen-specific cytolytic activity of long-term stimulated CAR-expressing T cells is assessed by monitoring fluorescence over time (indicative of tumor cytolysis). Tumor volume (normalized to time 0) was measured in Red Calibration Units (RCU) by the number of μm² areas. Figure 6A shows normalized tumor volumes of a549.cd19 (left panel) and Granta-519 tumor spheres (right panel) on day 9 co-cultured with CAR T cells that had been treated with compound 17 simultaneously during long term stimulation. Duplicate values of cell compositions prepared from two different healthy donors are shown. In 3D culture (fig. 6A), treatment with compound 17 during long-term stimulation reduced the cytolytic activity of CAR T cells to a small extent.

Supernatant was collected on day 5 from tumor spheroid cultures containing long-term stimulated CAR-expressing T cells as described above. Cytokine production was measured from the supernatant by Meso Scalediscovery (MSD) analysis of the collected cell supernatant. FIG. 6B shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNFα (right panel) after 5 days of 3D co-culture with K562.CD19 (circular) and Granta-519 tumor spheres. Duplicate values of cell compositions prepared from two different healthy donors are shown. As shown, treatment with compound 17 during long-term stimulation resulted in a dose-dependent increase in production of all three cytokines, similar to the results of two-dimensional tumor cell cultures.

Example 4 evaluation of Long term stimulated CAR-T cells after rescue treatment with Compound 17

Compound 17 was evaluated for its ability to rescue long-term stimulated CAR T cell depletion.

Anti-CD 19 CAR expressing T cells generated as described in example 2 were chronically stimulated with plate-bound CAR-specific anti-idiotype antibodies as described in example 3 for 7 days, but compound 17 was absent during the chronic stimulation. In contrast, long-term stimulated CAR T cells were treated with compound 17 during re-challenge with CD19 expressing tumor cells, and then evaluated for cytolytic activity and cytokine production.

A. two-dimensional CD19+ tumors

The chronically stimulated CAR-T cells were incubated with various CD19 expressing lymphoma cells at a 2.5:1 effector to target cell ratio in the presence of DGKi (0-1000 nM). Tumor cells were labeled with NucLight Red (NLR) and passed throughThe number of tumor cells was monitored. Figure 7A shows tumor cell numbers of k562.cd19 (left panel), granta-519 (middle panel) and Raji cells (right panel) during 2D co-culture with CAR T cells in the presence of compound 17. As shown in fig. 7A, the rescue treatment with high concentration of compound 17 during co-culture improved the cytolytic activity of CAR T cells against k562.cd19 and Granta-519 cells, but did not improve the cytolytic activity against Raji cells.

After 48 hours of co-culture of the long-term stimulated CAR-expressing T cells and CD 19-expressing tumor cells described above, the supernatant was collected. Cytokine production was measured from the supernatant by Meso Scalediscovery (MSD) analysis of the collected cell supernatant. FIG. 7B shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNFα (right panel) after 2D co-culture with K562.CD19 cells (circles), granta-519 cells (squares) and Raji cells (triangles) for 48 hours in the presence of compound 17. Duplicate values of cell compositions prepared from two different healthy donors are shown. As shown, the rescue treatment with compound 17 during co-culture resulted in a dose-dependent increase in production of all three cytokines, except for IL-2 production during 2D culture with Granta-519 cells.

B. three-dimensional spheroid CD19+ tumor

Cytolytic activity was also assessed in CD19 expressing tumor spheroid cultures incubated with long term stimulated CAR-T cells in the presence of DGKi. A549 CD19+ tumor spheres or Granta-519 tumor spheres were incubated with anti-CD 19 CAR expressing T cells at an effector to target cell ratio of 1:2 for up to 14 days. Tumor spheres were engineered to express red fluorescent dye to allow tumor cell lysis to be monitored by microscopy during the assay. Cytolytic activity was assessed by monitoring fluorescence over time, and tumor volume (normalized to time 0) was measured in Red Calibration Units (RCU) by number of areas of² μm. Figure 8A shows normalized tumor volumes of a549.cd19 (left panel) and Granta-519 tumor spheres (right panel) on day 9 co-culture with CAR T cells in the presence of compound 17. Duplicate values of cell compositions prepared from two different healthy donors are shown. As shown, the rescue treatment with compound 17 during co-culture improved the cytolytic activity of CAR T cells against a549.cd19 and Granta-519 tumor spheres.

Supernatants were collected from tumor spheroid cultures with long-term stimulated CAR-expressing T cells described above at day 5. Cytokine production was measured from the supernatant by Meso Scalediscovery (MSD) analysis of the collected cell supernatant. FIG. 8B shows the supernatant concentrations (in pg/mL) of IFNγ (left panel), IL-2 (middle panel) and TNF α (right panel) after 3D co-cultivation with K562.CD19 (round) and Granta-519 tumor spheres (square) for 5 days in the presence of compound 17. Duplicate values of cell compositions prepared from two different healthy donors are shown. Rescue treatment with compound 17 during tumor sphere co-culture resulted in a dose-dependent increase in production of all three cytokines.

C. Summary

Taken together, these results show that rescue treatment with compound 17 during CAR T cell re-challenge can improve cytokine function and cytokine production, particularly in tumor spheroid culture. In agreement with the results of the examples above, these results are consistent with the conclusion that delayed treatment with compound 17 can enhance or enhance the performance of T cells in response to CAR antigens and can reduce or reverse the depletion or long term stimulated state of CAR T cells.

Example 5 evaluation of treatment with Compound 17 on eTCR cells stimulated for longer term

Compound 17 was evaluated for its ability to rescue long-term stimulated T cell depletion engineered with T cell receptor (eTCR).

Primary human cd4+ and cd8+ T cells were isolated from healthy donors by an immunoaffinity-based method. The isolated cd4+ and cd8+ T cells were combined and stimulated with anti-CD 3/anti-CD 28 agents and then transduced with a lentiviral vector encoding anti-human papillomavirus 16 (anti-HPV 16) eTCR. The nucleotide sequences encoding the β -strand and the α -strand of eTCR are separated by a sequence encoding a 2A ribosomal jump element. Following transduction, the cells were cultured in medium containing human serum and cytokines for 9-10 days and then further evaluated.

To evaluate the effect of rescue treatment with compound 17 on cytolytic function, eTCR cells were stimulated with 10 μg/mL or 20 μg/mL plate-bound antibody to TCR vβ chains for 6 days in the absence of compound 17.

Following long-term stimulation, eTCR-T cells were co-cultured with HPV-expressing CaSki cells at a 2.5:1 effector to target cell ratio in the presence of compound 17 at 100 nM. As a control, long-term stimulated eTCR cells were co-cultured with CaSki cells in the absence of compound 17. CaSki cells were transduced with NucLight Red (NLR) and passed throughThe number of tumor cells was monitored.

FIG. 9 shows the number of tumor cells of CaSki cells monitored at various time points during co-culture with eTCR T cells stimulated over time in the presence of compound 17 (left panel, eTCR T cells stimulated over time with 10 μg/mL anti-V.beta.; right panel, eTCR T cells stimulated over time with 20 μg/mL anti-V.beta.). As shown, the rescue treatment with compound 17 was able to improve the cytolytic function of the long-term stimulated eTCR cells during culture with tumor cells. This result is consistent with the observations of CAR-expressing T cells shown in example 4, indicating that DGK inhibition is able to reverse or rescue the depleted or chronically stimulated state of T cells engineered with different antigen receptors.

In another experiment evaluating DGKi to rescue or reverse the activity of depletion, eTCR T cells were stimulated for a long period of 6 days as described above in the absence of compound 17. Then, eTCR T cells stimulated for a long period of time or fresh thawed eTCR T cells not stimulated for a long period of time were stimulated with 20 μg/mL of plate bound antibody directed against eTCR V β chain in the presence of 100nM compound 17 for additional 7 days. As a control, eTCR T cells stimulated for a longer period were stimulated for an additional 7 days in the absence of compound 17. Cell counts were determined at the end of 7 days of restimulation.

FIG. 10 shows the cell counts of eTCR T cells from two donors, shown in sequence from left to right, (1) eTCR T cells that were chronically stimulated, without compound treatment (control), (2) eTCR T cells that were chronically stimulated, with compound 17, or (3) fresh eTCR T cells that were not chronically stimulated. As expected, the proliferation capacity of long-term stimulated T cells was overall reduced compared to fresh eTCR T cells in the absence of compound 17 treatment (compare conditions (1) and (3)). However, treatment of chronically stimulated T cells with compound 17 resulted in increased proliferation of eTCR T cells. These results further demonstrate the ability of DGKi to reverse or rescue T cell depletion during subsequent antigen receptor dependent restimulation.

Example 6 evaluation of the dosing regimen of Compound 17 in combination with CAR-T cells

The in vivo anti-tumor effect of different dosing regimens of compound 17 in combination with anti-CD 19 CAR T cells was evaluated. To generate CD19 CAR T cells, T cells from healthy human donors were engineered with CARs against CD 19.

To evaluate antitumor effects in vivo, nod.cg.prkdc^scidIL2rg^tm1Wjl/SzJ (NSG) mice were intravenously injected with 0.5x10⁶ Raji lymphoma tumor cells (immortalized human B lymphocyte tumor cell line expressing CD 19) transduced with red-shifted firefly luciferase (Raji-rFluc cells). Tumor grafts were grown for 11 days, and then on day 1 of combination therapy, mice received a single intravenous injection of 1x10⁶ anti-CD 19 CAR expressing T cells (81.8% of total T cells administered) or no treatment. This dose of CAR-expressing T cells was not expected to achieve complete tumor rejection.

Following administration of CAR-T cells, mice received one of three different compound 17 dosing regimens or vehicle controls (n=8 per group). In particular, mice received compound 17 or vehicle control once daily on combination therapy (i) on days 1-14 ("early"), (ii) on days 14-42 ("delayed") or (iii) on days 1-28 ("sustained"). All doses of compound 17 were 0.3mg/kg and administered orally. To evaluate tumor burden by bioluminescence imaging, mice received intraperitoneal injections of fluorescein substrate resuspended in phosphate buffered saline. Tumor burden and survival of treated mice were assessed until day 50. In addition, flow cytometry for assessing the number of circulating CAR T cells was performed on days 15 and 29. Flow cytometry for evaluation of CD4 to CD8 ratio was performed on day 15. CAR expression was assessed using anti-idiotype (anti-ID) antibodies specific for the CAR.

Figures 11 and 12 show tumor burden and survival in mice receiving three different compound 17 dosing regimens in combination with anti-CD 19 CAR T cells, respectively. As shown in fig. 12, the administration of the sustained dosing regimen improved survival, whereas the early and delayed administration regimens did not improve survival. As indicated by the tumor control index values shown in fig. 13 and 14, co-administration of compound 17 (as in early and sustained regimens) during days 1-14 (e.g., during the period of time that CAR T cells can be expanded in vivo after administration) resulted in enhanced tumor suppression relative to vehicle-treated mice, whereas administration delay regimens did not result. The tumor control index score represents a composite parameter derived from tumor growth data.

Figure 15 shows the number of circulating CAR expressing cells and the CD4 to CD8 ratio of circulating T cells on day 15 in mice receiving either a delayed regimen or an early or sustained regimen. At this time point, mice receiving the delay regimen have received a previous dose of compound 17. As shown in fig. 15, the early or sustained administration regimen resulted in increased CAR T cell expansion and increased CD4 to CD8 ratio relative to vehicle treated mice, whereas a single dose of the administration delay regimen did not result. The number of circulating CAR expressing cells at day 29 is shown in figure 16.

Taken together, these results indicate that DGKi starts on the same day as the engineered cells are administered T cell therapy and that tumor burden is reduced and survival is improved once daily continuing on days 1-28 after T cell therapy administration. As demonstrated herein, the presence of DGKi (e.g., beginning on day 1) after T cell therapy administration can improve the expansion and/or persistence of CAR T cells (particularly cd4+ CAR T cells), which can contribute to the demonstrated anti-tumor effects. The continued presence of DGKi after day 14 may also contribute to the demonstrated anti-tumor effect. The presence of DGKi following administration of a T cell therapy may prevent or reduce the depletion of T cells in the T cell therapy, thereby increasing the antitumor activity and proliferation capacity of T cells following administration, without being limited by a particular mechanism of action.

Example 7 sustained compound 17 dosing regimen in combination with lower CAR T cell dose

The in vivo anti-tumor effect of the sustained compound 17 dosing regimen in combination with lower doses of CAR T cells against CD19 was evaluated. To generate CAR T cells against CD19, T cells from healthy human donors were engineered with CAR against CD 19.

To evaluate the anti-tumor effect in vivo, immunodeficient NSG mice were intravenously injected with 0.5x10⁶ Raji-rFluc cells. After 7 days of tumor cell injection, mice received a single intravenous injection of 0.5x10⁶ or 1x10⁶ anti-CD 19 CAR expressing T cells (88.5% of total administered T cells) or no treatment on day 1 of combination therapy. These doses of CAR expressing T cells failed to achieve complete tumor rejection.

Following CAR-T cell administration, mice receiving low doses of CAR T cells received 0.3mg/kg compound 17 once daily on days 1-30. Mice receiving high doses of CAR T cells received either vehicle control or 0.3mg/kg or 0.6mg/kg compound 17 (n=8 per group) once daily on days 1-30. All doses of compound 17 were administered orally. To evaluate tumor burden by bioluminescence imaging, mice received intraperitoneal injections of fluorescein substrate resuspended in phosphate buffered saline. Tumor burden and survival of treated mice were assessed until day 50. In addition, flow cytometry for evaluation of circulating CAR T cell numbers and CD4: CD8 expression was performed on days 5, 12, 19 and 26. CAR expression was assessed using anti-idiotype (anti-ID) antibodies specific for the CAR.

As shown in fig. 17 and 18, administration of CAR T cells in combination with compound 17 reduced tumor burden and improved survival in all groups, including groups receiving lower CAR T cell doses, compared to CAR T cells administered alone. As indicated by the tumor control index values shown in fig. 19, administration of CAR T cells in combination with compound 17 resulted in enhanced tumor suppression in all groups relative to CAR T cells administered alone.

As shown in fig. 20, administration of compound 17 increases CAR T cell expansion and/or persistence in all groups after administration of CAR T cells. CAR T cell expansion was delayed in the group receiving the lower CAR T cell dose and compound 17 combination relative to the group receiving the higher CAR T cell dose and compound 17 combination. The circulating CAR T cell count in the group receiving the lower CAR T cell dose in combination with compound 17 later exceeded the circulating CAR T cell count of the group receiving the higher CAR T cell dose alone.

Taken together, these results show that reduced tumor burden and improved survival and CAR T cell expansion and/or persistence can be achieved by combining CAR T cell administration with sustained DGKi administration (including administration of lower doses of CAR T cells).

Example 8 Effect of Compound 17 administration on expansion and/or persistence of CAR T cells from different donors

Compound 17 administration was evaluated for its effect on in vivo expansion and/or persistence of CAR T cells produced from different healthy donors. To generate anti-CD 19 CAR T cells, T cells from four healthy donors were engineered with CARs against CD 19.

Immunodeficient NSG mice were injected intravenously with 0.5X10⁶ Raji-rFluc cells. After 7 days of tumor cell injection, mice received a single injection of 1x10⁶ anti-CD 19 CAR expressing T cells (93.5% [ donor 1, 87.7% [ donor 2, 90.3% [ donor 3] or 97.6% [ donor 4] of total T cells administered) or no treatment on day 1 of combination therapy. This dose of CAR-expressing T cells was not expected to achieve complete tumor rejection.

Following CAR-T cell administration, mice received either vehicle control or 0.3mg/kg compound 17 (n=4 per group) once daily on days 1-7. All doses of compound 17 were administered orally. Flow cytometry for evaluation of circulating CAR T cell numbers was performed on days 7 and 14. CAR expression was assessed using anti-idiotype (anti-ID) antibodies specific for the CAR.

As shown in fig. 21, CAR T cells were expanded and/or persisted in all mice receiving compound 17 administration as compared to CAR T cells administered alone. These results indicate that DGKi administration can improve the expansion and/or persistence of CAR T cells from different donors, the differentiation status of which T cells and the number of naive T cells or naive-like T cells can vary.

Example 9 comparison of CAR-T cells with sustained Compound 17 dosing regimen with DGK alpha/zeta Single and double knockout CART cells

The in vivo anti-tumor effect of the combination anti-CD 19 CAR T cell therapy administration and sustained compound 17 dosing regimen was compared to the in vivo anti-tumor effect of the administration of dgkα single KO, dgkζsingle KO, or dgkα/ζdouble KO anti-CD 19 CAR T cells. To generate anti-CD 19 CAR T cells without DGK KO, T cells from healthy donors were engineered with a CAR against CD 19. Dgkα single KO, dgkζsingle KO and dgkα/ζdouble KO were introduced via double strand breaks for indel generation at the locus, yielding a non-functional gene.

To evaluate anti-tumor effects in vivo, immunodeficient NSG mice were intravenously injected with 0.5X10⁶ Raji-rFluc cells. 7 days after tumor cell injection, mice received a single intravenous injection of 1x10⁶ anti-CD 19 CAR expressing T cells (88.5% [ DGK KO free ], 87.8% [ DGK alpha single KO ], 88.2% [ DGK ζ single KO ] or 86.6% [ DGK alpha/ζ double KO ] of total T cells administered) or no treatment on day 1 of combination therapy. This dose of CAR-expressing T cells was not expected to achieve complete tumor rejection.

Following CAR-T cell administration, mice receiving CAR T cells without DGK KO received either vehicle control or 0.3mg/kg compound 17 (n=8 per group) once daily on days 1-30. All doses of compound 17 were administered orally. To evaluate tumor burden by bioluminescence imaging, mice received intraperitoneal injections of fluorescein substrate resuspended in phosphate buffered saline. Tumor burden and survival of treated mice were assessed until day 50. In addition, flow cytometry for evaluation of circulating CAR T cell numbers and CD4: CD8 expression was performed on days 5, 12, 19 and 26. CAR expression was assessed using anti-idiotype (anti-ID) antibodies specific for the CAR.

As shown in fig. 22, tumor burden was reduced in mice receiving the combination of CAR T cells with compound 17 and mice receiving dgkα/ζ double KO CAR T cells. As shown in fig. 23, improved survival was observed in mice receiving CAR T cells in combination with compound 17 and dgkζ single KO or dgkα/ζ double KO CAR T cells compared to mice receiving CAR T cells without DGK KO alone. As shown in figure 24, the tumor control index values indicate that administration of CAR T cells in combination with compound 17 and dgkα/ζ double KO CAR T cells showed improved anti-tumor effects relative to administration of CAR T cells without DGK KO alone. As shown in fig. 25, improved CAR T cell expansion and/or persistence was observed in mice receiving a combination of CAR T cells with compound 17 and mice receiving dgkα/ζ double KO CAR T cells.

Taken together, these results show that the combination of CAR T cells with sustained DGKi administration can result in improvements in tumor burden, survival, and CAR T cell expansion and/or persistence that are comparable to those achieved with dgkα/ζ double KO CAR T cells.

Example 10 CAR-T cell and persistent Compound 17 dosing regimen combination in a second CD19 expressing tumor model

In vivo anti-tumor effects of the combination of the sustained compound 17 dosing regimen with anti-CD 19 CAR T cells were evaluated in a second CD19 expressing tumor model. To generate anti-CD 19 CAR T cells, T cells from healthy human donors were engineered with CARs against CD 19.

To evaluate the anti-tumor effect in vivo, immunodeficient NSG mice were intravenously injected with 0.5x10⁶ Nalm6 leukemia tumor cells (immortalized human B cell precursor leukemia tumor cell line expressing CD 19) transduced with red-shifted firefly luciferase (Nalm 6-rFluc cells). In contrast to Raji cells, nalm6 cells do not express cell surface co-stimulatory molecules, making CAR T cells less likely to be activated in the presence of Nalm6 cells than Raji cells. After 4 days of tumor cell injection, mice received a single intravenous injection of 1x10⁶ anti-CD 19CAR expressing T cells (81.8% of total administered T cells) or no treatment on day 1 of combination therapy. This dose of CAR expressing T cells failed to achieve complete tumor rejection.

Following CAR-T cell administration, mice received either vehicle control or 0.3mg/kg compound 17 once daily on days 1-30. Other mice received 0.3mg/kg or 0.6mg/kg of compound 17 once daily on days 1-30, but did not previously administer CAR-T cells (n=8 per group). All doses of compound 17 were administered orally. To evaluate tumor burden by bioluminescence imaging, mice received intraperitoneal injections of fluorescein substrate resuspended in phosphate buffered saline. Tumor burden and survival of treated mice were assessed until day 50. In addition, flow cytometry for evaluation of circulating CAR T cell numbers and CD4: CD8 expression was performed on days 8, 16, 23 and 30. CAR expression was assessed using anti-idiotype (anti-ID) antibodies specific for the CAR.

As shown in fig. 26A, 26B, and 27, administration of the CAR T cell in combination with compound 17 reduced tumor burden and improved survival compared to CAR T cell administration alone or compound 17 administration. No single dose effect of compound 17 was observed. Figure 26B also shows historical tumor burden data from Nalm6-rFluc injected mice from a single previous experiment that also had received 1x10⁶ anti-CD 19 CAR expressing T cells generated from one of two healthy human donors (donor 1 and donor 2). In a separate prior experiment, mice did not receive administration of compound 17. As shown in fig. 26B, mice receiving the combination of CAR T cells with compound 17 in this experiment also had a lower tumor burden than mice in previous experiments alone. The tumor control index values as shown in fig. 28 indicate that the combination of CAR T cells with compound 17 resulted in enhanced tumor suppression relative to CAR T cell administration alone. As shown in fig. 29, administration of compound 17 after CAR T cell administration increases CAR T cell expansion and/or persistence.

Taken together, these results show that in the second CD19 expressing tumor model, reduced tumor burden and improved survival and CAR T cell expansion and/or persistence can be achieved by a combination of CAR T cell administration and sustained DGKi administration.

Example 11 tumor cell re-challenge after CAR-T cell and sustained Compound 17 dosing regimen combination

Mice receiving CAR T cells in example 10 in combination with a sustained compound 17 dosing regimen were re-challenged by intravenous injection of 5x10⁶ Nalm6-rFluc cells on day 71. For comparison, 5x10⁶ Nalm6-rFluc cells were injected intravenously into naive mice previously injected or administered Nalm6-rFluc cells, CAR T cells or compound 17 (n=4 per group). To evaluate tumor burden by bioluminescence imaging, mice received intraperitoneal injections of fluorescein substrate resuspended in phosphate buffered saline. Tumor burden was assessed 10 days after the additional challenge. The survival of the treated mice was assessed until 30 days after re-challenge.

Figure 30 shows that mice re-challenged with tumor cells have delayed tumor growth, particularly in long bones, compared to naive mice after administration of the CAR T cell in combination with compound 17. Survival benefits were also observed for re-challenged mice, as shown in figure 31. These results indicate that prior exposure to CAR T cells in combination with sustained DGKi administration can have long-term benefits in protection against late tumor cell re-challenge.

The scope of the invention is not intended to be limited to the particular disclosed embodiments provided (e.g., illustrating various aspects of the invention). Various modifications to the described compositions and methods will be apparent from the description and teachings herein. Such variations may be made without departing from the true scope and spirit of the disclosure and are intended to be within the scope of the disclosure.

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Claims (90)

Translated fromChinese

1.一种治疗方法，所述方法包括向罹患癌症的受试者施用组合疗法，其包括：1. A method of treatment comprising administering to a subject suffering from cancer a combination therapy comprising:(a)包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；以及(a) a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered on day 1 of the combination therapy; and(b)DGKα和/或DGKζ抑制剂，其中所述抑制剂的施用在所述组合疗法的第1-8天中任一天包括第1和第8天开始，以及所述抑制剂在或在约连续21和42天之间包括21和42天每天施用一次。(b) a DGKα and/or DGKζ inhibitor, wherein administration of the inhibitor begins on any of days 1-8 of the combination therapy, inclusive, and the inhibitor is administered once daily on or about between 21 and 42 consecutive days, inclusive.2.根据权利要求1所述的方法，其中所述抑制剂的施用在施用所述T细胞疗法之后开始。2. The method of claim 1, wherein administration of the inhibitor begins after administration of the T cell therapy.3.一种治疗方法，所述方法包括向罹患癌症的受试者施用DGKα和/或DGKζ抑制剂，其中所述抑制剂作为包括所述抑制剂和包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法的组合疗法的部分施用，其中：3. A method of treatment comprising administering a DGKα and/or DGKζ inhibitor to a subject suffering from cancer, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein:在开始施用所述抑制剂之前，所述受试者先前已经施用所述T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；Prior to initiating administration of the inhibitor, the subject had previously been administered the T cell therapy, wherein the T cell therapy was administered on day 1 of the combination therapy;所述抑制剂的施用在所述组合疗法的第1-8天中任一天包括第1和第8天开始；以及Administration of the inhibitor begins on any of days 1-8 of the combination therapy, including days 1 and 8; and所述抑制剂在或在约连续21和42天之间包括21和42天每天施用一次。The inhibitor is administered once daily on or about between 21 and 42 consecutive days, inclusive.4.一种治疗方法，所述方法包括向罹患癌症的受试者施用组合疗法，其包括：4. A method of treatment, the method comprising administering to a subject suffering from cancer a combination therapy comprising:(a)包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；以及(a) a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered on day 1 of the combination therapy; and(b)DGKα和/或DGKζ抑制剂，其中所述抑制剂的施用在所述组合疗法的第1-8天中任一天包括第1和第8天开始，以及连续施用所述抑制剂，至少直至在施用所述T细胞疗法后，所述受试者中所述T细胞疗法的工程化T细胞的数量达到峰值。(b) a DGKα and/or DGKζ inhibitor, wherein administration of the inhibitor begins on any one of days 1 to 8 of the combination therapy, including days 1 and 8, and administration of the inhibitor continues at least until the number of engineered T cells of the T cell therapy in the subject reaches a peak after administration of the T cell therapy.5.根据权利要求4所述的方法，其中所述抑制剂的施用在施用所述T细胞疗法之后开始。5. The method of claim 4, wherein administration of the inhibitor begins after administration of the T cell therapy.6.一种治疗方法，所述方法包括向罹患癌症的受试者施用DGKα和/或DGKζ抑制剂，其中所述抑制剂作为包括所述抑制剂和包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法的组合疗法的部分施用，其中：6. A method of treatment comprising administering a DGKα and/or DGKζ inhibitor to a subject suffering from cancer, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein:在开始施用所述抑制剂之前，所述受试者先前已经施用所述T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；Prior to initiating administration of the inhibitor, the subject had previously been administered the T cell therapy, wherein the T cell therapy was administered on day 1 of the combination therapy;所述抑制剂的施用在所述组合疗法的第1-8天中任一天包括第1和第8天开始；以及Administration of the inhibitor begins on any of days 1-8 of the combination therapy, including days 1 and 8; and连续施用所述抑制剂，至少直至在施用所述T细胞疗法后，所述受试者中所述T细胞疗法的工程化T细胞的数量达到峰值。The inhibitor is continuously administered at least until the number of engineered T cells of the T cell therapy in the subject reaches a peak after administration of the T cell therapy.7.根据权利要求4所述的方法，其中所述抑制剂的施用在组合疗法的第1天在施用所述T细胞疗法之前开始。7. The method of claim 4, wherein administration of the inhibitor begins on day 1 of the combination therapy prior to administration of the T cell therapy.8.一种治疗方法，所述方法包括向罹患癌症的受试者施用T细胞疗法，其包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞，其中所述T细胞疗法作为包括所述T细胞疗法和DGKα和/或DGKζ抑制剂的组合疗法的部分施用，其中：8. A method of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a DGKα and/or DGKζ inhibitor, wherein:所述T细胞疗法在所述组合疗法的第1天施用；The T cell therapy is administered on day 1 of the combination therapy;所述抑制剂的施用在所述组合疗法的第1天在施用所述T细胞疗法之前开始；以及Administration of the inhibitor begins on day 1 of the combination therapy prior to administration of the T cell therapy; and连续施用所述抑制剂，至少直至在施用所述T细胞疗法后，所述受试者中所述T细胞疗法的工程化T细胞的数量达到峰值。The inhibitor is continuously administered at least until the number of engineered T cells of the T cell therapy in the subject reaches a peak after administration of the T cell therapy.9.根据权利要求4-8中任一项所述的方法，其中所述抑制剂每天、每两天或每三天施用。9. The method of any one of claims 4-8, wherein the inhibitor is administered every day, every two days, or every three days.10.根据权利要求4-9中任一项所述的方法，其中所述抑制剂每天施用。10. The method of any one of claims 4-9, wherein the inhibitor is administered daily.11.根据权利要求4-10中任一项所述的方法，其中所述抑制剂在施用所述抑制剂的每天施用一次。11. The method of any one of claims 4-10, wherein the inhibitor is administered once per day of administration of the inhibitor.12.根据权利要求4-11中任一项所述的方法，其中施用所述抑制剂直至在施用所述T细胞疗法后，所述受试者中所述T细胞疗法的工程化T细胞的数量达到峰值。12. The method of any one of claims 4-11, wherein the inhibitor is administered until the number of engineered T cells of the T cell therapy in the subject reaches a peak after administration of the T cell therapy.13.根据权利要求4-12中任一项所述的方法，其中所述抑制剂在或在约21和42天之间包括21和42天的时间段施用。13. The method of any one of claims 4-12, wherein the inhibitor is administered over a period of time at or between about 21 and 42 days, inclusive.14.一种治疗方法，所述方法包括向罹患癌症的受试者施用组合疗法，其包括：14. A method of treatment comprising administering to a subject suffering from cancer a combination therapy comprising:(a)包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；以及(a) a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered on day 1 of the combination therapy; and(b)DGKα和/或DGKζ抑制剂，其中所述抑制剂的施用在施用所述组合疗法的第1-8天中任一天包括第1和第8天开始；以及所述抑制剂以治疗有效剂量在或在约连续21和42天之间的时间段施用。(b) a DGKα and/or DGKζ inhibitor, wherein administration of the inhibitor begins on any of days 1-8, including days 1 and 8, of administering the combination therapy; and the inhibitor is administered at a therapeutically effective dose for a period of between about 21 and 42 consecutive days.15.根据权利要求14所述的方法，其中所述抑制剂的施用在施用所述T细胞疗法之后开始。15. The method of claim 14, wherein administration of the inhibitor begins after administration of the T cell therapy.16.一种治疗方法，所述方法包括向罹患癌症的受试者施用DGKα和/或DGKζ抑制剂，其中所述抑制剂作为包括所述抑制剂和包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法的组合疗法的部分施用，其中：16. A method of treatment comprising administering a DGKα and/or DGKζ inhibitor to a subject suffering from cancer, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein:在开始施用所述抑制剂之前，所述受试者先前已经施用所述T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；The subject has previously been administered the T cell therapy prior to initiating administration of the inhibitor, wherein the T cell therapy is administered on day 1 of the combination therapy;所述抑制剂的施用在所述组合疗法的第1-8天中任一天包括第1和第8天开始，以及Administration of the inhibitor begins on any of days 1 to 8 of the combination therapy, including days 1 and 8, and所述抑制剂以治疗有效剂量在或在约21和42天之间的时间段施用。The inhibitor is administered at a therapeutically effective dose for a period of time between about 21 and 42 days.17.根据权利要求14所述的方法，其中所述抑制剂的施用在组合疗法的第1天在施用所述T细胞疗法之前开始。17. The method of claim 14, wherein administration of the inhibitor begins on day 1 of the combination therapy prior to administration of the T cell therapy.18.一种治疗方法，所述方法包括向罹患癌症的受试者施用包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法，其中所述T细胞疗法作为包括所述T细胞疗法和DGKα和/或DGKζ抑制剂的组合疗法的部分施用，其中：18. A method of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and an inhibitor of DGKα and/or DGKζ, wherein:所述T细胞疗法在所述组合疗法的第1天施用；The T cell therapy is administered on day 1 of the combination therapy;所述抑制剂的施用在所述组合疗法的第1天在施用所述T细胞疗法之前开始；以及Administration of the inhibitor begins on day 1 of the combination therapy prior to administration of the T cell therapy; and所述抑制剂以治疗有效剂量在或在约21和42天之间的时间段施用。The inhibitor is administered at a therapeutically effective dose for a period of time between about 21 and 42 days.19.根据权利要求14-18中任一项所述的方法，其中所述治疗有效剂量提供在或在约21和42天之间的时间段持续治疗有效的抑制剂剂量。19. The method of any one of claims 14-18, wherein the therapeutically effective dose provides a therapeutically effective dose of the inhibitor that lasts for a period of time of at or between about 21 and 42 days.20.根据权利要求14-19中任一项所述的方法，其中所述抑制剂每2天或3天施用。20. The method of any one of claims 14-19, wherein the inhibitor is administered every 2 or 3 days.21.一种治疗方法，所述方法包括向罹患癌症的受试者施用组合疗法，其包括：21. A method of treatment comprising administering to a subject suffering from cancer a combination therapy comprising:(a)包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；以及(a) a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered on day 1 of the combination therapy; and(b)DGKα和/或DGKζ抑制剂，其中所述抑制剂的施用在所述组合疗法的第1-8天中任一天包括第1和第8天开始，以及(b) a DGKα and/or DGKζ inhibitor, wherein administration of the inhibitor begins on any of days 1 to 8 of the combination therapy, including days 1 and 8, and所述抑制剂在多个周期中施用，每个周期包括施用所述抑制剂的施用期，随后是不施用所述抑制剂的休息期。The inhibitor is administered in multiple cycles, each cycle comprising an administration period during which the inhibitor is administered, followed by a rest period during which the inhibitor is not administered.22.根据权利要求21所述的方法，其中所述抑制剂的施用在施用所述T细胞疗法之后开始。22. The method of claim 21, wherein administration of the inhibitor begins after administration of the T cell therapy.23.一种治疗方法，所述方法包括向罹患癌症的受试者施用DGKα和/或DGKζ抑制剂，其中所述抑制剂作为包括所述抑制剂和包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法的组合疗法的部分施用，其中：23. A method of treatment comprising administering a DGKα and/or DGKζ inhibitor to a subject suffering from cancer, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein:在开始施用所述抑制剂之前，所述受试者先前已经施用所述T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；Prior to initiating administration of the inhibitor, the subject had previously been administered the T cell therapy, wherein the T cell therapy was administered on day 1 of the combination therapy;所述抑制剂的施用在所述组合疗法的第1-8天中任一天包括第1和第8天开始；以及Administration of the inhibitor begins on any of days 1-8 of the combination therapy, including days 1 and 8; and所述抑制剂在多个周期中施用，每个周期包括施用所述抑制剂的施用期，随后是不施用所述抑制剂的休息期。The inhibitor is administered in multiple cycles, each cycle comprising an administration period during which the inhibitor is administered, followed by a rest period during which the inhibitor is not administered.24.根据权利要求21-23中任一项所述的方法，其中在施用期期间，所述抑制剂每天、每两天或每三天施用。24. The method of any one of claims 21-23, wherein the inhibitor is administered every day, every two days, or every three days during the administration period.25.根据权利要求21-24中任一项所述的方法，其中在施用期期间，所述抑制剂每天施用。25. The method of any one of claims 21-24, wherein the inhibitor is administered daily during the administration period.26.根据权利要求21-25中任一项所述的方法，其中在施用期期间，所述抑制剂在施用所述抑制剂的每天施用一次。26. The method of any one of claims 21-25, wherein during the administration period, the inhibitor is administered once per day that the inhibitor is administered.27.根据权利要求21-26中任一项所述的方法，其中在所述多个周期内，施用所述抑制剂，直至在施用所述T细胞疗法后，所述受试者中所述T细胞疗法的工程化T细胞的数量达到峰值。27. The method of any one of claims 21-26, wherein the inhibitor is administered during the multiple cycles until the number of engineered T cells of the T cell therapy in the subject reaches a peak after administration of the T cell therapy.28.根据权利要求21-27中任一项所述的方法，其中在所述多个周期内，所述抑制剂在或在约21和42天之间包括21和42天的时间段施用。28. The method of any one of claims 21-27, wherein the inhibitor is administered during a period of time at or between about 21 and 42 days, inclusive, within the plurality of cycles.29.根据权利要求1-28中任一项所述的方法，其中所述抑制剂的施用在所述组合疗法的第1-6天中任一天包括第1和第6天、所述组合疗法的第1-4天中任一天包括第1和第4天、所述组合疗法的第1天或第2天和/或所述组合疗法的第1天开始。29. The method of any one of claims 1-28, wherein administration of the inhibitor begins on any of days 1-6 of the combination therapy, including days 1 and 6, any of days 1-4 of the combination therapy, including days 1 and 4, day 1 or day 2 of the combination therapy, and/or day 1 of the combination therapy.30.根据权利要求1或权利要求29所述的方法，其中所述抑制剂的施用在组合疗法的第1天在施用所述T细胞疗法之前开始。30. The method of claim 1 or claim 29, wherein administration of the inhibitor begins on day 1 of combination therapy prior to administration of the T cell therapy.31.一种治疗方法，所述方法包括向罹患癌症的受试者施用T细胞疗法，其包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞，其中所述T细胞疗法作为包括所述T细胞疗法和DGKα和/或DGKζ抑制剂的组合疗法的部分施用，其中：31. A method of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a DGKα and/or DGKζ inhibitor, wherein:所述T细胞疗法在所述组合疗法的第1天施用；The T cell therapy is administered on day 1 of the combination therapy;所述抑制剂的施用在所述组合疗法的第1天在施用所述T细胞疗法之前开始；以及Administration of the inhibitor begins on day 1 of the combination therapy prior to administration of the T cell therapy; and所述抑制剂在或在约连续21和42天之间包括21和42天每天施用一次。The inhibitor is administered once daily on or about between 21 and 42 consecutive days, inclusive.32.根据权利要求1-31中任一项所述的方法，其中所述抑制剂在或在约21和35天之间包括21和35天的时间段、在或在约21和28天之间包括21和28天的时间段、在或在约28和42天之间包括28和42天的时间段、在或在约28和35天之间包括28和35天的时间段、为或约28天的时间段；和/或为或约30天的时间段任选地经多个周期施用。32. The method of any one of claims 1-31, wherein the inhibitor is optionally administered over a period of at or between about 21 and 35 days, inclusive, at or between about 21 and 28 days, inclusive, at or between about 28 and 42 days, inclusive, at or between about 28 and 35 days, at or about 28 days; and/or at or about 30 days, over a period of optionally multiple cycles.33.一种治疗方法，所述方法包括向罹患癌症的受试者施用组合疗法，其包括：33. A method of treatment comprising administering to a subject suffering from cancer a combination therapy comprising:(a)包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；以及(a) a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered on day 1 of the combination therapy; and(b)DGKα和/或DGKζ抑制剂，其中所述抑制剂的施用在所述组合疗法的第1天开始，以及所述抑制剂在所述组合疗法的第1-28天包括第1和第28天每天施用一次。(b) a DGKα and/or DGKζ inhibitor, wherein administration of the inhibitor begins on day 1 of the combination therapy, and the inhibitor is administered once daily on days 1-28 of the combination therapy, inclusive.34.根据权利要求33所述的方法，其中所述抑制剂的施用在施用所述T细胞疗法之后开始。34. The method of claim 33, wherein administration of the inhibitor begins after administration of the T cell therapy.35.一种治疗方法，所述方法包括向罹患癌症的受试者施用DGKα和/或DGKζ抑制剂，其中所述抑制剂作为包括所述抑制剂和包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞的T细胞疗法的组合疗法的部分施用，其中：35. A method of treatment comprising administering a DGKα and/or DGKζ inhibitor to a subject suffering from cancer, wherein the inhibitor is administered as part of a combination therapy comprising the inhibitor and a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein:在开始施用所述抑制剂之前，所述受试者先前已经施用所述T细胞疗法，其中所述T细胞疗法在所述组合疗法的第1天施用；Prior to initiating administration of the inhibitor, the subject had previously been administered the T cell therapy, wherein the T cell therapy was administered on day 1 of the combination therapy;所述抑制剂的施用在所述组合疗法的第1天开始，以及Administration of the inhibitor begins on day 1 of the combination therapy, and所述抑制剂在所述组合疗法的第1-28天包括第1和第28天每天施用一次。The inhibitor is administered once daily on days 1-28, inclusive, of the combination therapy.36.根据权利要求33所述的方法，其中所述抑制剂的施用在施用所述T细胞疗法之前开始。36. The method of claim 33, wherein administration of the inhibitor begins prior to administration of the T cell therapy.37.一种治疗方法，所述方法包括向罹患癌症的受试者施用T细胞疗法，其包含表达针对由癌细胞表达的抗原的重组受体的活的工程化T细胞，其中所述T细胞疗法作为包括所述T细胞疗法和DGKα和/或DGKζ抑制剂的组合疗法的部分施用，其中：37. A method of treatment comprising administering to a subject suffering from cancer a T cell therapy comprising live engineered T cells expressing a recombinant receptor for an antigen expressed by a cancer cell, wherein the T cell therapy is administered as part of a combination therapy comprising the T cell therapy and a DGKα and/or DGKζ inhibitor, wherein:所述T细胞疗法在所述组合疗法的第1天施用；The T cell therapy is administered on day 1 of the combination therapy;所述抑制剂的施用在组合疗法的第1天在施用所述T细胞疗法之前开始；以及Administration of the inhibitor begins on day 1 of the combination therapy prior to administration of the T cell therapy; and所述抑制剂在所述组合疗法的第1-28天包括第1和第28天每天施用一次。The inhibitor is administered once daily on days 1-28, inclusive, of the combination therapy.38.根据权利要求33-37中任一项所述的方法，其中所述抑制剂在所述组合疗法的第1-30天包括第1和第30天每天施用一次。38. The method of any one of claims 33-37, wherein the inhibitor is administered once daily on days 1-30, inclusive, of the combination therapy.39.根据权利要求1-38中任一项所述的方法，其中所述抑制剂的施用在施用所述T细胞疗法12小时内或约12小时内、在施用所述T细胞疗法6小时内或约6小时内或在施用所述T细胞疗法4小时内或约4小时内开始。39. The method of any one of claims 1-38, wherein administration of the inhibitor is initiated within or about 12 hours of administration of the T cell therapy, within or about 6 hours of administration of the T cell therapy, or within or about 4 hours of administration of the T cell therapy.40.根据权利要求1-39中任一项所述的方法，其中所述抑制剂的施用在施用所述T细胞疗法2小时内或约2小时内开始。40. The method of any one of claims 1-39, wherein administration of the inhibitor is initiated within or about 2 hours of administering the T cell therapy.41.根据权利要求1-40中任一项所述的方法，其中所述抑制剂的施用在施用所述T细胞疗法1小时内或约1小时内开始。41. The method of any one of claims 1-40, wherein administration of the inhibitor begins within or about 1 hour of administering the T cell therapy.42.根据权利要求1、4、9-14、19-24、27-32和37-41中任一项所述的方法，其中所述抑制剂的施用在所述组合疗法的第1天与施用所述T细胞疗法同时开始。42. The method of any one of claims 1, 4, 9-14, 19-24, 27-32, and 37-41, wherein administration of the inhibitor is initiated on day 1 of the combination therapy simultaneously with administration of the T cell therapy.43.根据权利要求1、4、9-14、19-24、27-32和37-42中任一项所述的方法，其中所述抑制剂的施用在所述组合疗法的第1天在施用所述T细胞疗法期间开始。43. The method of any one of claims 1, 4, 9-14, 19-24, 27-32, and 37-42, wherein administration of the inhibitor begins on day 1 of the combination therapy during administration of the T cell therapy.44.根据权利要求1-43中任一项所述的方法，其中在施用所述T细胞疗法之前，所述受试者已经用包括施用氟达拉滨和/或环磷酰胺的淋巴细胞清除疗法预处理。44. The method of any one of claims 1-43, wherein prior to administration of the T cell therapy, the subject has been pretreated with lymphodepleting therapy comprising administration of fludarabine and/or cyclophosphamide.45.根据权利要求44所述的方法，其中所述方法进一步包括将淋巴细胞清除疗法施用至受试者。45. The method of claim 44, wherein the method further comprises administering lymphodepleting therapy to the subject.46.根据权利要求44或权利要求45所述的方法，其中所述淋巴细胞清除疗法包括施用：46. The method of claim 44 or claim 45, wherein the lymphodepleting therapy comprises administering:在或在约200和400mg/m²之间包括200和400mg/m²的环磷酰胺，每天施用持续在或在约2和4天之间包括2和4天；和/或At or between about 200 and 400 mg/m² , inclusive,^of cyclophosphamide, administered daily for or between about 2 and 4 days, inclusive; and/or在或在约20和40mg/m²之间包括20和40mg/m²的氟达拉滨，每天施用持续在或在约2和4天之间包括2和4天。At or between about 20 and 40 mg/m² , inclusive,^of fludarabine, administered daily for at or between about 2 and 4 days, inclusive.47.根据权利要求44-46中任一项所述的方法，其中所述淋巴细胞清除疗法包括施用为或约300mg/m²的环磷酰胺和为或约30mg/m²的氟达拉滨，每天施用持续或持续约3天。47. The method of any one of claims 44-46, wherein the lymphodepleting therapy comprises administering at or about 300 mg/^m2 of cyclophosphamide and at or about 30 mg/^m2 of fludarabine, administered daily for or about 3 days.48.根据权利要求44-47中任一项所述的方法，其中所述T细胞疗法在施用所述淋巴细胞清除疗法之后在或在约2和7天之间包括第2和第7天施用。48. The method of any one of claims 44-47, wherein the T cell therapy is administered on or between about 2 and 7 days, inclusive, after administration of the lymphodepleting therapy.49.根据权利要求1-48中任一项所述的方法，其中所述抑制剂为DGKα抑制剂而非DGKζ的显著抑制剂。49. The method of any one of claims 1-48, wherein the inhibitor is an inhibitor of DGKα but not a significant inhibitor of DGKζ.50.根据权利要求1-48中任一项所述的方法，其中所述抑制剂为DGKζ抑制剂而非DGKα的显著抑制剂。50. The method of any one of claims 1-48, wherein the inhibitor is a DGKζ inhibitor and is not a significant inhibitor of DGKα.51.根据权利要求1-48中任一项所述的方法，其中所述抑制剂为DGKα和DGKζ抑制剂。51. The method of any one of claims 1-48, wherein the inhibitor is a DGKα and DGKζ inhibitor.52.根据权利要求1-51中任一项所述的方法，其中所述抑制剂不为其他DGK的显著抑制剂。52. The method of any one of claims 1-51, wherein the inhibitor is not a significant inhibitor of other DGKs.53.根据权利要求1-52中任一项所述的方法，其中所述抑制剂为式(I)化合物：53. The method according to any one of claims 1 to 52, wherein the inhibitor is a compound of formula (I):或其药学上可接受的盐，其中：or a pharmaceutically acceptable salt thereof, wherein:R₁为H、F、Cl、Br、–CN、经0至4个R_1a取代的C_1–3烷基、经0至4个R_1a取代的C_3–4环烷基、经0至4个R_1a取代的C_1–3烷氧基、–NR_aR_a、–S(O)_nR_e或–P(O)R_eR_e；R₁ is H, F, Cl, Br, –CN, C_1-3 alkyl substituted with 0 to 4 R_1a , C_3-4 cycloalkyl substituted with 0 to 4 R_1a , C_1-3 alkoxy substituted with 0 to 4 R_1a , –NR_a R_a , –S(O)_n R_e or –P(O)R_e R_e ;每个R_1a独立地为F、Cl、–CN、–OH、–OCH₃或–NR_aR_a；Each R_1a is independently F, Cl, –CN, –OH, –OCH₃ or –NR_a R_a ;每个R_a独立地为H或C_1–3烷基；Each_Ra is independently H or_C1-3 alkyl;每个R_e独立地为经0至4个R_1a取代的C_3–4环烷基或C_1–3烷基；Each_Re is independently_C3-4 cycloalkyl or_C1-3 alkyl substituted with 0 to 4_R1a ;R₂为H、经0至4个R_2a取代的C_1–3烷基或经0至4个R_2a取代的C_3–4环烷基；R₂ is H, C_1-3 alkyl substituted by 0 to 4 R_2a , or C_3-4 cycloalkyl substituted by 0 to 4 R_2a ;每个R_2a独立地为F、Cl、–CN、–OH、–O(C_1–2烷基)、C_3–4环烷基、C_3–4烯基或C_3–4炔基；each R_2a is independently F, Cl, –CN, –OH, –O(C_1-2 alkyl), C_3-4 cycloalkyl, C_3-4 alkenyl, or C_3-4 alkynyl;R₃为H、F、Cl、Br、–CN、C_1–3烷基、C_1–2氟烷基、C_3–4环烷基、C_3–4氟环烷基或–NO₂；R₃ is H, F, Cl, Br, –CN, C_1–3 alkyl, C_1–2 fluoroalkyl, C_3–4 cycloalkyl, C_3–4 fluorocycloalkyl, or –NO₂ ;R₄为–CH₂R_4a、–CH₂CH₂R_4a、–CH₂CHR_4aR_4d、–CHR_4aR_4b或–CR_4aR_4bR_4c；R₄ is –CH₂ R_4a , –CH₂ CH₂ R_4a , –CH₂ CHR_4a R_4d , –CHR_4a R_4b or –CR_4a R_4b R_4c ;R_4a和R_4b独立地为：R_4a and R_4b are independently:(i)经0至4个独立地选自F、Cl、–CN、–OH、–OCH₃、–SCH₃、C_1–3氟烷氧基、–NR_aR_a、–S(O)₂R_e或–NR_aS(O)₂R_e的取代基取代的C_1–6烷基；(i) C 1-6 alkyl substituted with 0 to 4 substituents independently selected from F, Cl, —CN, —OH, —OCH₃ , —SCH₃ , C_1-3 fluoroalkoxy, —NR_a R_a , —S(O)₂ R_e or —NR_a S(O₎₂ R_e ;(ii)C_3–6环烷基、杂环基、苯基或杂芳基，各自经0至4个独立地选自F、Cl、Br、–CN、–OH、C_1–6烷基、C_1–3氟烷基、C_1–4羟基烷基、–(CH₂)_1–2O(C_1–3烷基)、C_1–4烷氧基、–O(C_1–4羟基烷基)、–O(CH)_1–3O(C_1–3烷基)、C_1–3氟烷氧基、–O(CH)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4烷基)、–C(O)OH、–C(O)O(C_1–4烷基)、–NR_cR_c、–NR_aS(O)₂(C_1–3烷基)、–NR_aC(O)(C_1–3烷基)、–NR_aC(O)O(C_1–4烷基)、–P(O)(C_1–3烷基)₂、–S(O)₂(C_1–3烷基)、–O(CH₂)_1–2(C_3–6环烷基)、–O(CH₂)_1–2(吗啉基)、环丙基、氰基环丙基、甲基氮杂环丁烷基、乙酰基氮杂环丁烷基、(叔丁氧羰基)氮杂环丁烷基、三唑基、四氢吡喃基、吗啉基、噻吩基、甲基哌啶基和R_d的取代基取代；或(ii) C_3-6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each substituted with 0 to 4 independently selected from F, Cl, Br, –CN, –OH, C_1-6 alkyl, C_1-3 fluoroalkyl, C_1-4 hydroxyalkyl, –(CH₂ )_1-2 O(C_1-3 alkyl), C_1-4 alkoxy, –O(C_1-4 hydroxyalkyl), –O(CH)_1-3 O(C_1-3 alkyl), C_1-3 fluoroalkoxy, –O(CH)_1-3 NR_c R_c , –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –C(O)(C_1-4 alkyl), –C(O)OH, –C(O)O(C_1-4 alkyl), –NR_c R_c , –NR_a S(O)₂ (C_1-3 alkyl), –NR_a C(O)(C_1-3 alkyl), –NR_a C(O)O(C_1–4 alkyl), –P(O)(C_1–3 alkyl)₂ , –S(O)₂ (C_1–3 alkyl), –O(CH₂ )_1–2 (C_3–6 cycloalkyl), –O(CH₂ )_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butyloxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, and a substituent of R_d ; or(iii)经一个选自C_3–6环烷基、杂环基、芳基和杂芳基的环状基团取代的C_1–4烷基，所述环状基团经0至3个独立地选自F、Cl、Br、–OH、–CN、C_1–6烷基、C_1–3氟烷基、C_1–3烷氧基、C_1–3氟烷氧基、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3烷基)、–NR_aC(O)(C_1–3烷基)、–NR_aC(O)O(C_1–4烷基)和C_3–6环烷基的取代基取代；(iii) C_1-4 alkyl substituted with a cyclic group selected from C_3-6 cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the cyclic group is substituted with 0 to 3 substituents independently selected from F, Cl, Br, –OH, –CN, C_1-6 alkyl, C_1-3 fluoroalkyl, C_1-3 alkoxy, C_1-3 fluoroalkoxy, –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –NR_c R_c , –NR_a S(O)₂ (C_1-3 alkyl), –NR_a C(O)(C_1-3 alkyl), –NR_a C(O)O(C_1-4 alkyl) and C_3-6 cycloalkyl;或R_4a和R_4b与其所连接的碳原子一起形成C_3–6环烷基或3-至6-元杂环基，各自经0至3个R_f取代；or R_4a and R_4b together with the carbon atom to which they are attached form a C_3-6 cycloalkyl or a 3- to 6-membered heterocyclyl, each substituted with 0 to 3 R_f ;每个R_f独立地为F、Cl、Br、–OH、–CN、C_1–6烷基、C_1–3氟烷基、C_1–3烷氧基、C_1–3氟烷氧基、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c，或选自C_3–6环烷基、3-至6-元杂环基、苯基、单环杂芳基和双环杂芳基的环状基团，每个环状基团经0至3个独立地选自F、Cl、Br、–OH、–CN、C_1–6烷基、C_1–3氟烷基、C_1–3烷氧基、C_1–3氟烷氧基和–NR_cR_c的取代基取代；each R_f is independently F, Cl, Br, –OH, –CN, C_1-6 alkyl, C_1-3 fluoroalkyl, C_1-3 alkoxy, C_1-3 fluoroalkoxy, –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –NR_c R_c , or a cyclic group selected from C_3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, Cl, Br, –OH, –CN, C_1-6 alkyl, C_1-3 fluoroalkyl, C_1-3 alkoxy, C_1-3 fluoroalkoxy, and –NR_c R_c ;R_4c为C_1–6烷基或C_3–6环烷基，各自经0至4个独立地选自F、Cl、–OH、C_1–2烷氧基、C_1–2氟烷氧基和–CN的取代基取代；R_4c is C_1-6 alkyl or C_3-6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, Cl, -OH, C_1-2 alkoxy, C_1-2 fluoroalkoxy and -CN;R_4d为–OCH₃；R_4d is –OCH₃ ;每个R_c独立地为H或C_1–2烷基；Each R_c is independently H or C_1-2 alkyl;R_d为经0至1个选自F、Cl、–CN、–CH₃和–OCH₃的取代基取代的苯基；R_d is phenyl substituted with 0 to 1 substituents selected from F, Cl, -CN, -CH₃ and -OCH₃ ;每个R₅独立地为–CN、经0至4个R_g取代的C_1–6烷基、经0至4个R_g取代的C_2–4烯基、经0至4个R_g取代的C_2–4炔基、经0至4个R_g取代的C_3–4环烷基、经0至4个R_g取代的苯基、经0至3个R_g取代的噁二唑基、经0至4个R_g取代的吡啶基、–(CH₂)_1–2(经0至4个R_g取代的杂环基)、–(CH₂)_1–2NR_cC(O)(C_1–4烷基)、–(CH₂)_1–2NR_cC(O)O(C_1–4烷基)、–(CH₂)_1–2NR_cS(O)₂(C_1–4烷基)、–C(O)(C_1–4烷基)、–C(O)OH、–C(O)O(C_1–4烷基)、–C(O)O(C_3–4环烷基)、–C(O)NR_aR_a或–C(O)NR_a(C_3–4环烷基)；each R₅ is independently –CN, C_1-6 alkyl substituted with 0 to 4 R_g , C_2-4 alkenyl substituted with 0 to 4 R_g , C 2-4 alkynyl substituted with 0 to 4 R_g , C_{3-4cycloalkyl} substituted with 0 to 4 R_g , phenyl substituted with 0 to 4 R g , oxadiazolyl substituted with 0 to 3 R_g , pyridyl substituted with 0 to 4 R_g , –(CH₂ )_1-2 (heterocyclyl substituted with 0 to 4_Rg ), –(CH₂ )_1-2 NR_c C(O)(C_1-4 alkyl), –(CH₂ )_1-2 NR_c C(O)O(C_1-4 alkyl), –(CH₂ )_1-2 NR_c S(O)₂ (C_1-4 alkyl), –C(O)(C_1–4 alkyl), –C(O)OH, –C(O)O(C_1–4 alkyl), –C(O)O(C_3–4 cycloalkyl), –C(O)NR_a R_a or –C(O)NR_a (C_3–4 cycloalkyl);每个R_g独立地为F、Cl、–CN、–OH、C_1–3烷氧基、C_1–3氟烷氧基、–O(CH₂)_1–2O(C_1–2烷基)或–NR_cR_c；each R_g is independently F, Cl, –CN, –OH, C_1-3 alkoxy, C_1-3 fluoroalkoxy, –O(CH₂ )_1-2 O(C_1-2 alkyl), or –NR_c R_c ;m为0、1、2或3；以及m is 0, 1, 2 or 3; andn为0、1或2。n is 0, 1 or 2.54.根据权利要求53所述的方法，其中所述抑制剂为式(I)化合物或其药学上可接受的盐，其中：54. The method of claim 53, wherein the inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:R₁为H、F、Cl、Br、–CN、经0至4个R_1a取代的C_1–3烷基、经0至3个R_1a取代的环丙基、经0至3个R_1a取代的C_1–3烷氧基、–NR_aR_a、–S(O)_nCH₃或–P(O)(CH₃)₂；R₁ is H, F, Cl, Br, –CN, C_1-3 alkyl substituted by 0 to 4 R_1a , cyclopropyl substituted by 0 to 3 R_1a , C_1-3 alkoxy substituted by 0 to 3 R_1a , –NR_a R_a , –S(O)_n CH₃ or –P(O)(CH₃ )₂ ;每个R_1a独立地为F、Cl或–CN；Each R_1a is independently F, Cl or –CN;每个R_a独立地为H或C_1–3烷基；Each_Ra is independently H or_C1-3 alkyl;R₂为H或经0至2个R_2a取代的C_1–2烷基；R₂ is H or C_1-2 alkyl substituted with 0 to 2 R_2a ;每个R_2a独立地为F、Cl、–CN、–OH、–O(C_1–2烷基)、环丙基、C_3–4烯基或C_3–4炔基；each R_2a is independently F, Cl, –CN, –OH, –O(C_1-2 alkyl), cyclopropyl, C_3-4 alkenyl or C_3-4 alkynyl;R₃为H、F、Cl、Br、–CN、C_1–2烷基、–CF₃、环丙基或–NO₂；R₃ is H, F, Cl, Br, –CN, C_1–2 alkyl, –CF₃ , cyclopropyl or –NO₂ ;R_4a和R_4b独立地为：R_4a and R_4b are independently:(i)经0至4个独立地选自F、Cl、–CN、–OH、–OCH₃、–SCH₃、C_1–3氟烷氧基和–NR_aR_a的取代基取代的C_1–4烷基；(i) C_1-4 alkyl substituted with 0 to 4 substituents independently selected from F, Cl, —CN, —OH, —OCH₃ , —SCH₃ , C_1-3 fluoroalkoxy, and —NR_a R_a ;(ii)C_3–6环烷基、杂环基、苯基或杂芳基，各自经0至4个独立地选自F、Cl、Br、–CN、–OH、C_1–6烷基、C_1–3氟烷基、–CH₂OH、–(CH₂)_1–2O(C_1–2烷基)、C_1–4烷氧基、–O(C_1–4羟基烷基)、–O(CH)_1–2O(C_1–2烷基)、C_1–3氟烷氧基、–O(CH)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、C(O)(C_1–4烷基)、–C(O)OH、–C(O)O(C_1–4烷基)、–NR_cR_c、–NR_aS(O)₂(C_1–3烷基)、–NR_aC(O)(C_1–3烷基)、–NR_aC(O)O(C_1–4烷基)、–P(O)(C_1–2烷基)₂、–S(O)₂(C_1–3烷基)、–O(CH₂)_1–2(C_3–4环烷基)、–O(CH₂)_1–2(吗啉基)、环丙基、氰基环丙基、甲基氮杂环丁烷基、乙酰基氮杂环丁烷基、(叔丁氧羰基)氮杂环丁烷基、三唑基、四氢吡喃基、吗啉基、噻吩基、甲基哌啶基和R_d的取代基取代；或(ii) C_3-6 cycloalkyl, heterocyclyl, phenyl or heteroaryl, each substituted with 0 to 4 independently selected from F, Cl, Br, –CN, –OH, C_1-6 alkyl, C_1-3 fluoroalkyl, –CH₂ OH, –(CH₂ )_1-2 O(C_1-2 alkyl), C_1-4 alkoxy, –O(C_1-4 hydroxyalkyl), –O(CH)_1-2 O(C_1-2 alkyl), C_1-3 fluoroalkoxy, –O(CH)_1-2 NR_c R_c , –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, C(O)(C_1-4 alkyl), –C(O)OH, –C(O)O(C_1-4 alkyl), –NR_c R_c , –NR_a S(O)₂ (C_1-3 alkyl), –NR_a C(O)(C_1-3 alkyl), –NR_a C(O)O(C_1–4 alkyl), –P(O)(C_1–2 alkyl)₂ , –S(O)₂ (C_1–3 alkyl), –O(CH₂ )_1–2 (C_3–4 cycloalkyl), –O(CH₂ )_1–2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butyloxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, and a substituent of R_d ; or(iii)经一个选自C_3–6环烷基、杂环基、苯基和杂芳基的环状基团取代的C_1–3烷基，所述环状基团经0至3个独立地选自F、Cl、Br、–OH、–CN、C_1–3烷基、C_1–2氟烷基、C_1–3烷氧基、C_1–2氟烷氧基、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3烷基)、–NR_aC(O)(C_1–3烷基)、–NR_aC(O)O(C_1–4烷基)和C_3–4环烷基的取代基取代；(iii) C_1-3 alkyl substituted with a cyclic group selected from C_3-6 cycloalkyl, heterocyclyl, phenyl and heteroaryl, wherein the cyclic group is substituted with 0 to 3 substituents independently selected from F, Cl, Br, –OH, –CN, C_1-3 alkyl, C_1-2 fluoroalkyl, C_1-3 alkoxy, C_1-2 fluoroalkoxy, –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –NR_c R_c , –NR_a S(O)₂ (C_1-3 alkyl), –NR_a C(O)(C_1-3 alkyl), –NR_a C(O)O(C_1-4 alkyl) and C_3-4 cycloalkyl;或R_4a和R_4b与其所连接的碳原子一起形成C_3–6环烷基或3-至6-元杂环基，各自经0至3个R_f取代；or R_4a and R_4b together with the carbon atom to which they are attached form a C_3-6 cycloalkyl or a 3- to 6-membered heterocyclyl, each substituted with 0 to 3 R_f ;每个R_f独立地为F、Cl、Br、–OH、–CN、C_1–4烷基、C_1–2氟烷基、C_1–3烷氧基、C_1–2氟烷氧基、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c或选自C_3–6环烷基、3-至6-元杂环基、苯基、单环杂芳基和双环杂芳基的环状基团，每个环状基团经0至3个独立地选自F、Cl、Br、–OH、–CN、C_1–4烷基、C_1–2氟烷基、C_1–3烷氧基、C_1–2氟烷氧基和–NR_cR_c的取代基取代；each R_f is independently F, Cl, Br, –OH, –CN, C_1-4 alkyl, C_1-2 fluoroalkyl, C_1-3 alkoxy, C_1-2 fluoroalkoxy, –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –NR_c R_c , or a cyclic group selected from C_3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, Cl, Br, –OH, –CN, C_1-4 alkyl, C_1-2 fluoroalkyl, C_1-3 alkoxy, C_1-2 fluoroalkoxy, and –NR_c R_c ;R_4c为C_1–4烷基或C_3–6环烷基，各自经0至4个独立地选自F、Cl、–OH、C_1–2烷氧基、C_1–2氟烷氧基和–CN的取代基取代；R_4c is C_1-4 alkyl or C_3-6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, Cl, -OH, C_1-2 alkoxy, C_1-2 fluoroalkoxy and -CN;以及每个R₅独立地为–CN、经0至4个R_g取代的C_1–5烷基、经0至4个R_g取代的C_2–3烯基、经0至4个R_g取代的C_2–3炔基、经0至4个R_g取代的C_3–4环烷基、经0至3个R_g取代的苯基、经0至3个R_g取代的噁二唑基、经0至3个R_g取代的吡啶基、–(CH₂)_1–2(经0至4个R_g取代的杂环基)、–(CH₂)_1–2NR_cC(O)(C_1–4烷基)、–(CH₂)_1–2NR_cC(O)O(C_1–4烷基)、–(CH₂)_1–2NR_cS(O)₂(C_1–4烷基)、–C(O)(C_1–4烷基)、–C(O)OH、–C(O)O(C_1–4烷基)、–C(O)O(C_3–4环烷基)、–C(O)NR_aR_a或–C(O)NR_a(C_3–4环烷基)。and each R₅ is independently –CN, C_1-5 alkyl substituted with 0 to 4 R_g substituted, C_2-3 alkenyl substituted with 0 to 4 R_g substituted, C 2-3 alkynyl substituted with 0 to 4 R_g substituted, C_{3-4cycloalkyl} substituted with 0 to 4 R_g substituted, phenyl substituted with 0 to 3 R_g substituted, oxadiazolyl substituted with 0 to 3 R_g substituted, pyridinyl substituted with 0 to 3 R_g substituted, –(CH₂ )_1-2 (heterocyclyl substituted with 0 to 4 R_g substituted), –(CH₂ )_1-2 NR_c C(O)(C_1-4 alkyl), –(CH₂ )_1-2 NR_c C(O)O(C_1-4 alkyl), –(CH₂ )_1-2 NR_c S(O)₂ (C_1-4 alkyl), –C(O)(C_1–4 alkyl), –C(O)OH, –C(O)O(C_1–4 alkyl), –C(O)O(C_3–4 cycloalkyl), –C(O)NR_a R_a or –C(O)NR_a (C_3–4 cycloalkyl).55.根据权利要求54所述的方法，其中所述抑制剂为式(I)化合物或其药学上可接受的盐，其具有结构：55. The method of claim 54, wherein the inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof having the structure:其中：in:R₁为–CN；_R1 is –CN;R₂为–CH₃；R₂ is –CH₃ ;R₃为H、F或–CN；_R3 is H, F or –CN;R₄为：_R4 is:56.根据权利要求53所述的方法，其中所述抑制剂为式(I)化合物或其药学上可接受的盐，其具有结构：56. The method of claim 53, wherein the inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof having the structure:57.根据权利要求1-52中任一项所述的化合物，其中所述抑制剂为式(II)化合物：57. The compound according to any one of claims 1-52, wherein the inhibitor is a compound of formula (II):或其盐，其中：or a salt thereof, wherein:R₁为H、F、Cl、Br、–CN、–OH、经0至4个R_1a取代的C_1–3烷基、经0至4个R_1a取代的C_3–4环烷基、经0至4个R_1a取代的C_1–3烷氧基、–NR_aR_a、–S(O)_nR_e或–P(O)R_eR_e；R₁ is H, F, Cl, Br, –CN, –OH, C_1-3 alkyl substituted with 0 to 4 R_1a , C_3-4 cycloalkyl substituted with 0 to 4 R_1a , C_1-3 alkoxy substituted with 0 to 4 R_1a , –NR_a R_a , –S(O)_n R_e or –P(O)R e R_e;每个R_1a独立地为F、Cl、–CN、–OH、–OCH₃或–NR_aR_a；Each R_1a is independently F, Cl, –CN, –OH, –OCH₃ or –NR_a R_a ;每个R_a独立地为H或C_1–3烷基；Each_Ra is independently H or_C1-3 alkyl;每个R_e独立地为经0至4个R_1a取代的C_3–4环烷基或C_1–3烷基；Each_Re is independently_C3-4 cycloalkyl or_C1-3 alkyl substituted with 0 to 4_R1a ;R₂为H、经0至4个R_2a取代的C_1–3烷基或经0至4个R_2a取代的C_3–4环烷基；R₂ is H, C_1-3 alkyl substituted by 0 to 4 R_2a , or C_3-4 cycloalkyl substituted by 0 to 4 R_2a ;每个R_2a独立地为F、Cl、–CN、–OH、–O(C_1–2烷基)、C_3–4环烷基、C_3–4烯基或C_3–4炔基；each R_2a is independently F, Cl, –CN, –OH, –O(C_1-2 alkyl), C_3-4 cycloalkyl, C_3-4 alkenyl, or C_3-4 alkynyl;R₄为–CH₂R_4a、–CH₂CH₂R_4a、–CH₂CHR_4aR_4d、–CHR_4aR_4b或–CR_4aR_4bR_4c；R₄ is –CH₂ R_4a , –CH₂ CH₂ R_4a , –CH₂ CHR_4a R_4d , –CHR_4a R_4b or –CR_4a R_4b R_4c ;R_4a和R_4b独立地为：R_4a and R_4b are independently:(i)–CN或经0至4个独立地选自F、Cl、–CN、–OH、–OCH₃、–SCH₃、C_1–3氟烷氧基、–NR_aR_a、–S(O)₂R_e或–NR_aS(O)₂R_e的取代基取代的C_1–6烷基；(i) -CN or C 1-6 alkyl substituted with 0 to 4 substituents independently selected from F, Cl, -CN, -OH, -OCH₃ , -SCH₃ , C_1-3 fluoroalkoxy, -NR_a R_a , -S(O)₂ R_eor -NR_a S(O)₂ R_e ;(ii)C_3–6环烷基、4-至10-元杂环基、苯基或5-至10-元杂芳基，各自经0至4个独立地选自F、Cl、Br、–CN、–OH、C_1–6烷基、C_1–3氟烷基、C_1–2溴烷基、C_1–2氰基烷基、C_1–4羟基烷基、–(CH₂)_1–2O(C_1–3烷基)、C_1–4烷氧基、C_1–3氟烷氧基、C_1–3氰基烷氧基、–O(C_1–4羟基烷基)、-O(CR_xR_x)_1-3O(C_1-3烷基)、C_1–3氟烷氧基、–O(CH₂)_1–3NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4烷基)、–C(O)OH、–C(O)O(C_1–4烷基)、–NR_cR_c、–CH₂NR_aNR_a、–NR_aS(O)₂(C_1–3烷基)、–NR_aC(O)(C_1–3烷基)、-(CR_xR_x)_0-2NR_aC(O)O(C_1-4烷基)、–P(O)(C_1–3烷基)₂、–S(O)₂(C_1–3烷基)、-(CR_xR_x)_1-2(C_3-4环烷基)、–(CR_xR_x)_1–2(吗啉基)、–(CR_xR_x)_1–2(二氟吗啉基)、–(CR_xR_x)_1–2(二甲基吗啉基)、–(CR_xR_x)_1–2(氧杂氮杂双环[2.2.1]庚烷基)、(CR_xR_x)_1–2(氧杂氮杂螺[3.3]庚烷基)、–(CR_xR_x)_1–2(甲基哌嗪酮基)、–(CR_xR_x)_1–2(乙酰基哌嗪基)、–(CR_xR_x)_1–2(哌啶基)、–(CR_xR_x)_1–2(二氟哌啶基)、–(CR_xR_x)_1–2(甲氧基哌啶基)、–(CR_xR_x)_1–2(羟基哌啶基)、–O(CR_xR_x)_0–2(C_3–6环烷基)、–O(CR_xR_x)_0–2(甲基环丙基)、–O(CR_xR_x)_0–2((乙氧基羰基)环丙基)、–O(CR_xR_x)_0–2(氧杂环丁烷基)、–O(CR_xR_x)_0–2(甲基氮杂环丁烷基)、–O(CR_xR_x)_0–2(四氢吡喃基)、–O(CR_xR_x)_1–2(吗啉基)、–O(CR_xR_x)_0–2(噻唑基)、环丙基、氰基环丙基、甲基氮杂环丁烷基、乙酰基氮杂环丁烷基、(叔丁氧羰基)氮杂环丁烷基、三唑基、四氢吡喃基、吗啉基、噻吩基、甲基哌啶基、二氧戊环基、吡咯烷酮基和R_d的取代基取代；或(ii) C_3-6 cycloalkyl, 4- to 10-membered heterocyclyl, phenyl or 5- to 10-membered heteroaryl, each substituted with 0 to 4 independently selected from F, Cl, Br, –CN, –OH, C_1-6 alkyl, C_1-3 fluoroalkyl, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1-4 hydroxyalkyl, –(CH₂ )_1-2 O(C_1-3 alkyl), C_1-4 alkoxy, C_1-3 fluoroalkoxy, C_1-3 cyanoalkoxy, –O(C_1-4 hydroxyalkyl), –O(CR_x R_x )_1-3 O(C_1-3 alkyl), C_1-3 fluoroalkoxy, –O(CH₂ )_1-3 NR_c R_c , –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –C(O)(C_{in 1-4} alkyl groups), –C(O)OH, –C(O)O(C_1–4 alkyl), –NR_c R_c , –CH₂ NR_a NR_a , –NR_a S(O)₂ (C_1–3 alkyl), –NR_a C(O)(C_1–3 alkyl), –(CR_x R_x )_0-2 NR_a C(O)O(C_1-4 alkyl), –P(O)(C_1–3 alkyl)₂ , –S(O)₂ (C_1–3 alkyl), –(CR_x R_x )_1-2 (C_3-4 cycloalkyl), –(CR_x R_x )_1–2 (morpholinyl), –(CR_x R_x )_1–2 (difluoromorpholinyl), –(CR_x R_x )_1–2 (dimethylmorpholinyl), –(CR_x R_x )_1–2 (oxaazabicyclo[2.2.1]heptyl), (CR_x R_x )_1–2 (oxazaspiro[3.3]heptanyl), –(CR_x R_x )_1–2 (methylpiperazinyl), –(CR x R_x )_1–2 (acetylpiperazinyl), –(CR_x R_x )_1–2 (piperidinyl), –(CR_x R_x )_1–2 (difluoropiperidinyl), –(CR_x R_x )_1–2 (methoxypiperidinyl), –(CR_x R_x )_1–2 (hydroxypiperidinyl), –O(CR_x R_x )_0–2 (C_{3–6cycloalkyl} ), –O(_{CR xRx} )_0–2 (methylcyclopropyl), –O(CR_x R_x )_0–2 ((ethoxycarbonyl)cyclopropyl), –O(CR_x R_x )_0–2 (oxetanyl), –O(CR_x R_x )_0–2 (methylazetidinyl), –O(CR_x R_x )_0–2 (tetrahydropyranyl), –O(CR_x R_x )_1–2 (morpholinyl), –O(CR_x R_x )_0–2 (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butyloxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, dioxolanyl, pyrrolidinyl, and a substituent of R_d ; or(iii)经一个选自C_3–6环烷基、4-至10-元杂环基、单环或双环芳基或5-至10-元杂芳基的环状基团取代的C_1–4烷基，所述环状基团经0至3个独立地选自F、Cl、Br、–OH、–CN、C_1–6烷基、C_1–3氟烷基、C_1–3烷氧基、C_1–3氟烷氧基、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3烷基)、–NR_aC(O)(C_1–3烷基)、–NR_aC(O)O(C_1–4烷基)和C_3–6环烷基的取代基取代；(iii) a C_1-4 alkyl group substituted with a cyclic group selected from C_3-6 cycloalkyl, a 4- to 10-membered heterocyclyl, a monocyclic or bicyclic aryl, or a 5- to 10-membered heteroaryl, wherein the cyclic group is substituted with 0 to 3 substituents independently selected from F, Cl, Br, –OH, –CN, C_1-6 alkyl, C_1-3 fluoroalkyl, C_1-3 alkoxy, C_1-3 fluoroalkoxy, –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –NR_c R_c , –NR_a S(O)₂ (C_1-3 alkyl), –NR_a C(O)(C_1-3 alkyl), –NR_a C(O)O(C_1-4 alkyl) and C_3-6 cycloalkyl;或R_4a和R_4b与它们所连接的碳原子一起形成C_3–6环烷基或3-至6-元杂环基，各自经0至3个R_f取代；or R_4a and R_4b together with the carbon atom to which they are attached form a C_3-6 cycloalkyl or a 3- to 6-membered heterocyclyl, each substituted with 0 to 3 R_f ;每个R_f独立地为F、Cl、Br、–OH、–CN、C_1–6烷基、C_1–3氟烷基、C_1–3烷氧基、C_1–3氟烷氧基、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c或选自C_3–6环烷基、3-至6-元杂环基、苯基、单环杂芳基和双环杂芳基的环状基团，每个环状基团经0至3个独立地选自F、Cl、Br、–OH、–CN、C_1–6烷基、C_1–3氟烷基、C_1–3烷氧基、C_1–3氟烷氧基和–NR_cR_c的取代基取代；each R_f is independently F, Cl, Br, –OH, –CN, C_1-6 alkyl, C_1-3 fluoroalkyl, C_1-3 alkoxy, C_1-3 fluoroalkoxy, –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –NR_c R_c , or a cyclic group selected from C_3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, Cl, Br, –OH, –CN, C_1-6 alkyl, C_1-3 fluoroalkyl, C_1-3 alkoxy, C_1-3 fluoroalkoxy, and –NR_c R_c ;R_4c为C_1–6烷基或C_3–6环烷基，各自经0至4个独立地选自F、Cl、–OH、C_1–2烷氧基、C_1–2氟烷氧基和–CN的取代基取代；R_4c is C_1-6 alkyl or C_3-6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, Cl, -OH, C_1-2 alkoxy, C_1-2 fluoroalkoxy and -CN;R_4d为–OCH₃；R_4d is –OCH₃ ;每个R_c独立地为H或C_1–2烷基；Each R_c is independently H or C_1-2 alkyl;R_d为经0或1个选自F、Cl、–CN、–CH₃和–OCH₃的取代基取代的苯基；R_d is phenyl substituted with 0 or 1 substituents selected from F, Cl, -CN, -CH₃ and -OCH₃ ;每个R₅独立地为–CN、经0至4个R_g取代的C_1–6烷基、经0至4个R_g取代的C_2–4烯基、经0至4个R_g取代的C_2–4炔基、经0至4个R_g取代的C_3–4环烷基、经0至4个R_g取代的苯基、经0至3个R_g取代的噁二唑基、经0至4个R_g取代的吡啶基、–(CH₂)_1–2(经0至4个R_g取代的4-至10-元杂环基)、–(CH₂)_1–2NR_cC(O)(C_1–4烷基)、–(CH₂)_1–2NR_cC(O)O(C_1–4烷基)、–(CH₂)_1–2NR_cS(O)₂(C_1–4烷基)、–C(O)(C_1–4烷基)、–C(O)OH、–C(O)O(C_1–4烷基)、–C(O)O(C_3–4环烷基)、–C(O)NR_aR_a或–C(O)NR_a(C_3–4环烷基)；each R₅ is independently –CN, C_1-6 alkyl substituted with 0 to 4 R_g , C_2-4 alkenyl substituted with 0 to 4 R_g , C 2-4 alkynyl substituted with 0 to 4 R_g , C_{3-4cycloalkyl} substituted with 0 to 4 R_g , phenyl substituted with 0 to 4 R_g , oxadiazolyl substituted with 0 to 3 R_g , pyridinyl substituted with 0 to 4 R_g , –(CH₂ )_1-2 (4- to 10-membered heterocyclyl substituted with 0 to 4 R_g ), –(CH 2 )_1-2 NR_c C(O)(C_1-4 alkyl), –(CH₂ )_1-2 NR_c C(O)O(C_1-4 alkyl), –(CH₂ )_1-2 NR_c S(O)₂ (C_1-4 alkyl), –C(O)(_C1–4 alkyl), –C(O)OH, –C(O)O(C_1–4 alkyl), –C(O)O(C_3–4 cycloalkyl), –C(O)NR_a R_a or –C(O)NR_a (C_3–4 cycloalkyl);每个R_g独立地为F、Cl、–CN、–OH、C_1–3烷氧基、C_1–3氟烷氧基、–O(CH₂)_1–2O(C_1–2烷基)或–NR_cR_c；each R_g is independently F, Cl, –CN, –OH, C_1-3 alkoxy, C_1-3 fluoroalkoxy, –O(CH₂ )_1-2 O(C_1-2 alkyl), or –NR_c R_c ;m为0、1、2或3；以及m is 0, 1, 2 or 3; andn为0、1或2。n is 0, 1 or 2.58.根据权利要求57所述的方法，其中所述抑制剂为式(II)化合物或其药学上可接受的盐，其中：58. The method of claim 57, wherein the inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein:R₁为H、F、Cl、Br、–CN、–OH、经0至4个R_1a取代的C_1–3烷基、经0至3个R_1a取代的环丙基、经0至3个R_1a取代的C_1–3烷氧基、–NR_aR_a、–S(O)_nCH₃或–P(O)(CH₃)₂；R₁ is H, F, Cl, Br, –CN, –OH, C_1-3 alkyl substituted with 0 to 4 R_1a , cyclopropyl substituted with 0 to 3 R_1a , C_1-3 alkoxy substituted with 0 to 3 R_1a , –NR_a R_a , –S(O)_n CH₃ or –P(O)(CH₃ )₂ ;R₂为H或经0至2个R_2a取代的C_1–2烷基；R₂ is H or C_1-2 alkyl substituted with 0 to 2 R_2a ;每个R_2a独立地为F、Cl、–CN、–OH、–O(C_1–2烷基)、环丙基、C_3–4烯基或C_3–4炔基；each R_2a is independently F, Cl, –CN, –OH, –O(C_1-2 alkyl), cyclopropyl, C_3-4 alkenyl or C_3-4 alkynyl;R_4a和R_4b独立地为：R_4a and R_4b are independently:(i)–CN或经0至4个独立地选自F、Cl、–CN、–OH、–OCH₃、–SCH₃、C_1–3氟烷氧基和–NR_aR_a的取代基取代的C_1–4烷基；(i) -CN or C_1-4 alkyl substituted with 0 to 4 substituents independently selected from F, Cl, -CN, -OH, -OCH₃ , -SCH₃ , C_1-3 fluoroalkoxy and -NR_a R_a ;(ii)C_3–6环烷基、4-至10-元杂环基、苯基或5-至10-元杂芳基，各自经0至4个独立地选自F、Cl、Br、–CN、–OH、C_1–6烷基、C_1–3氟烷基、C_1-2溴烷基、C_1-2氰基烷基、C_1-2羟基烷基、–CH₂NR_aR_a、–(CH₂)_1–2O(C_1–2烷基)、–(CH₂)_1–2NR_xC(O)O(C_1-2烷基)、C_1–4烷氧基、–O(C_1–4羟基烷基)、–O(CR_xR_x)_1–2O(C_1–2烷基)、C_1–3氟烷氧基、C_1–3氰基烷氧基、–O(CH₂)_1–2NR_cR_c、–OCH₂CH＝CH₂、–OCH₂C≡CH、–C(O)(C_1–4烷基)、–C(O)OH、–C(O)O(C_1–4烷基)、–NR_cR_c、–NR_aS(O)₂(C_1–3烷基)、–NR_aC(O)(C_1–3烷基)、–NR_aC(O)O(C_1–4烷基)、–P(O)(C_1–2烷基)₂、–S(O)₂(C_1–3烷基)、–(CH₂)_1–2(C_3–4环烷基)、–CR_xR_x(吗啉基)、–CR_xR_x(二氟吗啉基)、–CR_xR_x(二甲基吗啉基)、–CR_xR_x(氧杂氮杂双环[2.2.1]庚烷基)、–CR_xR_x(氧杂氮杂螺[3.3]庚烷基)、–CR_xR_x(甲基哌嗪酮基)、–CR_xR_x(乙酰基哌嗪基)、–CR_xR_x(哌啶基)、–CR_xR_x(二氟哌啶基)、–CR_xR_x(甲氧基哌啶基)、–CR_xR_x(羟基哌啶基)、–O(CH₂)_0–2(C_3–4环烷基)、–O(CH₂)_0–2(甲基环丙基)、–O(CH₂)_0–2((乙氧基羰基)环丙基)、–O(CH₂)_0–2(氧杂环丁烷基)、–O(CH₂)_0–2(甲基氮杂环丁烷基)、–O(CH₂)_1–2(吗啉基)、–O(CH₂)_0–2(四氢吡喃基)、–O(CH₂)_0–2(噻唑基)、环丙基、氰基环丙基、甲基氮杂环丁烷基、乙酰基氮杂环丁烷基、(叔丁氧羰基)氮杂环丁烷基、二氧戊环基、吡咯烷酮基、三唑基、四氢吡喃基、吗啉基、噻吩基、甲基哌啶基和R_d的取代基取代；或(ii) C_3-6 cycloalkyl, 4- to 10-membered heterocyclyl, phenyl or 5- to 10-membered heteroaryl, each independently selected from F, Cl, Br, –CN, –OH, C_1-6 alkyl, C_1-3 fluoroalkyl, C_1-2 bromoalkyl, C_1-2 cyanoalkyl, C_1-2 hydroxyalkyl, –CH₂ NR_a R_a , –(CH₂ )_1-2 O(C_1-2 alkyl), –(CH₂ )_1-2 NR_x C(O)O(C_1-2 alkyl), C_1-4 alkoxy, –O(C_1-4 hydroxyalkyl), –O(CR_x R_x )_1-2 O(C_1-2 alkyl), C_1-3 fluoroalkoxy, C_1-3 cyanoalkoxy, –O(CH₂ )_1-2 NR_c R_c , –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –C(O)(C_1–4 alkyl), –C(O)OH, –C(O)O(C_1–4 alkyl), –NR_c R_c , –NR_a S(O)₂ (C_1–3 alkyl), –NR_a C(O)(C_1–3 alkyl), –NR_a C(O)O(C_1–4 alkyl), –P(O)(C_1–2 alkyl)₂ , –S(O)₂ (C_1–3 alkyl), –(CH₂ )_1–2 (C_3–4 cycloalkyl), –CR_x R_x (morpholinyl), –CR_x R_x (difluoromorpholinyl), –CR_x R_x (dimethylmorpholinyl), –CR_x R_x (oxazabicyclo[2.2.1]heptyl), –CR_x R_x (oxazaspiro[3.3]heptyl), –CR_x R_x (methylpiperazinyl), –CR_x R_x (acetylpiperazinyl), –CR_x R_x (piperidinyl), –CR_x R_x (difluoropiperidinyl), –CR_x R x (methoxypiperidinyl), –CR_x R_x (hydroxypiperidinyl), –O(CH₂ )_0–2 (C_{3–4cycloalkyl} ), –O(_CH 2 )_0–2 (methylcyclopropyl), –O(CH₂ ) 0–2 ((ethoxycarbonyl)cyclopropyl), –O(CH₂ )_0–2 (oxetanyl), –O(CH₂ )_0–2 (methylazetidinyl), –O(CH₂ )_1–2 (_morpholinyl ), –O(CH₂ )_0–2 (tetrahydropyranyl)_{, –O(CH 2)0–2} R is substituted with a substituent selected from the group consisting of (thiazolyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butyloxycarbonyl)azetidinyl, dioxolanyl, pyrrolidinone, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl and R_d ; or(iii)经一个选自C_3–6环烷基、4-至10-元杂环基、单环或双环芳基或5-至10-元杂芳基的环状基团取代的C_1–3烷基，所述环状基团经0至3个独立地选自F、Cl、Br、–OH、–CN、C_1–3烷基、C_1–2氟烷基、C_1–3烷氧基、C_1–2氟烷氧基、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c、–NR_aS(O)₂(C_1–3烷基)、–NR_aC(O)(C_1–3烷基)、–NR_aC(O)O(C_1–4烷基)和C_3–4环烷基的取代基取代；(iii) a C_1-3 alkyl group substituted with a cyclic group selected from C_3-6 cycloalkyl, a 4- to 10-membered heterocyclyl, a monocyclic or bicyclic aryl, or a 5- to 10-membered heteroaryl, wherein the cyclic group is substituted with 0 to 3 substituents independently selected from F, Cl, Br, –OH, –CN, C_1-3 alkyl, C_1-2 fluoroalkyl, C_1-3 alkoxy, C_1-2 fluoroalkoxy, –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –NR_c R_c , –NR_a S(O)₂ (C_1-3 alkyl), –NR_a C(O)(C_1-3 alkyl), –NR_a C(O)O(C_1-4 alkyl) and C_3-4 cycloalkyl;或R_4a和R_4b与其连接的碳原子一起形成C_3–6环烷基或3-至6-元杂环基，各自经0至3个R_f取代；or R_4a and R_4b together with the carbon atom to which they are attached form a C_3-6 cycloalkyl or a 3- to 6-membered heterocyclyl, each substituted with 0 to 3 R_f ;每个R_f独立地为F、Cl、Br、–OH、–CN、C_1–4烷基、C_1–2氟烷基、C_1–3烷氧基、C_1–2氟烷氧基、–OCH₂CH＝CH₂、–OCH₂C≡CH、–NR_cR_c，或选自C_3–6环烷基、3-至6-元杂环基、苯基、单环杂芳基和双环杂芳基的环状基团，每个环状基团经0至3个独立地选自F、Cl、Br、–OH、–CN、C_1–4烷基、C_1–2氟烷基、C_1–3烷氧基、C_1–2氟烷氧基和–NR_cR_c的取代基取代；each R_f is independently F, Cl, Br, –OH, –CN, C_1-4 alkyl, C_1-2 fluoroalkyl, C_1-3 alkoxy, C_1-2 fluoroalkoxy, –OCH₂ CH＝CH₂ , –OCH₂ C≡CH, –NR_c R_c , or a cyclic group selected from C_3-6 cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted with 0 to 3 substituents independently selected from F, Cl, Br, –OH, –CN, C_1-4 alkyl, C_1-2 fluoroalkyl, C_1-3 alkoxy, C_1-2 fluoroalkoxy, and –NR_c R_c ;R_4c为C_1–4烷基或C_3–6环烷基，各自经0至4个独立地选自F、Cl、–OH、C_1–2烷氧基、C_1–2氟烷氧基和–CN的取代基取代；R_4c is C_1-4 alkyl or C_3-6 cycloalkyl, each substituted with 0 to 4 substituents independently selected from F, Cl, -OH, C_1-2 alkoxy, C_1-2 fluoroalkoxy and -CN;每个R₅独立地为–CN、经0至4个R_g取代的C_1–5烷基、经0至4个R_g取代的C_2–3烯基、经0至4个R_g取代的C_2–3炔基、经0至4个R_g取代的C_3–4环烷基、经0至3个R_g取代的苯基、经0至3个R_g取代的噁二唑基、经0至3个R_g取代的吡啶基、–(CH₂)_1–2(经0至4个R_g取代的4-至10-元杂环基)、–(CH₂)_1–2NR_cC(O)(C_1–4烷基)、–(CH₂)_1–2NR_cC(O)O(C_1–4烷基)、–(CH₂)_1–2NR_cS(O)₂(C_1–4烷基)、–C(O)(C_1–4烷基)、–C(O)OH、–C(O)O(C_1–4烷基)、–C(O)O(C_3–4环烷基)、–C(O)NR_aR_a或–C(O)NR_a(C_3–4环烷基)；each R₅ is independently –CN, C_1-5 alkyl substituted with 0 to 4 R_g , C_2-3 alkenyl substituted with 0 to 4 R_g , C 2-3 alkynyl substituted with 0 to 4 R_g , C_{3-4cycloalkyl} substituted with 0 to 4 R_g , phenyl substituted with 0 to 3 R_g , oxadiazolyl substituted with 0 to 3 R_g , pyridinyl substituted with 0 to 3 R_g , –(CH₂ )_1-2 (4- to 10-membered heterocyclyl substituted with 0 to 4 R_g ), –(CH 2 )_1-2 NR_c C(O)(C_1-4 alkyl), –(CH₂ )_1-2 NR_c C(O)O(C_1-4 alkyl), –(CH₂ )_1-2 NR_c S(O)₂ (C_1-4 alkyl), –C(O)(_C1–4 alkyl), –C(O)OH, –C(O)O(C_1–4 alkyl), –C(O)O(C_3–4 cycloalkyl), –C(O)NR_a R_a or –C(O)NR_a (C_3–4 cycloalkyl);每个R_x独立地为H或–CH₃；以及each R_x is independently H or -CH₃ ; andm为1、2或3。m is 1, 2 or 3.59.根据权利要求58所述的方法，其中所述抑制剂为式(II)化合物或其药学上可接受盐，其具有结构：59. The method of claim 58, wherein the inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof having the structure:R₁为–CN；_R1 is –CN;R₂为–CH₃；R₂ is –CH₃ ;R_5a为–CH₃或–CH₂CH₃；以及R_5a is -CH₃ or -CH₂ CH₃ ; andR_5c为–CH₃、–CH₂CH₃或–CH₂CH₂CH₃。R_5c is –CH₃ , –CH₂ CH₃ or –CH₂ CH₂ CH₃ .60.根据权利要求57所述的方法，其中所述抑制剂为式(II)化合物或其药学上可接受的盐，其具有结构：60. The method of claim 57, wherein the inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, which has the structure:61.根据权利要求57所述的方法，其中所述抑制剂为式(II)化合物或其药学上可接受的盐，其具有结构：61. The method of claim 57, wherein the inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof having the structure:62.根据权利要求1-13和21-61中任一项所述的方法，其中所述抑制剂以治疗有效量施用。62. The method of any one of claims 1-13 and 21-61, wherein the inhibitor is administered in a therapeutically effective amount.63.根据权利要求62所述的方法，其中所述治疗有效剂量提供在施用所述抑制剂的时间段持续治疗有效的抑制剂剂量。63. The method of claim 62, wherein the therapeutically effective dose provides a dose of the inhibitor that is therapeutically effective over the period of time during which the inhibitor is administered.64.根据权利要求1-63中任一项所述的方法，其中所述抑制剂以在或在约0.25和250mg之间包括0.25和250mg的每天量施用。64. The method of any one of claims 1-63, wherein the inhibitor is administered in an amount per day of between or about 0.25 and 250 mg, inclusive.65.根据权利要求1-64中任一项所述的方法，其中所述抑制剂以在或在约0.5和100mg之间包括0.5和100mg的每天量施用。65. The method of any one of claims 1-64, wherein the inhibitor is administered in an amount per day of between or about 0.5 and 100 mg, inclusive.66.根据权利要求1-65中任一项所述的方法，其中所述抑制剂口服施用。66. The method of any one of claims 1-65, wherein the inhibitor is administered orally.67.根据权利要求1-66中任一项所述的方法，其中所述重组受体为工程化T细胞受体(eTCR)。67. The method of any one of claims 1-66, wherein the recombinant receptor is an engineered T cell receptor (eTCR).68.根据权利要求1-66中任一项所述的方法，其中所述重组受体为嵌合抗原受体(CAR)。68. The method of any one of claims 1-66, wherein the recombinant receptor is a chimeric antigen receptor (CAR).69.根据权利要求68所述的方法，其中所述抗原为CD19，以及所述CAR为抗CD19 CAR。69. The method of claim 68, wherein the antigen is CD19 and the CAR is an anti-CD19 CAR.70.根据权利要求68或权利要求69所述的方法，其中所述CAR为(利基迈仑赛，lisocabtagene maraleucel)、TECARTUS^TM(布基奥仑赛，brexucabtagene autoleucel)、KYMRIAH^TM(司利弗明，tisagenlecleucel)或YESCARTA^TM(阿基仑赛，axicabtageneciloleucel)的CAR。70. The method of claim 68 or claim 69, wherein the CAR is CARs of TRIZOL (lisocabtagene maraleucel), TECARTUS^™ (brexucabtagene autoleucel), KYMRIAH^™ (tisagenlecleucel), or YESCARTA^™ (axicabtagene ciloleucel).71.根据权利要求68-70中任一项所述的方法，其中所述T细胞疗法为(利基迈仑赛，lisocabtagene maraleucel)、TECARTUS^TM(布基奥仑赛，brexucabtageneautoleucel)、KYMRIAH^TM(司利弗明，tisagenlecleucel)或YESCARTA^TM(阿基仑赛，axicabtagene ciloleucel)。71. The method according to any one of claims 68-70, wherein the T cell therapy is (lisocabtagene maraleucel), TECARTUS^™ (brexucabtagene autoleucel), KYMRIAH^™ (tisagenlecleucel) or YESCARTA^™ (axicabtagene ciloleucel).72.根据权利要求68所述的方法，其中所述抗原为BCMA，以及所述CAR为抗BCMACAR。72. The method of claim 68, wherein the antigen is BCMA and the CAR is anti-BCMA CAR.73.根据权利要求68或权利要求72所述的方法，其中所述CAR为(艾迪卡巴金，idecabtagene vicleucel)或CARVYKTI^TM(西达基奥仑赛，ciltacabtagene autoleucel)的CAR。73. The method of claim 68 or claim 72, wherein the CAR is (idecabtagene vicleucel) or CARVYKTI^™ (ciltacabtagene autoleucel).74.根据权利要求68、72和73中任一项所述的方法，其中所述T细胞疗法为(艾迪卡巴金，idecabtagene vicleucel)或CARVYKTI^TM(西达基奥仑赛，ciltacabtageneautoleucel)。74. The method according to any one of claims 68, 72 and 73, wherein the T cell therapy is (idecabtagene vicleucel) or CARVYKTI^™ (ciltacabtagene autoleucel).75.根据权利要求1-74中任一项所述的方法，其中所述癌症为实体肿瘤。75. The method of any one of claims 1-74, wherein the cancer is a solid tumor.76.根据权利要求1-74中任一项所述的方法，其中所述癌症为血液学(液体)肿瘤。76. The method of any one of claims 1-74, wherein the cancer is a hematological (liquid) tumor.77.根据权利要求1-76中任一项所述的方法，其中所述癌症为B细胞恶性肿瘤。77. The method of any one of claims 1-76, wherein the cancer is a B-cell malignancy.78.根据权利要求1-74、76和77中任一项所述的方法，其中所述癌症为白血病。78. The method of any one of claims 1-74, 76, and 77, wherein the cancer is leukemia.79.根据权利要求1-74、76和77中任一项所述的方法，其中所述癌症为淋巴瘤。79. The method of any one of claims 1-74, 76, and 77, wherein the cancer is lymphoma.80.根据权利要求1-74、76和77中任一项所述的方法，其中所述癌症为骨髓瘤。80. The method of any one of claims 1-74, 76, and 77, wherein the cancer is myeloma.81.根据权利要求80所述的方法，其中所述骨髓瘤为多发性骨髓瘤。81. The method of claim 80, wherein the myeloma is multiple myeloma.82.根据权利要求1-81中任一项所述的方法，其中所述癌症为复发或难治性的。82. The method of any one of claims 1-81, wherein the cancer is relapsed or refractory.83.根据权利要求1-82中任一项所述的方法，其中所述T细胞疗法包含在或在约0.1x10⁶和1,000x 10⁶之间包括0.1x 10⁶和1,000x 10⁶个总重组受体表达T细胞。83.^The method of any one of claims 1-82, wherein the T cell therapy comprises between or about between 0.1 x^{10 6and} 1,000 x 10⁶ total recombinant receptor-expressing T cells.84.根据权利要求1-83中任一项所述的方法，其中所述T细胞疗法包含在或在约10x 10⁶和1,000x 10⁶之间包括10x 10⁶和1,000x 10⁶个总重组受体表达T细胞。84.^{The method of any one of claims 1-83, wherein the T cell therapy comprises between or including about 10 x 10 6 and 1,000 x 10 6totalrecombinantreceptor} -expressing T cells.85.根据权利要求1-84中任一项所述的方法，其中所述T细胞疗法包含在或在约10x 10⁶和500x 10⁶之间包括10x 10⁶和500x 10⁶个总重组受体表达T细胞。85. The method of any one of claims 1-84, wherein the T cell therapy comprises between or including about 10 x¹⁰⁶ and 500 x^{10 6total} recombinant receptor-expressing T cells.86.根据权利要求83-85中任一项所述的方法，其中所述重组受体表达T细胞为活的重组受体表达T细胞。86. The method according to any one of claims 83-85, wherein the recombinant receptor-expressing T cells are live recombinant receptor-expressing T cells.87.根据权利要求1-86中任一项所述的方法，其中所述T细胞疗法静脉内施用。87. The method of any one of claims 1-86, wherein the T cell therapy is administered intravenously.88.根据权利要求1-87中任一项所述的方法，其中所述T细胞疗法的所述T细胞为受试者自体的。88. The method of any one of claims 1-87, wherein the T cells of the T cell therapy are autologous to the subject.89.根据权利要求1-87中任一项所述的方法，其中所述T细胞疗法的所述T细胞为受试者同种异体的。89. The method of any one of claims 1-87, wherein the T cells of the T cell therapy are allogeneic to the subject.90.根据权利要求1-89中任一项所述的方法，其中所述T细胞疗法的所述T细胞为人T细胞。90. The method of any one of claims 1-89, wherein the T cells of the T cell therapy are human T cells.