WO2025195483A1

Movatterモバイル変換

Info

Publication number: WO2025195483A1
Application number: PCT/CN2025/083921
Authority: WO
Inventors: Yangbing Zhao; Xiaojun Liu; Gengzhen ZHU
Original assignee: UTC Therapeutics Shanghai Co Ltd
Current assignee: UTC Therapeutics Shanghai Co Ltd
Priority date: 2024-03-21
Filing date: 2025-03-21
Publication date: 2025-09-25
Anticipated expiration: 2026-09-21

Abstract

Provided herein are anti-mesothelin antibodies and antigen-binding fragments, chimeric antigen receptors ( "CARs" ) having these anti-mesothelin antibodies and antigen-binding fragments ("mesothelin CARs" ) and genetically modified immune effector cells having such mesothelin CARs. Polynucleotides encoding the anti-mesothelin antibodies and antigen-binding fragments and mesothelin CARs are also provided herein. Compositions comprising anti-mesothelin antibodies and antigen-binding fragments and mesothelin CARs are also provided herein. The present disclosure also provides use of the anti-mesothelin antibodies and antigen-binding fragments and genetically modified immune effector cells having such mesothelin CARs in cancer treatment.

Description

MESOTHELIN TARGETTING ANTIBODIES, CHIMERIC ANTIGEN RECEPTORS, AND USES THEREOF

1. Field

The present invention relates to molecular biology, cell biology, and immuno-oncology. In particular, provided herein include anti-mesothelin antibodies, chimeric antigen receptors (CARs) comprising such anti-mesothelin antibodies ( “mesothelin CARs” ) , genetically engineered immune effector cells expressing such mesothelin CARs, and uses thereof in treating tumors or cancers.

2. Background

Cell-based immunotherapy is a therapy with curative potential for the treatment of cancer. T cells and other immune cells can be modified to target tumor antigens through the introduction of genetic material coding for artificial or synthetic receptors for antigen, such as Chimeric Antigen Receptors (CARs) , specific to selected antigens. Targeted T cell therapy using CARs (CARTs) has shown recent clinical success in treating some hematologic malignancies. However, translating CAR-expressing T cell therapy to solid tumors poses several obstacles that must be overcome to achieve clinical benefit. Accordingly, there are needs for novel therapeutic strategies to design CARs for treating cancers, particularly, solid tumors, which strategies capable of inducing potent tumor eradication with minimal toxicity and immunogenicity. Mesothelin is expressed in a variety of human cancers, such as mesothelioma, pancreatic cancer, and ovarian cancer. Current therapies targeting mesothelin, however, have only had limited success. Thus, additional mesothelin-targeting therapeutic options represent unmet needs. The compositions and methods provided herein meet these needs and provide other relative advantages.

3. Summary

The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) , comprising: (a) a light chain variable region (VL) comprising a light chain CDR1 (LCDR1) , a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3) have amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the LCDRs; and/or (b) a heavy chain variable region (VH) comprising a heavy chain CDR1 (HCDR1) , a heavy chain CDR2 (HCDR2) , and a heavy chain CDR3 (HCDR3) have amino acid sequences of SEQ ID NOs: 4, 5, and 6; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the HCDRs.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a LCDR1, a LCDR2, a LCDR3, a HCDR1, a HCDR2 and a HCDR3, wherein (a) the LCDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 2, and 3 respectively; and (b) the HCDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 4, 5, and 6 respectively.

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 7; and/or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 8.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NO: 7 and 8, respectively.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein are monoclonal antibodies or antigen-binding fragments.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein are selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

In some embodiments, the antibodies and antigen-binding fragments thereof provided herein selected from the group consisting of a Fab, a Fab’, a F (ab’) 2, a Fv, a scFv, a (scFv) 2, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .

In some embodiments, the antibody and antigen-binding fragment thereof provided herein is a scFv having the amino acid sequence of SEQ ID NO: 9.

Also provided herein are Chimeric Antigen Receptors (CARs) that specifically bind mesothelin, comprising, from N-terminus to C-terminus: (a) a mesothelin-binding domain that comprises the antibody or antigen-binding fragment thereof provided herein; (b) a transmembrane domain; and (c) a cytoplasmic domain.

In some embodiments of the CARs provide herein, the transmembrane domain is derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.

In some embodiments of the CARs provide herein, the transmembrane domain comprises CD8 transmembrane region.

In some embodiments of the CARs provide herein, the transmembrane domain is CD8 transmembrane region having the amino acid sequence of SEQ ID NO: 15 and nucleotide sequence of SEQ ID NO: 16.

In some embodiments of the CARs provide herein, the cytoplasmic domain comprises a signaling domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

In some embodiments of the CARs provide herein, the cytoplasmic domain further comprises a co-stimulatory domain derived from CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.

In some embodiments of the CARs provide herein, the cytoplasmic domain comprises a CD3ζsignaling domain and a 4-1BB co-stimulatory domain. In some embodiments, the cytoplasmic domain is CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 19 and nucleotide sequence of SEQ ID NO: 20. In some embodiments, the co-stimulatory domain is 4-1BB having the amino acid sequence of SEQ ID NO: 17 and nucleotide sequence of SEQ ID NO: 18.

In some embodiments, the CARs provided herein further comprising a CD8 hinge domain between the mesothelin-binding domain and the transmembrane domain. In some embodiments, the hinge domain is CD8 hinge domain having the amino acid sequence of SEQ ID NO: 13 and nucleotide sequence of SEQ ID NO: 14.

In some embodiments, the CARs provide herein further comprising a signal peptide domain derived from CD8 at N-terminus. In some embodiments, the signal peptide domain is CD8 having the amino acid sequence of SEQ ID NO: 11 and nucleotide sequence of SEQ ID NO: 12.

In some embodiments, the CARs provide herein comprising an amino acid sequence of SEQ ID NO: 10.

Also provided herein are polynucleotides that encode the antibody or antigen-binding fragment and the CARs thereof provided herein. In some embodiments, the polynucleotide is a mRNA.

Also provided herein are vectors that comprise the polynucleotide provided herein.

Also provided herein are cells that comprise the CARs provided herein or the polynucleotide encoding the CARs provided herein.

In some embodiments, the cells provided herein are immune effector cells.

In some embodiments, the cells provided herein are derived from cells isolated from peripheral blood or bone marrow.

In some embodiments, the cells provided herein are derived from cells differentiated in vitro from stem or progenitor cells selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.

In some embodiments, the cells provided herein are T cells or NK cells.

In some embodiments, the cells provided herein are cytotoxic T cells, helper T cells, gamma delta Ts, CD4+/CD8+ double positive T cells, CD4+ T cell, a CD8+ T cells, CD4-/CD8-double negative T cells, CD3+ T cells, naive T cells, effector T cells, helper T cells, memory T cells, regulator T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem memory TSCM cells, central memory TCM cells, effector memory TEM cells, or effector memory TEMRA cells.

In some embodiments, the cells provided herein recombinantly express a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein (i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and (ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

In some embodiments, the N-terminus of the first domain of the fusion protein is linked to the C-terminus of the second domain of the fusion protein. In some embodiments, the N-terminus of the second domain of the fusion protein is linked to the C-terminus of the first domain of the fusion protein. In some embodiments, the first domain and the second domain of the fusion protein are linked via a linker. In some embodiments, the linker are hinges derived from CD8, CD28, (GGGGS) n, (n=1, 2, 3, 4, 5…) or lgG4m. In some embodiments, the linker is CD28 hinge having an amino acid sequence of SEQ ID NO: 25.

In some embodiments, the fusion protein is at least 85%, 90%, 95%, 98%, 99%or 100%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-59 and 63-73. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 54. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 68.

Also provided herein are pharmaceutical compositions comprising the antibody or antigen-binding fragment provided herein, the CAR provided herein, the polynucleotide provided herein, the vector according provided herein and/or the cell of provided herein, and a pharmaceutically acceptable excipient.

Also provided herein are uses of the antibodies or antigen-binding fragments provided herein, the CARs provided herein, the polynucleotides provided herein, the vectors provided herein and/or the cells provided herein for the preparation of a medicament for the treatment of cancer.

Also provided herein are uses of the antibodies or antigen-binding fragments provided herein, the CARs provided herein, the polynucleotides provided herein, the vectors provided herein and/or the cells provided herein in cancer treatment.

Also provided herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibodies or antigen-binding fragments provided herein, the CARs provided herein, the polynucleotides provided herein, the vectors provided herein and/or the cells provided herein.

In some embodiments of the methods provided herein, the cells or population of the cells is autologous to the subject.

In some embodiments, the methods provided herein further comprise obtaining cells from the subject.

In some embodiments, the methods provided herein further comprise administering an additional therapy to the subject.

In some embodiments of the methods provided herein, the subjects are human.

In some embodiments of the methods provided herein, the cancer is a mesothelin-expressing cancer.

In some embodiments of the methods provided herein, the cancer is a solid tumor or a hematological cancer.

In some embodiments of the methods provided herein, the cancer is renal cancer, lung cancer, urothelial cancer, breast cancer, brain tumor, thyroid cancer, leukemia, lymphoma, and/or multiple myeloma.

In some embodiments of the methods provided herein, the cancer comprises renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, glioblastoma, anaplastic thyroid cancer, B-cell non-Hodgkin’s lymphoma, and/or chronic lymphoblastic leukemia.

Also provided herein are methods of preparing the cell provided herein, comprising transferring a polynucleotide encoding the antibody or antigen-binding fragment provided herein, a polynucleotide encoding the CAR provided herein, the polynucleotide provided herein, and/or the vector provided herein into a cell.

In some embodiments, wherein the method is for preparing a cell capable of expressing a CAR that specifically binds mesothelin, and the method comprises transferring the polynucleotide provided herein into the cell.

In some embodiments, wherein the the method is for preparing a cell capable of expressing a CAR that specifically binds mesothelin and expressing a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, and the method comprises transferring the polynucleotide of claim 18 or 19 into the cell and the polynucleotide encoding the fusion protein, wherein:
(i) the first domain of the fusion protein comprises (a) a ligand that binds an activation receptor
of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and
(ii) the second domain of the fusion protein comprises (a) a co-stimulatory receptor of the
immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

In some embodiments, wherein the polynucleotide is transferred via electroporation.

In some embodiments, wherein the polynucleotide is transferred via viral transduction. In some embodiments, the method comprises using a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus for the viral transduction.

In some embodiments, wherein the polynucleotide is transferred using a transposon system. In some embodiments, wherein the transposon system is Sleeping Beauty or PiggyBac.

In some embodiments, wherein the polynucleotide is transferred using gene-editing. In some embodiments, the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.

In some embodiments, wherein the cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

4. Brief Description of Drawings

FIG. 1. Flow cytometry results of MOLM14 stable transfected cell lines overexpressing human MSLN (huMSLN) using anti MSLN antibody.

FIG. 2. ELISA screening results. The value is the difference between the MOLM14-huMSLN reading minus the background cell MOLM14-muMSLN reading. Clones marked with a gray background (clone numbers: 100-186) were selected as candidate clones for subsequent PCR amplification of scFv sequences for sequencing.

FIG. 3A. pUTCK-anti-MSLN scFv. BBZ schematic diagram of lentiviral vector structure. FIG. 3B. Schematic diagram of membrane LACOSTIM (mLACO) and soluble LACOSTIM (sLACO) .

FIG. 4. A549 cells, A549 cells electroporated with 1 μg MSLN mRNA, and A549 cells electroporated with 10 μg MSLN mRNA. MSLN expression levels were stained and tested by flow cytometry after 24 hours.

FIG. 5. Anti-MSLN CAR-T and anti-MSLN/LACOSTIM CAR-T cells, flow cytometry results stained with MSLN-Fc antigen and anti-human IgG-Fc antibody. The staining results showed that the CAR molecules on anti-MSLN CAR-T and anti-MSLN/LACOSTIM CAR-T cells were effectively expressed.

FIG. 6. Flow cytometry results of anti-MSLN CAR-T and anti-MSLN/LACOSTIM CAR-T cells stained with CD40-Fc antigen and anti-human IgG-Fc antibody. The staining results showed that the expression level of LACOSTIM molecules on the CAR-T cells A4025-MH150 expressing LACOSTIM on the cell membrane was very high. LACOSTIM molecules were not detected on the cell membrane of secreted LACOSTIM-expressing CAR-T cells MH150-25-9.3, indicating that secreted LACOSTIM molecules may exist in soluble form outside the cells.

FIG. 7. Under the condition of E: T=3: 1, the killing effect results of co-incubation of control UTD cells and 3 types of anti-MSLN CAR-T cells on A549 cells and A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA.

FIG. 8. Under the condition of E: T=1: 1, the killing effect results of co-incubation of control UTD cells and 3 types of anti-MSLN CAR-T cells on A549 cells and A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA.

FIG. 9. Under the condition of E: T=0.3: 1, the killing effect results of co-incubation of control UTD cells and 3 types of anti-MSLN CAR-T cells on A549 cells and A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA.

FIG. 10. The killing effect of co-incubation of ASPC1 target cells with control UTD cells and 3 types of anti-MSLN CAR-T cells under 3 conditions: E: T=3: 1, E: T=1: 1, E: T=0.3: 1.

FIG. 11. ELISA was used to detect the IL2 release levels of UTD cells and 3 types of anti-MSLN CAR-T cells co-incubated with A549 cells, A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA, and ASPC1 target cells.

FIG. 12. ELISA was used to detect the IFN-γ release levels of UTD cells and 3 types of anti-MSLN CAR-T cells co-incubated with A549 cells, A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA, and ASPC1 target cells.

FIG. 13-FIG. 22. Under the condition of E: T=3: 1, the killing effect inCucyte analysis results of control UTD cells and 3 anti-MSLN CAR-T cells responded to HCC70 (FIG. 13) , SH-SY5Y (FIG. 14) , Huh-7 (FIG. 15) , U87 (FIG. 16) , U251 (FIG. 17) , A375 (FIG. 18) , 293T (FIG. 19) , PC3 (FIG. 20) , Supt1 (FIG. 21) and Molm14 (FIG. 22) Co-incubation of target cells.

FIG. 23. Results of the Tumor-Bearing Mouse Model Experiment Evaluating the Tumor-Killing Efficacy of CAR-T Cells

FIG. 24. Dose-Dependent Expansion of CAR-T Cells in Peripheral Blood of Tumor-Bearing Mice After Administration.

FIG. 25. Secretion Levels of sLACO in Peripheral Blood of Tumor-Bearing Mice After CAR-T Cell Administration.

FIG. 26. Results of the Tumor-Bearing Mouse Model Experiment Evaluating the Tumor-Killing Efficacy of CAR-T Cells.

FIG. 27. Flow cytometry detection of CAR-T cell proliferation in mice.

FIG. 28. Results of ELISA for the secretion level of sLACO in mouse peripheral blood.

5. Detailed Description

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

The present disclosure provides novel antibodies, including antigen-binding fragments that specifically bind mesothelin (e.g., human mesothelin) . Further, the present disclosure also provides chimeric antigen receptors (CARs) that comprise such antibodies or antigen-binding fragments that specifically bind mesothelin (e.g., human mesothelin) , as well as engineered immune effector cells (e.g., T cells) and populations of cells that recombinantly express a CAR (e.g., CAR-Ts) that specifically binds mesothelin (e.g., human mesothelin) . Pharmaceutical compositions comprising a therapeutically effective amount of such antibodies or antigen-binding fragments, and pharmaceutical compositions comprising a therapeutically effective amount of cells or population of cells are also disclosed herein. Also disclosed herein are uses of such pharmaceutical compositions for treating diseases and disorders relating to mesothelin expression (e.g., mesothelin expressing cancer) and related methods of treatment.
5.1 Definitions

Unless otherwise defined herein, scientific and technical terms used in the present disclosures shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

The present disclosure provides novel antibodies, including antigen-binding fragments that specifically bind mesothelin (e.g., human mesothelin) . The term “mesothelin” includes any variants or isoforms of mesothelin which are naturally expressed by cells. Accordingly, antibodies described herein can cross-react with mesothelin from species other than human (e.g., cynomolgus mesothelin) . Alternatively, the antibodies can be specific for human mesothelin and do not exhibit any cross-reactivity with other species. Mesothelin or any variants and isoforms thereof, can either be isolated from cells or tissues which naturally express them or be recombinantly produced using well-known techniques in the art and/or those described herein.

The term “antibody, ” and its grammatical equivalents as used herein refer to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , single-chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecule comprising an antigen-binding site (e.g., dual variable domain immunoglobulin molecules) as long as the antibodies exhibit the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) , based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. Unless expressly indicated otherwise, the term “antibody” as used herein include “antigen-binding fragment” of intact antibodies. The term “antigen-binding fragment” as used herein refers to a portion or fragment of an intact antibody that is the antigenic determining variable region of an intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F (ab’ ) 2, Fv, linear antibodies, single chain antibody molecules (e.g., scFv) , heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , disulfide-linked scFv (dsscFv) , diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD) , single variable domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , and single variable domain of heavy chain antibodies (VHH) , and bispecific or multispecific antibodies formed from antibody fragments.

The term “humanized antibody” as used herein refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulin. In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. In some instances, residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and/or binding capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability. The term “human antibody” as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The constant region can be one of five distinct types, referred to as alpha (a) , delta (δ) , epsilon (ε) , gamma (γ) and mu (μ) , based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (κ) of lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

The term “variable domain” or “variable region” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR) . The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. Numbering of amino acid positions used herein is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, D.C. ) 5thed. A variable region can be a human variable region.

A CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by a variety of methods/systems. These systems and/or definitions have been developed and refined over years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines the regions of most hypervariability within the antibody variable (V) domains (Kabat et al, J. Biol. Chem. 252: 6609-6616 (1977) ; Kabat, Adv. Prot. Chem. 32: 1-75 (1978) ) . The Chothia definition is based on the location of the structural loop regions, which defines CDR region sequences as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) ) . Both terminologies are well recognized in the art. Additionally, the IMGT system is based on sequence variability and location within the structure of the variable regions. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. Software programs (e.g., abYsis) are available and known to those of skill in the art for analysis of antibody sequence and determination of CDRs. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al, J. Mol. Biol. 273: 927-948 (1997) ; Morea et al, Methods 20: 267-279 (2000) ) . Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997) ) . Such nomenclature is similarly well known to those skilled in the art.

For example, CDRs defined according to either the Kabat (hypervariable) or Chothia (structural) designations, are set forth in the table below.

¹Residue numbering follows the nomenclature of Kabat et al., supra
²Residue numbering follows the nomenclature of Chothia et al., supra

One or more CDRs also can be incorporated into a molecule either covalently or noncovalently to make it an immuneadhesin. An immunoadhesin can incorporate the CDR (s) as part of a larger polypeptide chain, can covalently link the CDR (s) to another polypeptide chain, or can incorporate the CDR (s) noncovalently. The CDRs permit the immunoadhesin to bind to a particular antigen of interest. The CDR regions can be analyzed by, for example, abysis website.

Thus, unless otherwise specified, a CDR, or individual specified CDRs (e.g., LCDR1, LCDR2, LCDR3) , of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes, or other known schemes. For example, where it is stated that a particular CDR (e.g., a HCDR3) contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., HCDR3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes. In some embodiments, specific CDR sequences are specified, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan. Likewise, unless otherwise specified, a FR or individual specified FR (s) (e.g., VH FRl, VH FR2, VH FR3, VH FR4) , of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes.

The terms “epitope” and “antigenic determinant” are used interchangeably herein an refer to the site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, such as a localized region on the surface of an antigen. The target molecule can comprise, a protein, a peptide, a nucleic acid, a carbohydrate, or a lipid. An epitope having immunogenic activity is a portion of a target molecule that elicits an immune response in an animal. An epitope of a target molecule having antigenic activity is a portion of the target molecule to which an antibody binds, as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. The term, “epitope” includes linear epitopes and conformational epitopes. A region of a target molecule (e.g., a polypeptide) contributing to an epitope can be contiguous amino acids of the polypeptide or the epitope can come together from two or more non-contiguous regions of the target molecule. The epitope may or may not be a three-dimensional surface feature of the target molecule. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

The term “specifically binds, ” as used herein, means that a polypeptide or molecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. A binding moiety (e.g., antibody) that specifically binds a target molecule (e.g., antigen) can be identified, for example, by immunoassays, ELISAs, SPR (e.g., Biacore) , or other techniques known to those of skill in the art. Typically, a specific reaction will be at least twice background signal or noise and can be more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity. A binding moiety that specifically binds a target molecule can bind the target molecule at a higher affinity than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a target molecule can bind the target molecule with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a particular target molecule binds a different molecule at such a low affinity that binding cannot be detected using an assay described herein or otherwise known in the art. In some embodiments, “specifically binds” means, for instance, that a binding moiety binds a molecule target with a KD of about 0.1 mM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a KD of at about 10 μM or less or about 1 μM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a KD of at about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It is understood that, in some embodiments, a binding moiety (e.g., antibody) that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, i.e., binding to a single target. Thus, a binding moiety (e.g., antibody) can, in some embodiments, specifically bind more than one target. For example, an antibody can, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities.

The term “binding affinity” as used herein generally refers to the strength of the sum total of noncovalent interactions between a binding moiety and a target molecule (e.g., antigen) . The binding of a binding moiety and a target molecule is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (KD) . KD is the ratio of a dissociation rate (koff or kd) to the association rate (kon or ka) . The lower the KD of a binding pair, the higher the affinity. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following. In some embodiments, the “KD” or “KD value” can be measured by assays known in the art, for example by a binding assay. The KD may be measured in a radiolabeled antigen binding assay (RIA) (Chen, et al., (1999) J. Mol Biol 293: 865-881) . The KD or KD value may also be measured by using surface plasmon resonance assays by Biacore, using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 BIAcore, Inc., Piscataway, NJ) , or by biolayer interferometry using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA) .

The term “variant” as used herein in relation to a protein or a polypeptide with particular sequence features (the “reference protein” or “reference polypeptide” ) refers to a different protein or polypeptide having one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and/or additions as compared to the reference protein or reference polypeptide. The changes to an amino acid sequence can be amino acid substitutions. The changes to an amino acid sequence can be conservative amino acid substitutions. A functional fragment or a functional variant of a protein or polypeptide maintains the basic structural and functional properties of the reference protein or polypeptide.

The terms “polypeptide, ” “peptide, ” “protein, ” and their grammatical equivalents as used interchangeably herein refer to polymers of amino acids of any length, which can be linear or branched. It can include unnatural or modified amino acids or be interrupted by non-amino acids. A polypeptide, peptide, or protein can also be modified with, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

The terms “polynucleotide, ” “nucleic acid, ” and their grammatical equivalents as used interchangeably herein mean polymers of nucleotides of any length and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

The terms “identical, ” percent “identity, ” and their grammatical equivalents as used herein in the context of two or more polynucleotides or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

The term “vector, ” and its grammatical equivalents as used herein refer to a vehicle that is used to carry genetic material (e.g., a polynucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more polynucleotides are to be co-expressed, both polynucleotides can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding polynucleotides can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of polynucleotides into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotides are expressed in a sufficient amount to produce a desired product (e.g., an anti-mesothelin antibody or antigen-binding fragment as described herein) , and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

The term “genetic engineering” or its grammatical equivalents when used in reference to a cell is intended to mean alteration of the genetic materials of the cell that is not normally found in a naturally occurring cell. Genetic alterations include, for example, modifications introducing expressible polynucleotides, other additions, mutations/alterations, deletions and/or other functional disruption of the cell’s genes. Such modifications can be done in, for example, coding regions and functional fragments thereof of a gene. Additional modifications can be done in, for example, non-coding regulatory regions in which the modifications alter expression of a gene.

The term “transfer, ” “transduce, ” “transfect, ” and their grammatical equivalents as used herein refer to a process by which an exogenous polynucleotide is introduced into the host cell. A “transferred, ” “transfected, ” or “transduced” cell is one which has been transferred, transduced, or transfected with an exogenous polynucleotide. The cell includes the primary subject cell and its progeny. A polynucleotide can be “transferred” into a host cell using any type of approaches known in the art, including, e.g., a chemical method, a physical method, or a biological method. A polynucleotide is commonly “transduced” into a host cell using a virus. By contrast, a polynucleotide is commonly “transfected” into a host cell using a non-viral approach. These terms are used interchangeable at times, and a person of ordinary skill in the art would readily understand their meanings in different contexts.

As used herein, the term “encode” and its grammatical equivalents refer to the inherent property of specific sequences of nucleotides in a polynucleotide or a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

A polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

The term “immune effector cell” and its grammatical equivalents as used herein and understood in the art refer to cells that are of hematopoietic origin and play a direct role in the immune response against a target, such as a pathogen, a cancer cell, or a foreign substance. Immune effector cells include T cells, B cell, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils.

“Stimulation” of an immune effector cell means a primary response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event in the immune effector cell which can alter expression of certain genes and/or reorganization of cytoskeletal structures, and the like. A “stimulatory molecule” of an immune effector cell refers to a molecule on the immune effector cell that, upon binding with its cognate ligand, which is commonly present on an APC, can mediate signal transduction to promote the maturation, differentiation, proliferation, and/or activation of the immune effector cell. For example, a stimulatory molecule of the T cells, the TCR/CD3 complex triggers the activation of the T cells. The ligand for a stimulatory molecule, or “stimulatory ligand, ” means a ligand that is commonly present on an APC and can bind with a stimulatory molecule on the immune effector cell to mediate a primary response by the immune effector cell, including, but not limited to, maturation, differentiation, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, for example, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

A “co-stimulatory signal, ” as used herein and understood in the art, refers to a signal from a co-stimulatory receptor (e.g., CD28 or 4-1BB) , which in combination with a primary signal (e.g., TCR/CD3) promotes optimal clonal expansion, differentiation and effector functions of immune effector cells (e.g., T cells) . A “co-stimulatory receptor” of an immune effector cell, s used herein and understood in the art, refers to a molecule on the immune effector cell that specifically binds with a “co-stimulatory ligand” to mediate a co-stimulatory response by the immune effector cell, such as heightened activation or proliferation of the immune effector cell. Co-stimulatory receptors for immune effector cells include, but are not limited to, CD28, 4-1BB, ICOS, CD27, OX40, DAP10, CD30, 2B4, CD2, LIGHT, GITR, TLR, DR3, and CD43. A “functional fragment” of a co-stimulatory receptor is a fragment of the co-stimulatory receptor that retains its function to mediate a co-stimulatory signal and stimulate the immune effector cell. In some embodiments, a functional fragment of a co-stimulatory receptor retains the co-stimulatory domain of the co-stimulatory receptor. In some embodiments, the co-stimulatory domain is the cytoplasmic domain of the co-stimulatory receptor. In some embodiments, signals from co-stimulatory receptors of immune effector cells (e.g., T cells) lower the activation threshold for the immune effector cells. In some embodiments, signals from co-stimulatory receptors of T cells lead to the augmentation of TCR signaling events necessary for efficient cytokine production (via augmented transcriptional activity and messenger RNA stabilization) , cell cycle progression, survival, regulation of metabolism and T cell responses.

A “co-stimulatory ligand, ” as used herein and understood in the art, refers to a molecule that specifically binds a cognate co-stimulatory receptor on an immune effector cell, thereby providing a signal which, in addition to the primary signal provided by the stimulatory molecule, mediates a response in the immune effector cell, including, but not limited to, proliferation, activation, differentiation, and the like. The co-stimulatory ligand can be present on an APC (e.g., a dendritic cell) . Co-stimulatory ligands include, but are not limited to, CD58, mesothelin, CD83, CD80, CD86, CD137L (4-1BBL) , CD252 (OX40L) , CD275 (ICOS-L) , CD54 (ICAM-1) , CD49a, CD112 (PVRL2) , CD150 (SLAM) , CD155 (PVR) , CD265 (RANK) , CD270 (HVEM) , TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153 (CD30L) , CD48, CD160, CD200R (OX2R) , and CD44. A “receptor-binding fragment” of a co-stimulatory ligand refers to a fragment of the ligand that retains its capacity to bind its receptor.

The term “treat” and its grammatical equivalents as used herein in connection with a disease or a condition, or a subject having a disease or a condition refer to an action that suppresses, eliminates, reduces, and/or ameliorates a symptom, the severity of the symptom, and/or the frequency of the symptom associated with the disease or disorder being treated. For example, when used in reference to a cancer or tumor, the term “treat” and its grammatical equivalents refer to an action that reduces the severity of the cancer or tumor, or retards or slows the progression of the cancer or tumor, including (a) inhibiting the growth, or arresting development of the cancer or tumor, (b) causing regression of the cancer or tumor, or (c) delaying, ameliorating or minimizing one or more symptoms associated with the presence of the cancer or tumor.

The term “administer” and its grammatical equivalents as used herein refer to the act of delivering, or causing to be delivered, a therapeutic or a pharmaceutical composition to the body of a subject by a method described herein or otherwise known in the art. The therapeutic can be a compound, a polypeptide, an antibody, a cell, or a population of cells. Administering a therapeutic or a pharmaceutical composition includes prescribing a therapeutic or a pharmaceutical composition to be delivered into the body of a subject. Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV) , intramuscular (IM) , or intraperitoneal (IP) ; transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

The terms “effective amount, ” “therapeutically effective amount, ” and their grammatical equivalents as used herein refer to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to the subject. The therapeutically effective amount can be ascertained by measuring relevant physiological effects. The exact amount required vary from subject to subject, depending on the age, weight, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. An appropriate “effective amount” in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to a material that is suitable for drug administration to an individual along with an active agent without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.

The term “subject” as used herein refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. A subject can be a human. A subject can have a particular disease or condition.

The term “autologous” as used herein refers to any material derived from the same individual to which it is later to be re-introduced into the individual.

The term “allogeneic” as used herein refers to a graft derived from a different animal of the same species.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and/or SEQ ID NOs. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .
5.2 Anti-mesothelin antibodies and antigen-binding fragments

In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments provided herein are isolated. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments provided herein are substantially pure.

In some embodiments, the anti-mesothelin antibody or antigen-binding fragment provided herein comprises a multispecific antibody or antigen-binding fragment. In some embodiments, the anti-mesothelin antibody or antigen-binding fragment provided herein comprises a bispecific antibody or antigen-binding fragment. In some embodiments, provided herein is a Bi-specific T-cell engager (BiTE) . BiTEs are bispecific antibodies that bind to a T cell antigen (e.g., CD3) and a tumor antigen. BiTEs have been shown to induce directed lysis of target tumor cells and thus provide great potential therapies for cancers and other disorders. In some embodiments, provided herein are BiTEs that specifically bind CD3 and mesothelin. In some embodiments, the BiTEs comprises an anti-mesothelin antibody or antigen-binding fragment provided herein. In some embodiments, the BiTEs comprises an anti-mesothelin scFv provided herein.

In some embodiments, the anti-mesothelin antibody or antigen-binding fragment provided herein comprises a monovalent antigen-binding site. In some embodiments, an anti-mesothelin antibody or antigen-binding fragment comprises a monospecific binding site. In some embodiments, an anti-mesothelin antibody or antigen-binding fragment comprises a bivalent binding site.

In some embodiments, an anti-mesothelin antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment. Monoclonal antibodies can be prepared by any method known to those of skill in the art. One exemplary approach is screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228: 1315-1317; and WO 92/18619. In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries expressing variable regions or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art.

In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to one of skill in the art. For example, using a hybridoma method, a mouse, rat, rabbit, hamster, or other appropriate host animal, is immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a human protein or a fragment thereof.

Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. The hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas that produce monoclonal antibodies directed to a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore) , and radioimmunoassay) . Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution or other techniques. The hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal. The monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.

In some embodiments, monoclonal antibodies are made using recombinant DNA techniques as known to one skilled in the art. For example, the polynucleotides encoding an antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

In some embodiments, a monoclonal antibody is modified by using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light chain and heavy chain of a mouse monoclonal antibody are replaced with the constant regions of a human antibody to generate a chimeric antibody. In some embodiments, the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region (s) is used to optimize specificity and/or affinity of a monoclonal antibody.

In some embodiments, an anti-mesothelin antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment. Various methods for generating humanized antibodies are known in the art. Methods are known in the art for achieving high affinity binding with humanized antibodies. A non-limiting example of such a method is hypermutation of the variable region and selection of the cells expressing such high affinity antibodies (affinity maturation) . In addition to the use of display libraries, the specified antigen (e.g., recombinant mesothelin or an epitope thereof) can be used to immunize a non-human animal, e.g., a rodent. In certain embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be generated and isolated using methods known in the art and/or disclosed herein. In some embodiments, a mouse can be immunized with an antigen (e.g., recombinant mesothelin or an epitope thereof) .

In some embodiments, an anti-mesothelin antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from an immunized individual. In any case, cells that produce an antibody directed against a target antigen can be generated and isolated. In some embodiments, a human antibody is selected from a phage library, where that phage library expresses human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable region gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are well-known in the art. Once antibodies are identified, affinity maturation strategies known in the art, including but not limited to, chain shuffling and site-directed mutagenesis, can be employed to generate higher affinity human antibodies. In some embodiments, human antibodies are produced in transgenic mice that contain human immunoglobulin loci. Upon immunization these mice are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

The specific CDR sequences defined herein are generally based on a combination of Kabat definition. However, it is understood that reference to a heavy chain CDR or CDRs and/or a light chain CDR or CDRs of a specific antibody encompass all CDR definitions as known to those of skill in the art.

Anti-mesothelin antibodies or antigen-binding fragments provided herein include the MH150 clone, and the sequence features are described below.

In some embodiments, anti-mesothelin antibodies or antigen-binding fragments provided herein (e.g., human mesothelin) comprise one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein. In some embodiments, anti-mesothelin antibodies or antigen-binding fragments provided herein comprise a VL comprising one, two, and/or three, LCDRs from Table 1. In some embodiments, anti-mesothelin antibodies or antigen-binding fragments provided herein comprise a VH comprising one, two, and/or three HCDRs from Table 2. In some embodiments, anti-mesothelin antibodies or antigen-binding fragments provided herein comprise one, two, and/or three LCDRs from Table 1 and one, two, and/or three HCDRs from Table 2.

Table 1 Amino acid sequences of light chain variable region CDRs (LCDRs) of anti-mesothelin

Table 2 Amino acid sequences of heavy chain variable region CDRs (HCDRs) of anti-mesothelin

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) , comprising a light chain variable region (VL) comprising (1) a light chain CDR1 (LCDR1) having an amino acid sequence of SEQ ID NO: 1; (2) a light chain CDR2 (LCDR2) having an amino acid sequence consisting of SEQ ID NO: 2; or (3) a light chain CDR3 (LCDR3) having an amino acid sequence of SEQ ID NO: 3; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the LCDRs. In some embodiments, the variant has about 5 amino acid substitutions, additions, and/or deletions in the LCDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (HCDR1) having an amino acid sequence of SEQ ID NO: 4; (2) a heavy chain CDR2 (HCDR2) having an amino acid sequence of SEQ ID NO: 5; or (3) a heavy chain CDR3 (HCDR3) having an amino acid sequence of SEQ ID NO: 6; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the HCDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the HCDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) having a VL and a VH. In some embodiments, the VL and VH are connected by a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 21) . For example, the linker may have the amino acid sequence of GGGGS. In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5. For example, the linker may have the amino acid sequence of EAAAK (SEQ ID NO: 22) . In some embodiments, the linker has the amino acid sequence of (PA) nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 23. In some embodiments, the linker has the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 24) .

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) having a VL and a VH, wherein the VL comprises LCDR1, CDR2 and CDR3 and the VH comprises HCDR1, CDR2 and CDR3, and wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, and 6, respectively, or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) comprising a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 7. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) comprising a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 8.

Table 3 Amino acid sequences of light chain variable regions (VL) and heavy chain variable region (VH) of anti-mesothelin antibody

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity sequence identity to an amino acid sequence of SEQ ID NO: 7; and (b) a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity sequence identity to an amino acid sequence of SEQ ID NO: 8.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NO: 7 and 8, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) comprising (a) a VL comprising LCDRs from a VL having an amino acid sequence of SEQ ID NO: 7; and/or (b) a VH comprising HCDRs from a VH having an amino acid sequence of SEQ ID NO: 8.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human mesothelin) comprising a VL and a VH, wherein the VL comprises LCDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 7, and the VH comprises HCDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 8.

In some embodiments, provided herein are also antibodies or antigen-binding fragments that compete with the antibody or antigen-binding fragment provided above for binding to mesothelin (e.g., human mesothelin) . Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, can be determined using known competition experiments, e.g., BIACORE surface plasmon resonance (SPR) analysis. In some embodiments, an anti-mesothelin antibody or antigen-binding fragment competes with, and inhibits binding of another antibody or antigen-binding fragment to mesothelin by at least 50%, 60%, 70%, 80%, 90%or 100%. Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: l0. H0l/pdb. prot4277 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with MH150 for binding to mesothelin.

The present disclosure further contemplates additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate biological properties of the antibody, including but not limited to, specificity, thermostability, expression level, effector function (s) , glycosylation, immunogenicity, and/or solubility. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of an antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

Variations can be a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native antibody or polypeptide sequence. In some embodiments, amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Insertions or deletions can be in the range of about 1 to 5 amino acids. In some embodiments, the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule. In some embodiments, variations in the amino acid sequence that are biologically useful and/or relevant can be determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parent protein.

In some embodiments, variants can include addition of amino acid residues at the amino-and/or carboxyl-terminal end of the antibody or polypeptide. The length of additional amino acids residues can range from one residue to a hundred or more residues. In some embodiments, a variant comprises an N-terminal methionyl residue. In some embodiments, the variant comprises an additional polypeptide/protein (e.g., Fc region) to create a fusion protein. In some embodiments, a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g., a fluorescent tag or an enzyme) .

The variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

In some embodiments, a variant of an anti-mesothelin antibody or antigen-binding fragment disclosed herein can retain the ability to bind mesothelin to a similar extent, the same extent, or to a higher extent, as the parent antibody or antigen-binding fragment. In some embodiments, the variant can be at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%or more identical in amino acid sequence to the parent antibody or antigen-binding fragment. In certain embodiments, a variant of an anti-mesothelin antibody or antigen-binding fragment comprises the amino acid sequence of the parent anti-mesothelin antibody or antigen-binding fragment with one or more conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties.

In some embodiments, a variant of an anti-mesothelin antibody or antigen-binding fragment comprises the amino acid sequence of the parent antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions. In some embodiments, a variant of an anti-mesothelin antibody or antigen-binding fragment comprises the amino acid sequence of the parent binding antibody or antigen-binding fragment with one or more non-conservative amino acid substitution, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant (e.g., mesothelin binding) . In certain embodiments, the one or more conservative amino acid substitutions and/or the one or more non-conservative amino acid substitutions can enhance a biological activity of the variant, such that the biological activity of the functional variant is increased as compared to the parent binding moiety.

In some embodiments, the variant can have 1, 2, 3, 4, or 5 amino acid substitutions in the CDRs (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of the binding moiety.

In some embodiments, anti-mesothelin antibodies or antigen-binding fragments described herein are chemically modified naturally or by intervention. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications can be carried out by known techniques. The anti-mesothelin antibodies or antigen-binding fragments can comprise one or more analogs of an amino acid (including, for example, unnatural amino acids) , as well as other modifications known in the art.

The anti-mesothelin antibodies or antigen-binding fragments of the present disclosure can be analyzed for their physical, chemical and/or biological properties by various methods known in the art. In some embodiments, an anti-mesothelin antibody is tested for its ability to bind mesothelin (e.g., human mesothelin) . Binding assays include, but are not limited to, SPR (e.g., Biacore) , ELISA, and FACS. In addition, antibodies can be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.

Epitope mapping is a method of identifying the binding site, region, or epitope on a target protein where an antibody binds. A variety of methods are known in the art for mapping epitopes on target proteins. These methods include mutagenesis, including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology) ; display methods (e.g., phage display, microbial display, and ribosome/mRNA display) ; methods involving proteolysis and mass spectroscopy; and structural determination (e.g., X-ray crystallography and NMR) . In some embodiments, anti-mesothelin antibodies or antigen-binding fragments described herein are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography, and papain digestion.

In some embodiments, an anti-mesothelin antibody or antigen-binding fragment is conjugated to a cytotoxic agent or moiety. In some embodiments, an anti-mesothelin antibody or antigen-binding fragment is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate) . In some embodiments, the cytotoxic moiety is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs) , or other intercalating agents. In some embodiments, the cytotoxic moiety is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4) , and tubulysins. In some embodiments, the cytotoxic moiety is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In some embodiments, an antibody is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065.

In some embodiments, an anti-mesothelin antibody or antigen-binding fragment described herein is conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and/or detection. A detectable substance can include, but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine (s) ; fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC) , rhodamine, tetramethylrhodamine isothiocyanate (TRITC) , dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3) , and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials, such as 212Bi, 14C, 57Co, 51Cr, 67Cu, 18F, 68Ga, 67Ga, 153Gd, 159Gd, 68Ge, 3H, 166Ho, 131I, 125I, 123I, 121I, 115In, 113In, 112In, 111In, 140La, 177Lu, 54Mn, 99Mo, 32P, 103Pd, 149Pm, 142Pr, 186Re, 188Re, 105Rh, 97Ru, 35S, 47Sc, 75Se, 153Sm, 113Sn, 117Sn, 85Sr, 99mTc, 201Ti, 133Xe, 90Y, 69Yb, 175Yb, 65Zn; positron emitting metals; and magnetic metal ions positron emitting metals; and magnetic metal ions.

An anti-mesothelin antibody or antigen-binding fragment described herein can be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, an immobilized anti-mesothelin antibody or antigen-binding fragment is used in an immunoassay. In some embodiments, an immobilized anti-mesothelin antibody or antigen-binding fragment is used in purification of the target antigen (e.g., human mesothelin) .
5.3 CARs, TCRs and Genetically Engineered Immune Effector Cells

The anti-mesothelin antibodies or antigen-binding fragments described herein can be used as part of a chimeric antigen receptor (CAR) or a T-Cell Receptor (TCR) that can be expressed in an immune effector cell for cancer treatment. As such, provided herein are also CARs and TCRs that specifically bind mesothelin, immune effector cells that express such CARs or TCRs, and the uses of such cells.
5.3.1 TCRs

Provided herein are T cell receptors (TCRs) that specifically bind mesothelin ( “mesothelin TCR” ) . TCRs are antigen-specific molecules that are responsible for recognizing antigenic peptides presented in the context of a product of the MHC on the surface of APCs or any nucleated cells. This system endows T cells, via their TCRs, with the potential ability to recognize the entire array of intracellular antigens expressed by a cell (including virus proteins) that are processed into short peptides, bound to an intracellular MHC molecule, and delivered to the surface as a peptide-MHC complex. This system allows foreign protein (e.g., mutated cancer antigen or virus protein) or aberrantly expressed protein to serve a target for T cells (e.g., Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544) .

As such, in some embodiments, provided herein are TCRs comprising an anti-mesothelin antibody or antigen-binding fragment described herein. The anti-mesothelin antibody or antigen-binding fragment can be any anti-mesothelin antibody or antigen-binding fragment described herein. For illustrative purposes, in some embodiments, the TCRs provided herein can comprise an anti-mesothelin antibody or antigen-binding fragment having a VL and a VH, wherein the VL comprises LCDR1, CDR2 and CDR3 and the VH comprises HCDR1, CDR2 and CDR3, and wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NO:1, 2, 3, 4, 5, and 6.

In some embodiments, the TCRs provided herein can comprise an anti-mesothelin antibody or antigen-binding fragment that is the scFv designated as MH150.
5.3.2 CARs

The term “chimeric antigen receptor” or “CAR” as used herein refers to an artificially constructed hybrid protein or polypeptide containing a binding moiety (e.g., an antibody) linked to immune cell (e.g., T cell) signaling or activation domains. In some embodiments, CARs are synthetic receptors that retarget T cells to tumor surface antigens (Sadelain et al., Nat. Rev. Cancer 3 (l) : 35-45 (2003) ; Sadelain et al., Cancer Discovery 3 (4) : 388-398 (2013) ) . CARs can provide both antigen binding and immune cell activation functions onto an immune cell such as a T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition can give T-cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a mechanism of tumor escape.

The typical structure of a CAR molecule includes an extracellular antigen-binding domain (e.g., scFv) , a transmembrane domain (TM) and an intracellular signaling domain. The extracellular antigen-binding domain of a CAR is usually derived from a monoclonal antibody (mAb) or from receptors or their ligands. Antigen binding by the CARs triggers phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the intracellular domain, initiating a signaling cascade required for cytolysis induction, cytokine secretion, and proliferation. CAR-expressing T cells (“CART” s) can be classified into three generations according to the presence of intracellular co-stimulatory signals.

In some embodiments, provided herein are CARs that specifically binds mesothelin ( “mesothelin CAR” ) . In some embodiments, the CAR can be a “first generation, ” “second generation” or “third generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3 (4) : 388-398 (2013) ; Jensen et al., Immunol. Rev. 257: 127-133 (2014) ; Sharpe et al., Dis. Model Mech. 8 (4) : 337-350 (2015) ; June et al (2018) , Science 359 (6382) : 1361-1365) .

“First generation” CARs are typically composed of an extracellular antigen binding domain, for example, a single-chain variable fragment (scFv) , fused to a transmembrane domain, which is fused to a cytoplasmic/intracellular domain of the T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3ζ-chain, which is the primary transmitter of signals from endogenous T cell receptors (TCRs) . “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. “Second-generation” CARs comprise a cancer antigen-binding domain fused to an intracellular signaling domain capable of activating immune effector cells such as T cells and a co-stimulatory domain designed to augment immune effector cell, such as T cell, potency and persistence (Sadelain et al., Cancer Discov. 3: 388-398 (2013) ) . CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically borne by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co-stimulatory receptors, for example, CD28, 4-1BB, ICOS, OX40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell. “Second generation” CARs provide both co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3ζ signaling domain. Studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of T cells. In 2017, FDA approved two anti-CD19 CAR T cell products for the treatment of relapsed B-cell precursor acute lymphoblastic leukemia (B-ALL) and B-cell Non-Hodgkin Lymphoma. “Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4-1BB domains, and activation, for example, by comprising a CD3ζactivation domain.

As such, provided herein are CARs that specifically binds mesothelin, comprising, from N-terminus to C-terminus: (a) a mesothelin binding domain comprising an anti-mesothelin antibody or antigen-binding fragment provided herein, (b) a transmembrane domain, and (c) a cytoplasmic domain. The anti-mesothelin antibody or antigen-binding fragment can be any anti-mesothelin antibody or antigen-binding fragment described herein. For illustrative purposes, in some embodiments, the CARs provided herein can comprise an anti-mesothelin antibody or antigen-binding fragment having a VL and a VH, wherein the VL comprises LCDR1, CDR2 and CDR3 and the VH comprises HCDR1, CDR2 and CDR3, and wherein the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, and 6, respectively.

In some embodiments, the CARs provided herein can comprise an anti-mesothelin antibody or antigen-binding fragment that is the scFv designated as MH150.

In some embodiments, the transmembrane domain of the CARs provided herein comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of the CAR provided herein can be derived from a protein or polypeptide that is naturally expressed in an immune effector cell. A transmembrane domain derived from a protein or polypeptide means that the transmembrane domain comprises the entire transmembrane region of the protein or polypeptide, or a fragment thereof. In some embodiments, the CAR provided herein can have a transmembrane domain derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T-cell receptor (TCR) α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, or other polypeptides expressed in the immune effector cell. In some embodiments, the transmembrane domain of CARs provided herein comprises the transmembrane region of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T-cell receptor (TCR) α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, or other polypeptides expressed in the immune effector cell.

In some embodiments, the transmembrane domain of CARs provided herein is derived from CD8. In some embodiments, the transmembrane domain comprises the transmembrane region of CD8. In some embodiments, the transmembrane domain is derived from CD28. In some embodiments, the transmembrane domain comprises the transmembrane region of CD28. In some embodiments, the transmembrane domain is derived from CD3ζ. In some embodiments, the transmembrane domain comprises the transmembrane region of CD3ζ.

In some embodiments, the transmembrane domain can be synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine. Optionally, the transmembrane domain can be derived from a polypeptide that is not naturally expressed in the immune effector cell, so long as the transmembrane domain can function in transducing signal from antigen bound to the CAR to the intracellular signaling and/or co-stimulatory domains. In some embodiments, the transmembrane domain can comprise a triplet of phenylalanine, tryptophan and valine at each end. Optionally, a short oligo-or polypeptide linker, preferably between 2 and 10 amino acids in length can form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

Cytoplasmic domains of CARs provided herein can contain a signaling domain that functions in the immune effector cell expressing the CAR. Such a signaling domain can be, for example, derived from CD3ζ, Fc receptor γ, FcγRIIa, FcRβ (FcεR1b) , CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, or DAP12. A signaling domain can also be a combination of signaling domains derived from molecules selected from CD3ζ, Fc receptor γ, FcγRIIa, FcRβ (FcεR1b) , CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. A signaling domain derived from a protein or polypeptide refers to the domain of the protein or polypeptide that is responsible for activating the immune effector cell (e.g., a T cell) , or a fragment thereof that retains its activation function. In general, the signaling domain induces persistence, trafficking and/or effector functions in the transduced immune effector cells such as T cells (Sharpe et al., Dis. Model Mech. 8: 337-350 (2015) ; Finney et al., J. Immunol. 161: 2791-2797 (1998) ; Krause et al., J. Exp. Med. 188: 619-626 (1998) ) . The signaling domain of a protein or polypeptide can be the intracellular domain of the protein or polypeptide. In some embodiments, the signaling domain comprises the intracellular domain of CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

In some embodiments, the cytoplasmic domain of CARs provided herein comprises a signaling domain derived from CD3ζ. In some embodiments, the signaling domain comprises the intracellular domain of CD3ζ.

In some embodiments, the cytoplasmic domain of CARs provided herein further comprises a co-stimulatory domain. In some embodiments, the cytoplasmic domain of CARs provided herein further comprises two co-stimulatory domains. Such a co-stimulatory domain can provide increased activation of an immune effector cell (e.g., T cell) . A co-stimulatory signaling domain can be derived from, for example, CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, TIGIT, GITR, TLR, DR3, or CD43. A co-stimulatory domain derived from a protein or polypeptide refers to the domain of the protein or polypeptide that is responsible for providing increased activation of an immune effector cell (e.g., T cell) , or a fragment thereof that retains its activation function. In some embodiments, the co-stimulatory domain of CARs provided herein comprises the intracellular domain of CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, TIGIT, GITR, TLR, DR3, or CD43.

In some embodiments, the cytoplasmic domain of CARs provided herein comprises a co-stimulatory domain derived from CD28. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD28. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from 4-1BB. In some embodiments, the co-stimulatory domain comprises the intracellular domain of 4-1BB.

CARs comprising an intracellular domain that comprises a co-stimulatory domain derived from 4-1BB, ICOS or DAP-10 have been described previously (see U.S. 7,446,190, which is incorporated herein by reference, which also describes representative sequences for 4-1BB, ICOS and DAP-10) . In some embodiments, the cytoplasmic domain of a CAR can comprise two co-stimulatory domains derived from two co-stimulatory receptors, such as CD28 and 4-1BB (see Sadelain et al., Cancer Discov. 3 (4) : 388-398 (2013) ) , or CD28 and OX40, or other combinations of co-stimulatory ligands, as disclosed herein.

The extracellular domain of a CAR can be fused to a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum and subsequent translocation to the cell surface. It is understood that, once a polypeptide containing a signal peptide is expressed at the cell surface, the signal peptide has generally been proteolytically removed during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, a polypeptide such as a CAR is generally expressed at the cell surface as a mature protein lacking the signal peptide, whereas the precursor form of the polypeptide includes the signal peptide. A signal peptide or leader can be essential if a CAR is to be glycosylated and/or anchored in the cell membrane. The signal sequence or leader is a peptide sequence generally present at the N-terminus of newly synthesized proteins that directs their entry into the secretory pathway. The signal peptide is covalently joined to the N-terminus of the extracellular antigen-binding domain of a CAR as a fusion protein. Any suitable signal peptide, as are well known in the art, can be applied to a CAR to provide cell surface expression in an immune cell (see Gierasch Biochem. 28: 923-930 (1989) ; von Heijne, J. Mol. Biol. 184 (1) : 99–105 (1985) ) . Particularly useful signal peptides can be derived from cell surface proteins naturally expressed in the immune cell provided herein, including any of the signal peptides of the polypeptides disclosed herein. Thus, any suitable signal peptide can be utilized to direct a CAR to be expressed at the cell surface of an immune effector cell provided herein.

In some embodiments, a CAR can also comprise a spacer region or sequence that links the domains of the CAR to each other. For example, a spacer can be included between a signal peptide and an antigen binding domain, between the antigen binding domain and the transmembrane domain, between the transmembrane domain and the intracellular domain, and/or between domains within the intracellular domain, for example, between a stimulatory domain and a co-stimulatory domain. The spacer region can be flexible enough to allow interactions of various domains with other polypeptides, for example, to allow the antigen binding domain to have flexibility in orientation in order to facilitate antigen recognition. The spacer region can be, for example, the hinge region from an IgG, the CH2CH3 (constant) region of an immunoglobulin, and/or portions of CD3 (cluster of differentiation 3) or some other sequence suitable as a spacer. In some embodiments, a CAR disclosed herein comprises a hinge domain that connects the mesothelin binding domain and the transmembrane domain. In some embodiments, the hinge domain comprises human CD8 hinge domain. In some embodiments, the hinge domain comprises human CD28 hinge domain.

Provided below are some exemplary molecules from which domains of the CARs provided herein can be derived.

CD3ζ. CD3ζ comprises 3 Immune-receptor-Tyrosine-based-Activation-Motifs (ITAMs) , and transmits an activation signal to the cell, for example, a cell of the lymphoid lineage such as a T cell, after antigen is bound. A CD3ζ polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_932170 (NP_932170.1, GI: 37595565; see below) , SEQ ID NO: 28, or fragments thereof. In some embodiments, a CD3ζ signaling domain has an amino acid sequence of amino acids 52 to 164 of the CD3ζ polypeptide sequence provided below, or a fragment thereof that is sufficient for signaling activity. See GenBank NP_932170 for reference to domains within CD3ζ, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 30; transmembrane region, amino acids 31 to 51; intracellular domain, amino acids 52 to 164. In some embodiments, a CAR can have a transmembrane domain derived from CD3ζ. The transmembrane domain can comprise the transmembrane region of CD3ζ (e.g., amino acids 31 to 51 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from CD3ζ. In some embodiments, a signaling domain of CD3ζ can comprise the intracellular domain of CD3ζ (e.g., amino acids 52 to 164 of the sequence below) , or a fragment thereof. It is understood that sequences of CD3ζ that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

4-1BB. 4-1BB, also referred to as tumor necrosis factor receptor superfamily member 9, can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. A 4-1BB polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P41273 (P41273.1, GI: 728739) or NP_001552 (NP_001552.2, GI: 5730095) . In some embodiments, a CAR can comprise a transmembrane domain derived from 4-1BB.

CD8. Cluster of differentiation 8 (CD8) is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR) . CD8 binds to a major histocompatibility complex (MHC) molecule and is specific for the class I MHC protein. In some embodiments, a CAR can comprise a transmembrane domain derived from CD8. A CD8 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_001139345.1 (GI: 225007536) , or fragments thereof. See GenBank NP_001139345.1 for reference to domains within CD8, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 182; transmembrane domain amino acids, 183 to 203; intracellular domain, amino acids 204 to 235. In some embodiments, a CAR can comprise a hinge domain derived from CD8.

As such, for exemplary purposes, a CAR disclosed herein can comprise, from N-terminus to the C-terminus, an anti-mesothelin antibody or antigen-binding fragment (e.g., scFvs disclosed herein) , a hinge (e.g., CD8 hinge) , a transmembrane region (e.g., CD8 transmembrane region) , a costimulatory domain (e.g., the intracellular domain of 4-1BB) , and a signaling domain (e.g., the T cell signaling domain of CD3ζ) .
5.4 LACOSTIM fusion proteins

In some embodiments, cells provided herein recombinantly express a fusion protein. In some embodiments, cells are genetically modified to express the fusion protein. In some embodiments, cells provided herein are genetically modified to express the fusion protein provided herein and the CAR provided herein.

In some embodiments, the fusion protein is a membrane protein. In some embodiments, the fusion protein is a soluble protein. In some embodiments, the fusion protein is a bispecific antibody.

In some embodiments, the fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein (i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and (ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

In some embodiments, the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof. In some embodiments, the co-stimulatory ligand is selected from the group consisting of CD58, mesothelin, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44. In some embodiments, the second domain is an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the second domain is a monoclonal antibody. In some embodiments, the second domain is a chimeric, humanized, or human antibody.

In some embodiments, the first domain of the fusion protein comprises a CD40L ECD, and the second domain of the fusion protein comprises a CD28 cytoplasmic domain. In some embodiments, the first domain of the fusion protein comprises a CD40L. In some embodiments, the first domain of the fusion protein comprises a CD40L ECD and the second domain of the fusion protein comprises an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain of the fusion protein comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain of the fusion protein comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

In some embodiments, the fusion protein further comprises a signal peptide domain. In some embodiments, the C-terminus of the signal peptide is linked to the N-terminus of the first domain. In some embodiments, the signal peptide comprises a signal peptide derived from CD8. In some embodiments, the signal peptide comprises a signal peptide derived from CD40L.

In some embodiments, cells provided herein recombinantly express a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein: (i) the first domain comprises an anti-CD40 antibody or antigen-binding fragment thereof; and (ii) the second domain comprises a CD28 transmembrane region and a CD28 cytoplasmic domain. In some embodiments, cells provided herein express CARs provided herein and express the fusion protein. Expression of the fusion proteins disclosed herein can not only promote the proliferation and activation of immune effector cells (e.g., T cells) , but also stimulate the maturation and epitope spreading activities of antigen-presenting cells. In some embodiments, expression of the fusion proteins in genetically engineered T cell disclosed herein helps them overcome immunosuppression in tumor microenvironment mediated by such as the PD1/PD-L1 signaling, regulatory T cells (Tregs) and TGF-beta signaling, and enhances their anti-tumor activities. The fusion proteins of the present disclosure are also referred to as Lymphocytes-Antigen presenting cells Co-stimulators ( “LACOSTIMs” or “LACO” ) . For example, the fusion protein described herein comprises Lymphocytes-Antigen presenting cells Co-stimulators described in US Patent No. 11667723, the disclosure of US Patent No. 11667723 is hereby incorporated by reference.

CD40: CD40 is a 48 kD transmembrane glycoprotein surface receptor that is a member of the Tumor Necrosis Factor Receptor superfamily (TNFRSF) . Exemplary amino acid sequences of human CD40 are described (see, e.g., Accession: ALQ33424.1, GenBank NP_001241.1, GI: 957949089) , and SEQ ID NO: 35 is the amino acid sequence of human CD40. CD40 was initially characterized as a co-stimulatory receptor expressed on APCs that played a central role in B and T cell activation. The ligand for CD40, CD154 (also known as TRAP, T-BAM, CD40 Ligand or CD40L) is a type II integral membrane protein. See GenBank NP_001241.1 for reference to domains within CD40, for example, signal peptide, amino acids 1 to 20; extracellular domain, amino acids 21 to 193; transmembrane domain, amino acids 194 to 215; intracellular domain, amino acids 216 to 277.

CD28: Cluster of Differentiation 28 (CD28) is a protein expressed on T cells that provides co-stimulatory signals for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. A CD28 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P10747 (P10747.1, GI: 115973) or NP_006130 (NP_006130.1, GI: 5453611) , the amino acid sequence of SEQ ID NO: 37, or fragments thereof. See GenBank NP_006130 for reference to domains within CD28, for example, signal peptide, amino acids 1 to 18; extracellular domain, amino acids 19 to 152; transmembrane domain, amino acids 153 to 179; intracellular domain, amino acids 180 to 220.

In some embodiments, the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an anti-CD40 scFv. In some embodiments, the first domain comprises an anti-CD40 scFv having amino acid sequences of SEQ ID NOs: 38-48 as listed in table 4.

Table 4: CD40 scFvs

In some embodiments, the second domain is an antibody or an antigen-binding fragment that binds CD28. In some embodiments, the second domain comprises a CD28 cytoplasmic domain. In some embodiments, the second domain comprises a CD28 cytoplasmic domain having amino acid sequences of SEQ ID NO: 36. In some embodiments, the second domain comprises a CD28 scFv. In some embodiments, the second domain comprises a CD28 scFv having VH (SEQ ID NO: 60) and/or VL (SEQ ID NO: 61) . In some embodiments, the second domain comprises a CD28 scFv having an amino acid sequence of SEQ ID NO: 62.

Table 5: CD28 scFv

In some embodiments, the fusion protein is at least 85%, 90%, 95%, 98%, 99%or 100%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-59 and 63-73. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 54. In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 68.
5.5 Polynucleotides and Vectors

Also provided herein are polynucleotides that encode a polypeptide (e.g., an anti-mesothelin antibody or antigen-binding fragment or a CAR that specifically binds mesothelin, or a fusion protein) described herein. The term “polynucleotides that encode a polypeptide” encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. Single-stranded DNA can be the coding strand or non-coding (anti-sense) strand. The polynucleotides of the disclosure can be mRNA.

Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments, provided herein are vectors comprising a polynucleotide provided herein. The vectors can be expression vectors. In some embodiments, vectors provided herein comprise a polynucleotide encoding an anti-mesothelin antibody or antigen-binding fragment described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a polypeptide that is part of an anti-mesothelin antibody or antigen-binding fragment described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a CAR or TCR described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a polypeptide that is part of a CAR or TCR described herein. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, lentivirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

Provided herein are cells comprising the polynucleotides disclosed herein. In some embodiments, provided herein are cells comprising a polynucleotide that encodes a polypeptide disclosed herein. In some embodiments, provided herein are cells comprising a vector having a polynucleotide disclosed herein. In some embodiments, provided herein are cells recombinantly expressing a polypeptide disclosed herein. The polypeptide can be an anti-mesothelin antibody or antigen-binding fragment. The polypeptide can be mesothelin CAR. The polypeptide can be a mesothelin TCR. In some embodiments, the cells provided herein can further express the fusion proteins disclosed herein or comprise a polynucleotide encoding a fusion protein disclosed herein. The fusion protein can be any LACOSTIM molecules disclosed herein.

In some embodiments, the cells provided herein express a CAR disclosed herein and a fusion protein disclosed herein. In some embodiments, cells provided herein comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a CAR. In some embodiments, the cells express a TCR disclosed herein and a fusion protein disclosed herein. In some embodiments, the cells comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a TCR.

In some embodiments, cells co-expressing the first and second fragment have the amino acid sequences or the nucleoside sequences of SEQ ID NOs: 74-97, In some embodiments, the first and second fragment have the amino acid sequence listed in table 6.

Table 6: CAR AND LACOSTIM sequences in Co-expressing LACOSTIM CAR-T Cells:

In some embodiments, the immune effector cells provided herein can be genetically engineered. In some embodiments, the genetically engineered immune effector cells provided herein are isolated. In some embodiments, the genetically engineered immune effector cells provided herein are substantially pure.

As such, in some embodiments, provided herein are immune effector cells recombinantly expressing a polypeptide (e.g., an antibody or a CAR) disclosed herein. Provided herein are also immune effector cells (e.g., T cells) comprising a polynucleotide encoding a polypeptide (e.g., an antibody or a CAR) disclosed herein, or a vector having a polynucleotide disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) comprising a polynucleotide that encodes an anti-mesothelin antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) recombinantly expressing an anti-mesothelin antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are immune effector cells comprising a polynucleotide that encodes a mesothelin CAR disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) recombinantly expressing a mesothelin CAR disclosed herein (e.g., mesothelin CART cell) . In some embodiments, provided herein are immune effector cells comprising a polynucleotide that encodes a mesothelin TCR disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) recombinantly expressing a mesothelin TCR disclosed herein (e.g., mesothelin TCRT cell) .

In some embodiments, the immune effector cell provided herein is a T cell. The T cell can be a cytotoxic T cell, a helper T cell, or a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a cytotoxic T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, an effector memory TEMRA cell, or a gamma delta T cell. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the T cell is genetically engineered. In some embodiments, the T cells provided herein are isolated. In some embodiments, the T cells provided herein are substantially pure.

In some embodiments, genetically engineered cells provided herein are derived from cells isolated from a subject. As used herein, a genetically engineered cell that is “derived from” a source cell means that the genetically engineered cell is obtained by taking the source cell and genetically manipulating the source cell. The source cell can be from a natural source. For example, the source cell can be a primary cell isolated from a subject. The subject can be an animal or a human. The source cell can also be a cell that has undergone passages or genetically manipulation in vitro.

In some embodiments, genetically engineered cells provided herein are derived from cells isolated from a human. Immune effector cells (e.g., T cells) can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cell lines available in the art can be used. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from peripheral blood. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from bone marrow. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from peripheral blood mononuclear cells (PBMC) .

In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a stem or progenitor cell. In some embodiments, the stem or progenitor cell is selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a T cell progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a hematopoietic stem and progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a hematopoietic multipotent progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from an embryonic stem cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from an induced pluripotent cell.

In some embodiments, provided herein are a population of cells comprising a cell disclosed herein. The cells disclosed herein can comprise a polynucleotide that encodes a polypeptide disclosed herein or recombinantly express a polypeptide disclosed herein. The polypeptide can be an anti-mesothelin antibody or antigen-binding fragment, a mesothelin CAR, or a mesothelin TCR. In some embodiments, cells disclosed herein can further express the fusion proteins disclosed herein or comprise a polynucleotide encoding a fusion protein disclosed herein. The fusion protein can be any fusion protein disclosed herein. In some embodiments, the population of cells provided herein express a CAR disclosed herein and a fusion protein disclosed herein. In some embodiments, the population of cells provided herein comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a CAR. In some embodiments, the genetically engineered immune effector cells provided herein express a TCR disclosed herein and a fusion protein disclosed herein. In some embodiments, the population of cells provided herein comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a TCR. In some embodiments, the population of cells provided herein comprise a polynucleotide that comprises a first fragment encoding a CAR and a second fragment encoding a fusion protein. In some embodiments, the population of cells provided herein comprise a polynucleotide that comprises a first fragment encoding a TCR and a second fragment encoding a fusion protein. In some embodiments, the polynucleotide has the first fragment and the second fragment from N-terminus to the C-terminus. In some embodiments, the polynucleotide has the second fragment and the first fragment, from N-terminus to the C-terminus.

Provided herein are also pharmaceutical compositions comprising the anti-mesothelin antibodies or antigen-binding fragments disclosed herein and/or the cells (e.g., CAR-T cells) disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the anti-mesothelin antibodies or antigen-binding fragments disclosed herein and a pharmaceutically acceptable carrier. The anti-mesothelin antibodies or antigen-binding fragments can be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise soluble anti-mesothelin antibodies or antigen-binding fragments provided herein at 1-1000 mg/ml. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the cells disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions comprising the cells (e.g., CAR-T cells) disclosed herein can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of cells in a cell population using various well-known methods, as described herein. The ranges of purity in cell populations comprising genetically engineered cells provided herein can be from about 20%to about 100%.

In some embodiments, the pharmaceutical compositions are useful in immunotherapy. In some embodiments, the pharmaceutical compositions are useful in immuno-oncology. In some embodiments, the pharmaceutical compositions are useful in inhibiting tumor growth in a subject (e.g., a human patient) . In some embodiments, the pharmaceutical compositions are useful in treating cancer in a subject (e.g., a human patient) .

Provided herein are also kits for preparation of pharmaceutical compositions having the anti-mesothelin antibodies or antigen-binding fragments disclosed herein. In some embodiments, the kit comprises the anti-mesothelin antibodies or antigen-binding fragments disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kits can comprise anti-mesothelin antibodies or antigen-binding fragments disclosed herein for administration to a subject. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the anti-mesothelin antibodies or antigen-binding fragments.

Provided herein are also kits for preparation of the cells (e.g., T cells) disclosed herein. In some embodiments, the kits comprise one or more vectors for generating a genetically engineered cell, such as a T cell, that expresses the anti-mesothelin antibodies or antigen-binding fragments and/or the anti-mesothelin CAR disclosed herein. The kits can be used to generate genetically engineered immune effector cells (e.g., T cells) from autologous or non-autologous cells to be administered to a compatible subject. In another embodiment, the kits can comprise immune effector cells disclosed herein for administration to a subject. In specific embodiments, the kits comprise the immune effector cells disclosed herein in one or more containers. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the immune effector cells.

In some embodiments, provided herein is a pharmaceutical composition comprising anti-mesothelin antibodies or antigen-binding fragments or cells provided herein wherein the composition is suitable for local administration. In some embodiments, local administration comprises intratumoral injection, peritumoral injection, juxta-tumoral injection, intralesional injection and/or injection into a tumor draining lymph node, or essentially any tumor-targeted injection where the antitumor agent is expected to leak into primary lymph nodes adjacent to targeted solid tumor.

Pharmaceutically acceptable carriers that can be used in compositions provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) . Depending on the route of administration, the active ingredient (i.e., anti-mesothelin antibodies or antigen-binding fragments or immune effector cells provided herein) , can be coated in a material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.

Provided herein are also pharmaceutical compositions or formulations that improve the stability of the anti-mesothelin antibodies or antigen-binding fragments to allow for their long-term storage. In some embodiments, the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years or more. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4℃, 25℃, or 40℃.

The pharmaceutical compositions disclosed herein can further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, an antioxidant, a wetting agent, an emulsifying agent, a dispersing agent and/or a surfactant, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well-known to the skilled person. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In some embodiments, the pharmaceutical composition is an aqueous formulation. Such a formulation is typically a solution or a suspension, but can also include colloids, dispersions, emulsions, and multi-phase materials. In some embodiments, the pharmaceutical compositions disclosed herein are freeze-dried, to which the physician or the patient adds solvents and/or diluents prior to use.

The amount of active ingredient which can be combined with a carrier material in the pharmaceutical compositions or formulations disclosed herein can vary. In some embodiments, the amount of active ingredient which can be combined with a carrier material is the amount that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

The pharmaceutical compositions disclosed herein can be prepared with carriers that protect the active ingredient against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.

In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments or immune effector cells (e.g., T cells) described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the activate ingredient described herein cross the BBB (if desired, e.g., for brain cancers) , they can be formulated, for example, in liposomes.
5.8 Methods and Uses

The present disclosure also provides methods of uses of the anti-mesothelin antibodies or antigen-binding fragments, mesothelin CARs, mesothelin TCRs, polynucleotides encoding such anti-mesothelin antibodies or antigen-binding fragments and mesothelin CARs/TCRs, vectors comprising such polynucleotides, mesothelin CAR/TCR-expressing cells or pharmaceutical compositions having such cells disclosed herein in treating cancer. Without being bound by theory, the anti-mesothelin antibodies or antigen-binding fragments and the mesothelin CAR/TCR-expressing cells disclosed herein can specifically target mesothelin expressing cancer cells in vivo, thereby delivering their therapeutic effect of eliminating, lysing and/or killing cancer cells. In some embodiments, the methods include administering a therapeutically effective amount of the anti-mesothelin antibodies or antigen-binding fragments disclosed herein to a subject in need thereof. In some embodiments, the methods include administering a therapeutically effective amount of mesothelin CAR-expressing immune effector cells disclosed herein to a subject in need thereof. In one embodiment, the methods can include administering a therapeutically effective amount of mesothelin CARTs disclosed herein to a subject in need thereof.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-mesothelin antibodies or antigen-binding fragments disclosed herein. In some embodiments, provided herein are uses of the anti-mesothelin antibodies or antigen-binding fragments disclosed herein in the treatment of tumor or cancer. In some embodiments, provided herein are uses of the anti-mesothelin antibodies or antigen-binding fragments provided herein for the preparation of a medicament for the treatment of tumor or cancer.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immune effector cells (e.g., mesothelin CARTs) disclosed herein. In some embodiments, provided herein are uses of the immune effector cells disclosed herein (e.g., mesothelin CARTs) in treatment of tumor or cancer. In some embodiments, provided herein are uses of the immune effector cells (e.g., mesothelin CARTs) provided herein for the preparation of a medicament for the treatment of tumor or cancer. In some embodiments, a population of cells comprising the immune effector cell disclosed herein is used in the treatment. The population of cells can be homogenous. The population of cells can be heterogenous.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, provided herein are uses of the pharmaceutical composition disclosed herein in treatment of tumor or cancer. In some embodiments, provided herein are uses of the pharmaceutical composition provided herein for the preparation of a medicament for the treatment of tumor or cancer.

Actual dosage levels of the active ingredients (i.e., the anti-mesothelin antibodies or antigen-binding fragments or the immune effector cells provided herein) in the pharmaceutical compositions described herein can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions 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.

The anti-mesothelin antibodies or antigen-binding fragments can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the anti-mesothelin antibodies or antigen-binding fragments in the patient. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease.

In some embodiments, immune effector cells provided herein that recombinantly express the mesothelin CARs or TCRs disclosed herein can be used in the therapeutic methods disclosed herein. When a cell therapy is adopted, the cells provided herein can be administered as a dose based on cells per kilogram (cells/kg) of body weight of the subject to which the cells are administered. The cell doses can be in the range of about 10⁴ to about 10¹⁰ cells/kg of body weight, for example, about 10⁵ to about 10⁹, about 10⁵ to about 10⁸, about 10⁵ to about 10⁷, or about 10⁵ to 10⁶ cells/kg of body weight, depending on the mode and location of administration. In general, in the case of systemic administration, a higher dose is used than in regional administration, where the immune effector cells are administered in the region of a tumor. The precise determination of what would be considered an effective dose can be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject, as described above. Dosages can be readily determined by those skilled in the art based on the disclosure herein and knowledge in the art.

Proliferation of the cells provided herein is generally done ex vivo, prior to administration to a subject, and can be desirable in vivo after administration to a subject (see Kaiser et al., Cancer Gene Therapy 22: 72-78 (2015) ) . Cell proliferation should be accompanied by cell survival to permit cell expansion and persistence, such as with T cells.

Diseases that can be treated using the anti-mesothelin antibodies or antigen-binding fragments, the immune effector cells, or the pharmaceutical compositions provided herein include any disease or disorder associated with mesothelin, and any disease or disorder in which mesothelin is specifically expressed and/or in which mesothelin has been targeted for treatment (collectively “mesothelin-associated diseases or disorders” ) . Mesothelioma, for example, is a disease associated with mesothelin.

Malignant mesothelioma is a type of cancer that occurs in the thin layer of cells lining the body's internal organs, known as the mesothelium. There are three recognized types of mesothelioma. Pleural mesothelioma (e.g., malignant pleural mesothelioma, or MPM) is the most common form of the disease, accounting for roughly 70%of cases, and occurs in the lining of the lung known as the pleura. Peritoneal mesothelioma occurs in the lining of the abdominal cavity, known as the peritoneum. Pericardial mesothelioma originates in the pericardium, which lines the heart. In some embodiments, anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions disclosed herein can be used to treat mesothelioma. In some embodiments, the mesothelioma is pleural mesothelioma. In some embodiments, the mesothelioma is peritoneal mesothelioma. In some embodiments, the mesothelioma is pericardial mesothelioma.

Symptoms of pleural mesothelioma include, e.g., lower back pain or side chest pain, and shortness of breath. Other symptoms include difficulty swallowing, persistent cough, fever, weight loss or fatigue. Additional symptoms that some patients experience include muscle weakness, loss of sensory capability, coughing up blood, facial and arm swelling, and hoarseness. In the early stages of the disease, such as stage 1 mesothelioma, symptoms can be mild. Patients usually report pain in one area of the chest that never seems to go away, weight loss and fever.

Peritoneal mesothelioma originates in the abdomen and as a result, symptoms often include abdominal pain, weight loss, nausea, and vomiting. Fluid buildup may occur in the abdomen as well as a result of the cancer. Peritoneal mesothelioma originates in the abdomen and will frequently spread to other organs in area including the liver, spleen or bowel. Severe abdominal pain is the most common complaint that patients first experience. There can also be a discomfort level with fluid buildup in the abdomen as well. Other symptoms of peritoneal mesothelioma may include difficult bowel movements, nausea and vomiting, fever and swollen feet.

Pericardial mesothelioma is the least common form of mesothelioma. Pericardial mesothelioma involves the heart. This rare type of mesothelioma cancer invades the pericardium. As the cancer progresses, the heart is not able to deliver oxygen as efficiently to the body causing further decline in health at an increasingly rapid rate. The symptoms most commonly associated with pericardial mesothelioma mimic those of a heart attack: nausea, pain in the chest and shortness of breath.

In some embodiments, methods provided herein can treat a subject having mesothelioma. In some embodiments, methods provided herein can treat a subject at risk to develop mesothelioma. A subject can be at risk to develop mesothelioma if the subject has been exposed to asbestos. Exposure to asbestos and the inhalation of asbestos particles can cause mesothelioma. In most cases, mesothelioma symptoms will not appear in a subject exposed to asbestos until many years after the exposure has occurred. In some embodiments, methods provided herein can treat a subject suspected of having mesothelioma, e.g., as evidenced by the presence of one or more of the symptoms described herein and/or exposure to asbestos. In some embodiments, the subject that can be treated with the methods disclosed herein can have a precancerous condition such as, e.g., pleural plaques, benign mesothelioma or mesothelial hyperplasia.

Another example of a disease or disorder associated with mesothelin is pancreatic cancer. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein can be used to treat pancreatic cancer. Pancreatic cancers that can be treated with methods described herein include, but are not limited to, exocrine pancreatic cancers and endocrine pancreatic cancers. Exocrine pancreatic cancers include, but are not limited to, adenocarcinomas, acinar cell carcinomas, adenosquamous carcinomas, colloid carcinomas, undifferentiated carcinomas with osteoclast-like giant cells, hepatoid carcinomas, intraductal papillarymucinous neoplasms, mucinous cystic neoplasms, pancreatoblastomas, serous cystadenomas, signet ring cell carcinomas, solid and pseuodpapillary tumors, pancreatic ductal carcinomas, and undifferentiated carcinomas. In some embodiments, the exocrine pancreatic cancer is pancreatic ductal carcinoma. Endocrine pancreatic cancers include, but are not limited to, insulinomas and glucagonomas.

In some embodiments, the pancreatic cancer is any of early stage pancreatic cancer, non-metastatic pancreatic cancer, primary pancreatic cancer, resected pancreatic cancer, advanced pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer, unresectable pancreatic cancer, pancreatic cancer in remission, recurrent pancreatic cancer, pancreatic cancer in an adjuvant setting, or pancreatic cancer in a neoadjuvant setting. In some embodiments, the pancreatic cancer is locally advanced pancreatic cancer, unresectable pancreatic cancer, or metastatic pancreatic ductal carcinoma. In some embodiments, the pancreatic cancer is resistant to the gemcitabine-based therapy. In some embodiments, the pancreatic cancer is refractory to the gemcitabine-based therapy.

Ovarian cancer can also be associated with mesothelin expression. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein can be used to treat ovarian cancer. Ovarian cancer is classified according to the histology of the tumor. Surface epithelial-stromal tumor, also known as ovarian epithelial carcinoma, is the most common type of ovarian cancer. It includes serous tumor (including serous papillary cystadenocarcinoma) , endometrioid tumor and mucinous cystadenocarcinoma.

The methods described herein can be used to treat various stages of ovarian cancer, e.g., stage I, stage II, stage III or stage IV. Staging can be performed, e.g., when the ovarian cancer is removed. Ovarian cancer is staged as follows: Stage I cancer is confined to one or both ovaries. The cancer is stage II if either one or both of the ovaries is involved and has spread to the uterus and/or the fallopian tubes or other sites in the pelvis. The cancer is stage III cancer if one or both of the ovaries is involved and has spread to lymph nodes or other sites outside of the pelvis but is still within the abdominal cavity, such as the surface of the intestine or liver. The cancer is stage IV cancer if one or both ovaries are involved and the cancer has spread outside the abdomen or to the inside of the liver.

In some embodiments, the ovarian cancer is resistant to one or more chemotherapeutic agent. In some embodiments, the ovarian cancer is refractory to the one or more chemotherapeutic agent.

Other cancers that can be treated with the anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein include, e.g., brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer (e.g., lung adenocarcinoma) , melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, uterine cancer, and any combination thereof.

In certain diseases and conditions, mesothelin is expressed on malignant cells and cancers. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematopoietic cancer. In some embodiments, the cancer is a renal cancer, lung cancer, urothelial cancer, breast cancer, brain tumor, thyroid cancer, leukemia, lymphoma, and/or multiple myeloma. In some embodiments, the cancer is renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, glioblastoma, anaplastic thyroid cancer, B-cell non-Hodgkin’s lymphoma, and/or chronic lymphoblastic leukemia.

In some embodiments, the methods include identifying a subject who has, is suspected to have, or is at risk for developing a mesothelin-associated disease or disorder. Hence, provided are methods for identifying subjects with diseases or disorders associated with elevated mesothelin expression and selecting them for treatment with the anti-mesothelin antibodies or antigen-binding fragments, the immune effector cells, or the pharmaceutical compositions provided herein.

In some embodiments, a subject can be screened for the presence of a disease or disorder associated with elevated mesothelin expression, such as a mesothelin-expressing cancer. In some embodiments, the methods include screening for or detecting the presence of a mesothelin-associated disease, e.g., a tumor. Thus, in some embodiments, a sample can be obtained from a patient suspected of having a disease or disorder associated with elevated mesothelin expression and assayed for the expression level of mesothelin. In some embodiments, a subject who tests positive for a mesothelin-associated disease or disorder can be selected for treatment by the present methods, and can be administered a therapeutically effective amount of the anti-mesothelin antibodies or antigen-binding fragments, the immune effector cells, or the pharmaceutical compositions provided herein.

In cancer treatment, eliminating cancer or tumor cells in a subject can occur, but any clinical improvement constitutes a benefit. An anti-tumor effect can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An anti-tumor effect can also be manifested by the ability of the cells or pharmaceutical compositions provided herein in prevention of the occurrence of tumor in the first place. In some embodiments, an “anti-tumor effect” can be manifested by the reduction in cancer-induced immunosuppression. Clinical improvement comprises decreased risk or rate of progression or reduction in pathological consequences of the cancer or tumor. It is also understood that a method of treating cancer can include any effect that ameliorates a sign or symptom associated with cancer. Such signs or symptoms include, but are not limited to, reducing tumor burden, including inhibiting growth of a tumor, slowing the growth rate of a tumor, reducing the size of a tumor, reducing the number of tumors, eliminating a tumor, all of which can be measured using routine tumor imaging techniques well known in the art. Other signs or symptoms associated with cancer include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers.

In some embodiments, the methods or uses provided herein can reduce tumor burden. Thus, administration of the anti-mesothelin antibodies or antigen-binding fragments, cells or pharmaceutical compositions disclosed herein can reduce the number of tumor cells, reduce tumor size, and/or eradicate the tumor in the subject. Methods for monitoring patient response to administration of a pharmaceutical composition disclosed herein are known in the art and can be employed in accordance with methods disclosed herein.

In the methods disclosed herein, a therapeutically effective amount of the anti-mesothelin antibodies or antigen-binding fragments, cells or pharmaceutical compositions disclosed herein is administered to a subject in need of cancer treatment. The subject can be a mammal. In some embodiments, the subject is a human. In some embodiments, these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts and are suitably defined for different types of cancers. Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.

In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another mesothelin-specific antibody and/or mesothelin CART and/or other therapy, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogeneic HSCT or autologous HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another mesothelin-targeted therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.

In some embodiments, the subject is one that is eligible for a transplant, such as is eligible for a hematopoietic stem cell transplantation (HSCT) , e.g., allogeneic HSCT or autologous HSCT. In some embodiments, the subject has not previously received a transplant, despite being eligible, prior to administration of the mesothelin-binding molecules, including the anti-mesothelin antibodies or antigen-binding fragments, the immune effector cells, or the pharmaceutical compositions provided herein. In some embodiments, the subject is one that is not eligible for a transplant, such as is not eligible for a hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT or autologous HSCT.

In some embodiments, the methods provided herein include adoptive cell therapy, whereby genetically engineered immune effector cells expressing the provided recombinant receptors comprising a mesothelin-binding molecule (e.g., mesothelin CARs provided herein) are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in a mesothelin-targeted manner, such that the cells of the disease or disorder are targeted for destruction. Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject such as one having, or at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in a mesothelin-expressing cancer.

Methods for administration of cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al.; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8 (10) : 577-85) . See, e.g., Themeli et al. (2013) Nat Biotechnol. 31 (10) : 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438 (1) : 84-9; Davila et al. (2013) PLoS ONE 8 (4) : e61338.

In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some embodiments, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are 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.

Anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein can be administered with medical devices known in the art. For example, in some embodiments, a needleless hypodermic injection device can be used, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use described herein include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

Combination therapy using agents with different mechanisms of action can result in additive or synergetic effects. Combination therapy can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent disclosed herein. Combination therapy can decrease the likelihood that resistant cancer cells will develop. In some embodiments, the additional therapy results in an increase in the therapeutic index of the cells or pharmaceutical compositions described herein. In some embodiments, the additional therapy results in a decrease in the toxicity and/or side effects of cells or pharmaceutical compositions described herein. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions described herein can be administered in combination with an additional therapy. In some embodiments, the additional therapy can be surgical resection, radiotherapy, or chemotherapy.

The additional therapy can be administered prior to, concurrently with, or subsequent to administration of the anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions described herein. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. A person skilled in the art can readily determine appropriate regimens for administering a pharmaceutical composition described herein and an additional therapy in combination, including the timing and dosing of an additional agent to be used in a combination therapy, based on the needs of the subject being treated.
5.9 Methods of production

With respect to generating cells recombinantly expressing an antibody or antigen-binding fragment thereof disclosed herein, a chimeric antigen receptor disclosed herein, and/or a fusion protein disclosed herein, one or more polynucleotides is introduced into the target cell using a suitable expression vector. The target immune effector cells (e.g., T cells) are transferred with one or more polynucleotides encoding a fusion protein, or a CAR/TCR/BiTE and a fusion protein. The CAR/TCR/BiTE and fusion protein encoding polynucleotides can be on separate vectors or on the same vector, as desired. For example, a polynucleotide encoding a CAR or a fusion protein disclosed herein can be cloned into a suitable vector, such as a viral vector, and introduced into the target cell using well known molecular biology techniques (see Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999) ) . Any vector suitable for expression in a cell, particularly a human cell, can be used. The vectors contain suitable expression elements such as promoters that provide for expression of the encoded nucleic acids in the target cell. In the case of a retroviral vector, cells can optionally be activated to increase transduction efficiency (see Parente-Pereira et al., J. Biol. Methods 1 (2) e7 (doi 10.14440/jbm. 2014.30) (2014) ; Movassagh et al., Hum. Gene Ther. 11: 1189-1200 (2000) ; Rettig et al., Mol. Ther. 8: 29-41 (2003) ; Agarwal et al., J. Virol. 72:3720-3728 (1998) ; Pollok et al., Hum. Gene Ther. 10: 2221-2236 (1998) ; Quinn et al., Hum. Gene Ther. 9: 1457-1467 (1998) ; see also commercially available methods such as Dynabeads^TM human T cell activator products, Thermo Fisher Scientific, Waltham, MA) .

In one embodiment, the vector is a retroviral vector, for example, a gamma retroviral or lentiviral vector, which is employed for the introduction of a fusion protein and/or a CAR, TCR, or BiTE into the target cell. For genetic modification of the cells to express a fusion protein and/or a CAR, TCR, or BiTE, a retroviral vector can be employed for transduction. However, it is understood that any suitable viral vector or non-viral delivery system can be used. Combinations of a retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., Mol. Cell. Biol. 5: 431-437 (1985) ) ; PA317 (Miller et al., Mol. Cell. Biol. 6: 2895-2902 (1986) ) ; and CRIP (Danos et al., Proc. Natl. Acad. Sci. USA 85: 6460-6464 (1988) ) . Non-amphotropic particles are suitable too, for example, particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art (Relander et al., Mol. Therap. 11: 452-459 (2005) ) . Possible methods of transduction also include direct co-culture of the cells with producer cells (for example, Bregni et al., Blood 80: 1418-1422 (1992) ) , or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations (see, for example, Xu et al., Exp. Hemat. 22: 223-230 (1994) ; Hughes, et al. J. Clin. Invest. 89: 1817-1824 (1992) ) .

Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus derived vector, or a herpes virus, such as Epstein-Barr Virus (see, for example, Miller, Hum. Gene Ther. 1 (1) : 5-14 (1990) ; Friedman, Science 244: 1275-1281 (1989) ; Eglitis et al., BioTechniques 6: 608-614 (1988) ; Tolstoshev et al., Current Opin. Biotechnol. 1: 55-61 (1990) ; Sharp, Lancet 337: 1277-1278 (1991) ; Cornetta et al., Prog. Nucleic Acid Res. Mol. Biol. 36: 311-322 (1989) ; Anderson, Science 226: 401-409 (1984) ; Moen, Blood Cells 17: 407-416 (1991) ; Miller et al., Biotechnology 7: 980-990 (1989) ; Le Gal La Salle et al., Science 259: 988-990 (1993) ; and Johnson, Chest 107: 77S-83S (1995) ) . Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med. 323: 370 (1990) ; Anderson et al., U.S. Pat. No. 5,399,346) . Generally, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8: 423-430 (1997) ; Kido et al., Current Eye Research 15: 833-844 (1996) ; Bloomer et al., J. Virol. 71: 6641-6649 (1997) ; Naldini et al., Science 272: 263-267 (1996) ; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319-10323 (1997) ) .

Particularly useful vectors for expressing a fusion protein disclosed herein and/or CAR/TCR/BiTE include vectors that have been used in human gene therapy. In one non-limiting embodiment, a vector is a retroviral vector. The use of retroviral vectors for expression in T cells or other immune effector cells, including engineered T cells, has been described (see Scholler et al., Sci. Transl. Med. 4: 132-153 (2012; Parente-Pereira et al., J. Biol. Methods 1 (2) : e7 (1-9) (2014) ; Lamers et al., Blood 117 (1) : 72-82 (2011) ; Reviere et al., Proc. Natl. Acad. Sci. USA 92: 6733-6737 (1995) ) . In one embodiment, the vector is an SGF retroviral vector such as an SGF γ-retroviral vector, which is Moloney murine leukemia-based retroviral vector. SGF vectors have been described previously (see, for example, Wang et al., Gene Therapy 15: 1454-1459 (2008) ) .

The vectors used herein employ suitable promoters for expression in a particular host cell. The promoter can be an inducible promoter or a constitutive promoter. In some embodiments, the promoter of an expression vector provides expression in a stem cell, such as a hematopoietic stem cell. In some embodiments, the promoter of an expression vector provides expression in an immune effector cell, such as a T cell. Non-viral vectors can be used as well, so long as the vector contains suitable expression elements for expression in the target cell. Some vectors, such as retroviral vectors, can integrate into the host genome.

In some embodiments, provided herein are methods of genetically engineering an immune effector cell by transferring a polynucleotide provided herein into the cell using a non-viral delivery system. For example, physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. In some embodiments, RNA electroporation can be used (Van Driessche et al. Folia histochemica et cytobiologica 43: 4 213-216 (2005) ) . In some embodiments, DNA transfection and transposon can be used. In some embodiments, the Sleeping Beauty system or PiggyBac system is used (e.g., Ivics et al., Cell, 91 (4) : 501-510 (1997) ; et al. (2007) Nucleic Acids Research. 35 (12) : e87) . Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle) .

In some embodiments, provided herein are methods of genetically engineering an immune effector cell by transferring a polynucleotide provided herein into the cell using gene-editing. If desired, targeted integration can be implemented using technologies such as a nuclease, transcription activator-like effector nucleases (TALENs) , Zinc-finger nucleases (ZFNs) , clustered regularly interspaced short palindromic repeats (CRISPRs) , homologous recombination, non-homologous end joining, microhomology-mediated end joining, homology-mediated end joining and the like (Gersbach et al., Nucl. Acids Res. 39: 7868-7878 (2011) ; Vasileva, et al. Cell Death Dis. 6: e1831. (Jul 23 2015) ; Sontheimer, Hum. Gene Ther. 26 (7) : 413-424 (2015) ; Yao et al. Cell Research volume 27, 801-814 (2017) ) .

Immune effector cells provided herein can be obtained from a subject. Sources for the immune effector cells provided herein include, but are not limited to, peripheral blood, umbilical cord blood, bone marrow, or other sources of hematopoietic cells. Immune effector cells (e.g., T cells) can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, cell lines available in the art can be used. Immune effector cells provided herein can be isolated by methods well known in the art, including commercially available isolation methods (see, for example, Rowland-Jones et al., LYMPHOCYTES: A PRACTICAL APPROACH, Oxford University Press, New York (1999) ) . Various methods for isolating immune effector cells have been described previously, and can be used, including but not limited to, using peripheral donor lymphocytes (Sadelain et al., Nat. Rev. Cancer 3: 35-45 (2003) ; Morgan et al., Science 314: 126-129 (2006) , and using selectively in v/Yro-expanded antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or dendritic cells (Dupont et al., Cancer Res. 65: 5417-5427 (2005) ; Papanicolaou et al., Blood 102: 2498-2505 (2003) ) .

The immune effector cells can be autologous or non-autologous to the subject to which they are administered in the methods of treatment disclosed herein. Autologous cells are isolated from the subject to which the engineered cells are to be administered. Optionally, the cells can be obtained by leukapheresis, where leukocytes are selectively removed from withdrawn blood, made recombinant, and then retransfused into the donor. Alternatively, allogeneic cells from a non-autologous donor that is not the subject can be used. In the case of a non-autologous donor, the cells are typed and matched for human leukocyte antigen (HLA) to determine an appropriate level of compatibility, as is well known in the art. The cells can optionally be cryopreserved after isolation and/or genetic engineering, and/or expansion of genetically engineered cells (see Kaiser et al., supra, 2015) ) . Methods for cyropreserving cells are well known in the art (see, for example, Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, 4^th ed., Wiley-Liss, New York (2000) ; Harrison and Rae, GENERAL TECHNIQUES OF CELL CULTURE, Cambridge University Press (1997) ) .

In some embodiments, isolated immune effector cells are genetically engineered ex vivo for recombinant expression of a fusion protein. In some embodiments, isolated immune effector cells are genetically engineered ex vivo for recombinant expression of a fusion protein and a CAR/TCR/BiTE. In some embodiments, immune effector cells provided herein are obtained by in vitro sensitization, wherein the sensitization can occur before or after the immune effector cells are genetically engineered to recombinantly express the fusion protein disclosed herein. In an embodiment where the sensitized immune effector cells, such T cells, are isolated from in vivo sources, it will be self-evident that genetic engineering occurs of the already-sensitized immune effector cells.
5.10 Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i. m., intramuscular (ly) ; i. p., intraperitoneal (ly) ; s. c., subcutaneous (ly) ; and the like.

Materials and Methods

Primary human lymphocytes. Primary human CD4+ T cells and CD8+ T cells were isolated from healthy volunteer donors. T cells were stimulated with anti-CD3/CD28 Dynabeads (Life Technologies, Grand Island, NY) and cultured in the R10 medium (RPMI-1640 medium supplemented with 10%fetal bovine serum, 1%HEPES, 1%GutaMAX, 1%penicillin and streptomycin, 1%MEM NEAA, and 1%sodium pyruvate) supplemented with 100 IU/mL IL-2.

Cell lines. The following cell lines were cultured in R10 medium and used in related experiments: A549 (human lung cancer cells) , ASPC1 (human pancreatic tumor cells) , H226 (human lung carcinoma cells) , OVCAR3 (human adenocarcinoma cells) , U87 (human malignant gliomas cells) , U251 (human malignant glioblastoma multiforme cells) , Caski (human cervical cancer cells) , SH-SY5Y (human neuroblastoma cells) , Siha (human squamous cell carcinoma cells) , HepG2 (human hepatocellular carcinoma cells) , PC3 (human prostate cancer cells) , HCC70 (human breast cancer cells) , THP1 (human leukemia cells) , Jeko1 (human lymphoma cells) , Nalm6 (human lymphoblastic leukemia cells) , Molm14 (human myeloid leukemia cells) , Raji (human lymphoma cells) , RPMI8226 (human myeloma cells) , A498 (human carcinoma cells) , Huh-7 (hepatocyte-derived carcinoma cells) , A375 (Malignant Melanoma cells) , 293T (human embryonic kidney (HEK) cells) , Supt1 (Lymphoblastic Lymphoma cells) .

Lentivirus production and transduction. Lentiviral particles were produced from HEK293T cells transfected with transfer plasmids and packaging plasmids pRSV. REV, pMD2. G and pMDLg. pRRE. Lentiviral particles were harvested after 24 and 48 hours and concentrated by ultracentrifugation.

T cells were stimulated by CD3/CD28 dynabeads on day 0. Lentivirus was added to culture medium on day 1. The cells were fed with R10 medium with 100 IU/ml IL-2 every day or every two days.

Production of anti-mesothelin CAR mRNA. In vitro transcription of mRNA was performed using the Ambion mMessage mMACHINE T7 Ultra kit (Life Technologies, Carlsbad, CA) .

Electroporation of anti-mesothelin CAR mRNA. mRNA was added to 0.1 ml T cells (0.5×10⁸ cells/ml) that were washed twice and resuspended with OPTI-MEM. Electroporation was performed in a 0.2 cm cuvette with a BTX830 (Harvard Apparatus BTX) at 500 V and 0.7 ms.

Tumor killing assay using IncuCyte real-time cell analyzer. Tumor cells and T cells were washed with R10 medium twice, and then were resuspended in R10 medium. Both tumor cells and CAR+ T cells were seeded at 4,000 cells/well in a 96 well flat-bottom plate. Total integrated intensity was recorded every 8 hours.

ELISA. 0.1 million of CAR-T cells and 0.1 million of tumor cells were co-cultured at 37 ℃ for 24 h, then the supernatant was collected and the ELISA assay was performed to measure IL-2 and IFNγ secretion levels following the instruction manual of ELISA kits (R&D Systems) .
5.10.1 Example 1: Construction of MOLM14-huMSLN stable cell line

The full-length huMSLN gene sequence was cloned into the lentiviral vector pUTCK, and the lentivirus was packaged and infected into MOLM14 cells. On the 3rd day after infection, the cells were stained with anti-huMSLN flow cytometry antibody. MOLM14 cells stained positive by huMSLN antibody were sorted using flow cytometry and continued to be expanded and cultured. As shown in FIG. 1, the sorted MOLM14-huMSLN cell line expresses high levels of huMSLN protein.
5.10.2 Example 2: anti-MSLN antibody phage display library screening

According to the initial OD value of about 0.1, an appropriate amount of original phage library liquid was inoculated into a 500ml Erlenmeyer flask with 2×YT medium, and 100 μg/mL ampicillin and 2%glucose were added. Set the speed to 250 rpm, the temperature to 37℃, and shake the bacteria for about 3 hours until the OD600 value reaches approximately 0.5. Add about 3×10¹² M13KO7 helper phage to the above bacterial solution and let it stand at 37 ℃ for 30 minutes. After 30 minutes, transfer the infected bacterial solution to a 50 ml centrifuge tube, centrifuge at 2400 g, 12 ℃ for 30 minutes, and discard the supernatant to remove the inhibitory effect of glucose on antibody expression.

Resuspend the pellet to 600 ml 2×YT and add ampicillin at a final concentration of 100 μg/mL, kanamycin at 60 μg/mL, and IPTG at 100 μM. Shake the culture overnight at 30℃, 200 rpm. The next day, collect the bacterial liquid, centrifuge at 2400g, 12℃ for 30 minutes. After centrifugation, transfer 36 ml of the supernatant to a new 50 ml centrifuge tube, then add 9 ml of PEG/NaCl, mix thoroughly, and place on ice for 1-2 hours. After 1-2 hours, centrifuge the mixture at 4000 rpm and 4℃ for 30 minutes.

After centrifugation, gently discard the supernatant, resuspend the phage pellet in 3 ml of 1×PBS/5%FBS solution, and store it at 4 ℃ for later use. Then, mix the purified phage library and MOLM14-huMSLN (resuspended in 5%FBS/PBS) evenly, and spin at 10 rpm on a mixer in a refrigerator at 4 ℃. After 1.5 hours, wash with 5%FBS/PBS and remove as much supernatant as possible. Next, use 500 μl of 0.1M, pH2.2 Glycine to elute the phage and let it stand at room temperature for 10 minutes. After centrifugation, transfer the supernatant to a new tube and add 50 μl of pH9.5 Tris-HCL to neutralize the pH.

Add the eluted phage to 1E7/2.5 ml MOLM14 (resuspended in 5%FBS/PBS) , mix well, rotate at 10 rpm, and combine for about 0.5 hours. Centrifuge and repeat the above steps two more times.

After the last centrifugation, transfer the supernatant to a new 15 ml tube and use it to infect TG1. Repeat the panning operation twice more, and the clones finally obtained are tested by whole-cell phage ELISA (the results are shown in FIG. 2) . The positive clones are amplified by PCR to obtain the scFv sequence (the gray background in FIG. 2 is the PCR identification clone) . Finally, the scFv sequence identified by sequencing is cloned into the pUTCK-MSLN CAR vector (FIG. 3A) .
5.10.3 Example 3: Tumor cell culture

Tumor cell lines are cultured in RPMI-1640 or DMEM medium containing 10%fetal bovine serum and 1%Penicillin-Streptomycin antibiotics, and are passaged once every 2-3 days.
5.10.4 Example 4: A549 tumor cell mRNA electroporation

1. Collect A549 tumor cells and wash them three times with Opti-MEM medium.

2. Resuspend the cells in Opti-MEM medium and adjust the cell concentration to 5×10e7/ml.

3. Mix the required volume of mRNA and 100 μl of tumor cells gently, and electroporate immediately.

4. Set parameters on the BTX ECM 830 machine: 360V voltage, 1 millisecond pulse duration.

5. Add the mRNA/cell mixture into the BTX electroporation cup and tap gently to avoid air bubbles.

6. Perform electroporation, then transfer the electroporated cells to 1 ml of preheated culture medium, mix evenly, and then place them in a 37 ℃ incubator for further culturing.

7. The protein expression levels were detected on the second day after electroporation. The results are shown in FIG. 4. Both 1 μg and 10 μg of MSLN mRNA electroporated A549 were expressed to varying degrees.
5.10.5 Example 5: Preparation of lentiviral CAR-T cells

Clone 2 candidate MSLN CAR sequences and 3 MSLN CAR/LACOSTIM sequences into the pUTCK vector respectively (as shown in FIG. 3A) . Then, perform plasmid extraction, lentivirus packaging, ultracentrifugation, and titer determination. Finally, freeze them in a -80℃ refrigerator for later use. Preparation of CAR-T cells: On day 1, activate T cells with anti-CD3/CD28 Dynabeads, and adjust the cell density to 1e6 cells/ml. On day 2, add lentivirus to T cells at an MOI of 3 and culture for another two days. On day 5, remove the anti-CD3/CD28 Dynabeads, perform cell counting, passage and rehydration. On day 9, take 3e5 T cells from each group, stain them with MSLN-Fc/anti-human IgG Fc and CD40-Fc/anti-human IgG Fc antibodies respectively, conduct flow cytometry analysis to detect the CAR positivity rate (as shown in FIG. 5) , and assess the expression of LACOSTIM (as shown in FIG. 6) .

Continue to culture the CAR-T cells until about day 13, harvest them, and use them directly for functional experiments, or freeze them for later use.
5.10.6 Example 6: In vitro cytotoxicity testing of CAR-T cell preparation

1. Twelve hours prior to co-culture for cytotoxicity experiments with CAR-T cells, seed tumor cells into a flat-bottom 96-well plate at a density of 3,000-10,000 cells/100 μl per well.

2. After 8-12 hours, allow the cells to fully adhere to the well surface. Dilute various anti-MSLN CAR-T cells to appropriate cell densities and co-incubate them with tumor cells at different effector-to-target ratios (such as E: T=3: 1, 1: 1, and 0.3: 1) .

3. Place the 96-well plate into the InCucyte S3 machine and set the scanning parameters.

4. After 3 days of scanning, analyze the total green fluorescence cumulative intensity (GCU xμm²/well) to calculate tumor cell killing efficiency. The results are shown in FIGs 7-10. Compared with UTD cells, MH150 CAR-T cells, and 2 types of MSLN CAR/LACOSTIM CAR-T cells, such as A4025-MH150 and MH150-25-9.3 CAR-T cells, have minimal killing effect on A549 cells with low MSLN expression levels. However, they exhibit high killing efficiency against A549 cells and ASPC1 cells with high MSLN expression levels. ELISA analysis of the supernatant of tumor and CAR-T cells cocultured for 24 hours also confirms these experimental findings (see FIG. 11 and FIG. 12) .

Furthermore, in subsequent studies, a variety of different tumor cells that do not express MSLN, such as HCC70, SH-SY5Y, Huh-7, U87, U251, A375, 293T, were screened as target cells (see FIGs 13-22) . Various candidate sequences demonstrated good specificity.
5.10.7 Example 7: Detection of tumor suppression in tumor-bearing animal models in vivo

Conducting animal model experiments using MSLN CAR/LACOSTIM CAR-T cells (e.g. MH150-25-9.3 CAR-T cells) . OVCAR3 cells in the expansion phase were subcutaneously injected into immunodeficient M-NSG mice at a dose of 10×10⁶ cells per mouse to establish an OVCAR3 subcutaneous tumor-bearing model. When tumor volumes reached 70-100 mm³, the tumor-bearing mice were divided into three groups according to Table 7 and administered CAR-T cells at doses of 1×10⁶, 2×10⁶, and 3×10⁶ CAR-positive cells, respectively. The grouping and dosing regimens are detailed in Table 7.

Table 7 Grouping Scheme for Animal Model Experiments

The mice were continuously maintained under standardized conditions, with tumor volume, body weight, and survival status recorded throughout the study. Results are shown in FIG. 23. The experimental data from the mouse model demonstrated that following a single injection of CAR-T cells, both the medium-dose and high-dose groups exhibited significant tumor-killing efficacy compared to the low-dose group. In the high-dose group, complete tumor regression was observed in some mice by day 21 post-injection, and all mice achieved complete tumor regression by day 31. In contrast, tumor growth was markedly suppressed in individual mice within the low-dose group, while tumors in other mice in the same group continued to progress.

Detection of CAR-T Cells Change in Tumor-Bearing Animal Models. The proportion of CAR+ T cells in the peripheral blood of tumor-bearing mice was detected by flow cytometry after administration, and the expansion of CAR-T cells in the mice was analyzed. The results are shown in FIG. 24. The results indicate that after a single injection of CAR-T cells, the proportion of CAR-T cells in the peripheral blood of each dose group gradually increased, reaching a peak at day 21-28. This demonstrates that CAR-T cells undergo significant expansion during the process of tumor killing.

Detection of sLACO Secretion Levels in Tumor-Bearing Animal Models. The secretion levels of sLACO in the peripheral blood of tumor-bearing mice were detected using the enzyme-linked immunosorbent assay (ELISA) method after administration. The results are shown in FIG. 25. The results indicate that after a single injection of CAR-T cells, the secretion levels of sLACO in the peripheral blood of each dose group gradually increased with the expansion of CAR-T cells, and the secretion levels were largely consistent with the expansion of CAR-T cells.
5.10.8 Example 8: Comparison of In Vivo Experimental Results in Tumor-Bearing Animal
Models Using Cells from Different Manufacturing Processes

Animal model experiments were conducted using MSLN CAR/LACOSTIM CAR-T cells (e.g. MH150-25-9.3 CAR-T cells) . Meanwhile, T cells from the same donor (NTD) were used as control T cells. OVCAR3 cells in the expansion phase were subcutaneously injected into immunodeficient M-NSG mice at a dose of 10×10⁶ cells per mouse to establish an OVCAR3 subcutaneous tumor model. When the tumor size reached 70-100 mm3, the tumor-bearing mice were divided into three groups according to Table 8 and injected with NTD cells, CAR-T cells 1 (2×10⁶ CAR-positive cells) , and CAR-T cells 2 (2×10⁶ CAR-positive cells) , respectively. CAR-T cells 1 and CAR-T cells 2 were CAR-T cells produced from two different manufacturing processes using the same donor. The grouping and dosing regimen are shown in Table 8.

Table 8 Grouping Scheme for Animal Model Experiments

*Process 1: Use human AB serum as a supplement in the expansion medium (e.g. Gemini, 100-912) .
*Process 2: Use human platelet lysate as a supplement in the expansion medium (e.g. Compass
Biomedical, PLSA, 081023) .

The mice were continuously monitored, and tumor volume, body weight, and survival status were recorded. The results are shown in FIG. 26. The experimental results from the mouse model indicate that after a single injection of CAR-T cells, both CAR-T cells 1 and CAR-T cells 2 demonstrated significant tumor-killing efficacy, with complete tumor regression observed in some mice starting at 21 days post-injection. In contrast, tumors in mice injected with control T cells continued to grow.

Detection of CAR-T Cells Change in Tumor-Bearing Animal Models. The number of CAR+T cells in the peripheral blood of tumor-bearing mice from this example was detected by flow cytometry after administration, and the expansion of CAR-T cells in the mice was analyzed. The results are shown in FIG. 27. The results indicate that after a single injection of CAR-T cells, the number of CAR-T cells in the peripheral blood of different process groups gradually increased, reaching a peak at day 22-28. No expansion was observed in the T cell control group. This demonstrates that CAR-T cells undergo significant expansion during the process of tumor killing.

Detection of sLACO Secretion Levels in Tumor-Bearing Animal Models. The secretion levels of sLACO in the peripheral blood of tumor-bearing mice from this example were detected using the enzyme-linked immunosorbent assay (ELISA) method after administration. The results are shown in FIG. 28. The results indicate that after a single injection of CAR-T cells, the secretion levels of sLACO in the peripheral blood of different process groups gradually increased, peaking at day 14-22. The secretion peak occurred approximately 7 days earlier than the peak of CAR-T cell expansion.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

An antibody or antigen-binding fragment thereof that specifically binds mesothelin, comprising:
(a) a light chain variable region (VL) comprising a light chain CDR1 (LCDR1) , light chain CDR2 (LCDR2) , a light chain CDR3 (LCDR3) having an amino acid sequence of SEQ ID NOs: 1, 2, and 3 respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the LCDRs; and/or
(b) a heavy chain variable region (VH) comprising a heavy chain CDR1 (HCDR1) , a heavy chain CDR2 (HCDR2) , a heavy chain CDR3 (HCDR3) having an amino acid sequence of SEQ ID NOs: 4, 5, and 6 respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the HCDRs.
The antibody or antigen-binding fragment thereof of claim 1, comprising a LCDR1, a LCDR2, a LCDR3, a HCDR1, a HCDR2 and a HCDR3, wherein
(a) the LCDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 2, and 3 respectively; and
(b) the HCDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 4, 5, and 6 respectively.
The antibody or antigen-binding fragment thereof of any one of claims 1-2, comprising:
(a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 7; and/or
(b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence of SEQ ID NO: 8.
The antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising a VL and a VH, wherein the VL and the VH have the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.
The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment.
The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.
The antibody or antigen-binding fragment thereof of any one of claims 1-6 that is selected from the group consisting of a Fab, a Fab’, a F (ab’) ₂, a Fv, a scFv, a (scFv) ₂, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .
The antibody or antigen-binding fragment thereof of any one of claims 1-7 that is a scFv having the amino acid sequence of SEQ ID NO: 9.
A Chimeric Antigen Receptor (CAR) that specifically binds mesothelin, comprising, from N-terminus to C-terminus:
(a) a mesothelin-binding domain that comprises the antibody or antigen-binding fragment thereof of any one of claims 1-8;
(b) a transmembrane domain; and
(c) a cytoplasmic domain.
The CAR of claim 9, wherein the transmembrane domain is derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.
The CAR of any one of claims 9-10, wherein the transmembrane domain comprises CD8 transmembrane region.
The CAR of any one of claims 9-11, wherein the cytoplasmic domain comprises a signaling domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.
The CAR of any one of claims 9-12, wherein the cytoplasmic domain further comprises a co-stimulatory domain derived from CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.
The CAR of any one of claims 9-13, wherein the cytoplasmic domain comprises a CD3ζsignaling domain and a 4-1BB co-stimulatory domain.
The CAR of any one of claims 9-14, further comprising a CD8 hinge domain between the mesothelin-binding domain and the transmembrane domain.
The CAR of any one of claims 9-15, further comprising a signal peptide domain derived from CD8 at N-terminus.
The CAR of any one of claims 9-16, comprising an amino acid sequence of SEQ ID NO: 10.
A polynucleotide that encodes the antibody or antigen-binding fragment of any one of claims 1-8 or the CAR of any one of claims 9-17.
The polynucleotide of claim 18, wherein the polynucleotide is a DNA or mRNA.
A vector that comprises the polynucleotide of claim 19.
A cell comprising the antibody or antigen-binding fragment of any one of claims 1-8, the CAR of any one of claims 9-17, the polynucleotide encoding the CAR of any one of claims 18-19, or the vector of claim 20.
The cell of claim 21, wherein the cell is an immune effector cell.
The cell of any one of claims 21-22, wherein the cell is derived from a cell isolated from peripheral blood or bone marrow.
The cell of any one of claims 21-23, wherein the cell is derived from a cell differentiated in vitro from a stem or progenitor cell is selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.
The cell of any one of claims 21-24, wherein the cell is a T cell or a NK cell.
The cell of any one of claims 21-25, wherein the cell is a cytotoxic T cell, a helper T cell, a gamma delta T, a CD4⁺/CD8⁺ double positive T cell, a CD4⁺ T cell, a CD8⁺ T cell, a CD4^-/CD8^-double negative T cell, a CD3⁺ T cell, a naive T cell, an effector T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, or an effector memory TEMRA cell.
The cell of any one of claims 21-26, wherein the cell recombinantly expresses a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein
(i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and
(ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.
The cell of claim 27, wherein the N-terminus of the first domain is linked to the C-terminus of the second domain.
The cell of claim 27, wherein the N-terminus of the second domain is linked to the C-terminus of the first domain.
The cell of any one of claims 27-29, wherein the first domain and the second domain are linked via a linker.
The cell of any one of claims 27-30, wherein the APC is selected from the group consisting of a dendritic cell, a macrophage, a myeloid derived suppressor cell, a monocyte, a B cell, a T cell, and a Langerhans cell.
The cell of any one of claims 27-31, wherein the activation receptor of the APC is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN.
The cell of claim 32, wherein the first domain comprises a ligand that binds CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN, or a receptor-binding fragment thereof.
The cell of claim 33, wherein the first domain comprises a receptor-binding fragment of CD40 Ligand (CD40L) .
The cell of any one of claims 27-31, wherein the first domain comprises an antibody that binds the activation receptor of the APC, or an antigen-binding fragment thereof.
The cell of claim 35, wherein the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof.
The cell of any one of claims 27-36, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.
The cell of any one of claims 27-37 wherein the second domain comprises a cytoplasmic domain of the co-stimulatory receptor.
The cell of claim 38, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.
The cell of claim 38, wherein the co-stimulatory receptor is CD28.
The cell of any one of claims 27-37, wherein the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof.
The cell of claim 41, wherein the co-stimulatory ligand is selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44.
The cell of any one of claims 27-37, wherein the second domain is an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof.
The cell of claim 43, wherein the second domain is an scFv.
The cell of claim 43 or 44, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.
The cell of claim 43 or 44, wherein the co-stimulatory receptor is CD28.
The cell of claim 27, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof.
The cell of claim 27, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises a CD28 cytoplasmic domain.
The cell of any one of claim 47 to 48, wherein the fusion protein is at least 85%, 90%, 95%, 98%, or 99%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-59 and 63-73.
The cell of any one of claims 27-49 that is an immune effector cell.
The cell of claim 50 that is derived from a cell isolated from peripheral blood or bone marrow.
The cell of claim 50 that is derived from a cell differentiated in vitro from a stem or progenitor cell selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.
The cell of any one of claims 27 to 52 that is a T cell or a NK cell.
The cell of claim 53 that is a cytotoxic T cell, a helper T cell, a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, or an effector memory TEMRA cell.
The cell of claim 53 that is a cytotoxic T cell.
A population of the cells comprising the cell of any one of claims 27-49, wherein the population of cells are derived from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) .
A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-8, the CAR of any one of claims 9-17, the polynucleotide of any one of claims 18-19, the vector of claim 20 and/or the cell or cell population of any one of claims 21-56, and a pharmaceutically acceptable carrier or excipient.
Use of the antibody or antigen-binding fragment of any one of claims 1-8, the CAR of any one of claims 9-17, the polynucleotide of any one of claims 18-19, the vector of claim 20, the cell or cell population of any one of claims 21-56 and/or the pharmaceutical composition of claim 57 for the preparation of a medicament for the treatment of cancer.
Use of the antibody or antigen-binding fragment of any one of claims 1-8, the CAR of any one of claims 9-17, the polynucleotide of any one of claims 18-19, the vector of claim 20, the cell or cell population of any one of claims 21-56 and/or the pharmaceutical composition of claim 57 in cancer treatment.
The use of claim 58 or 59, wherein the cell, population of cells, or pharmaceutical composition is used in combination with an additional therapy.
A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-8, the CAR of any one of claims 9-17, the polynucleotide of any one of claims 18-19, the vector of claim 20, the cell of or cell population of any one of claims 21-56 and/or the pharmaceutical composition of claim 57.
The method of claim 61, wherein the cell or population of cells is autologous to the subject.
The method of claim 61, further comprising obtaining cells from the subject.
The method of any one of claims 61-63, further comprising administering an additional therapy to the subject.
The method of any one of claims 61-64, wherein the subject is a human.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer is a mesothelin-expressing cancer.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer is a solid tumor or a hematological cancer.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer is renal cancer, lung cancer, urothelial cancer, breast cancer, brain tumor, thyroid cancer, leukemia, lymphoma, and/or multiple myeloma.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer comprises renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, glioblastoma, anaplastic thyroid cancer, B-cell non-Hodgkin’s lymphoma, and/or chronic lymphoblastic leukemia.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer is mesothelioma.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer is pleural mesothelioma, peritoneal mesothelioma, or pericardial mesothelioma.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer is pancreatic cancer.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer is pancreatic ductal carcinoma.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer is ovarian cancer.
The use of any one of claims 58-60 or the method of any one of claims 61-65, wherein the cancer is ovarian epithelial carcinoma.
A method of preparing the cell of any one of claims 21-55, comprising transferring a polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1-8, a polynucleotide encoding the CAR of any one of claims 9-17, the polynucleotide of any one of claims 18-19, and/or the vector of claim 20 into a cell.
The method of claim 76, wherein the method is for preparing a cell capable of expressing a CAR that specifically binds mesothelin, and the method comprises transferring the polynucleotide of claim 18 or 19 into the cell.
The method of any one of claims 76-77, wherein the method is for preparing a cell capable of expressing a CAR that specifically binds mesothelin and expressing a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, and the method comprises transferring the polynucleotide of claim 18 or 19 into the cell and the polynucleotide encoding the fusion protein, wherein:
(i) the first domain of the fusion protein comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and
(ii) the second domain of the fusion protein comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.
The method of any one of claims 76-78, wherein the polynucleotide is transferred via electroporation.
The method of any one of claims 76-78, wherein the polynucleotide is transferred via viral transduction.
The method of claim 80, comprising using a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus for the viral transduction.
The method of any one of claims 76-78, wherein the polynucleotide is transferred using a transposon system.
The method of claim 82, wherein the transposon system is Sleeping Beauty or PiggyBac.
The method of any one of claims 76-78, wherein the polynucleotide is transferred using gene-editing.
The method of claim 84, wherein the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.
The method of any one of claims 76-85, wherein the cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell.