WO2018081531A2 - Methods for human t-cell activation - Google Patents

Abstract

The present invention provides, among other things, methods of increasing T-cell activation in a subject, comprising administering to the subject a composition that inhibits hematopoietic progenitor kinase 1 (HPKl). The composition may comprise, for example, a small molecule HPKl inhibitor. The method may further comprise administering to the subject an anti-CTLA-4 antibody and/or an anti-PD-1 antibody.

Description

METHODS FOR HUMAN T-CELL ACTIVATION

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent

Application serial number 62/414,345 filed on October 28, 2016, the entirety of which is hereby incorporated by reference.

SEQUENCE LISTING

[0002] The present specification makes reference to a Sequence Listing, submitted electronically as a .txt file named "ARD-483WO SL" on October 27, 2017. The .txt file was generated October 25, 2017 and is 943 bytes in size. The entire contents of the Sequence Listing are herein incorporated by reference.

BACKGROUND

[0003] Patients who respond to checkpoint inhibitory antibodies display durable, treatment-free remissions in various cancers. Only a fraction of patients who receive checkpoint inhibitory antibodies respond, however. Methods of increasing the efficacy of checkpoint inhibitory antibodies are desirable.

SUMMARY OF THE INVENTION

[0004] Checkpoint inhibitory antibodies function by blocking inhibitory checkpoints such as signaling through inhibitory checkpoint receptors. This strategy aims to decrease the checkpoint-mediated inhibition of immune cells, especially T-cells. Complementary methods of activating T-cells may be useful to treat patients who do not respond to checkpoint inhibitory antibodies.

[0005] The human programmed death receptor- 1 (PD-1) blocking antibody nivolumab (OPDIVO®), for example, binds to human PD-1, which is an inhibitory checkpoint receptor present on T-cells. Nivolumab sterically blocks the binding of PD-1 ligands PD-L1 and PD-L2 to PD-1, thereby suppressing PD-1 -mediated signaling and reducing T-cell inhibition. This reduction of T-cell inhibition enables T-cells to mount an effective immune response against cancer cells in a subset of patients.

[0006] Nivolumab 's FDA approved indications include specific patient populations with melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, and squamous cell carcinoma of the head and neck. The percentage of patients who respond to nivolumab remains low, however, and methods of increasing the efficacy of nivolumab and similar pharmaceuticals are desirable. In general, a pharmaceutical agent that increases T- cell activation could be useful to treat a variety of different patient populations.

[0007] In some aspects, the present invention provides methods of increasing T-cell activation in a human patient, comprising administering to the patient a composition that inhibits hematopoietic progenitor kinase 1 (HPKl). The method may further comprise administering to the patient an anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibody. The anti-CTLA-4 antibody may be selected from ipilimumab and tremelimumab. The method may further comprise administering to the patient an anti-PD-1 (programmed cell death protein 1) antibody. The anti-PD-1 antibody may be selected from nivolumab and pembrolizumab.

[0008] The method may further comprise administering to the patient an anti-TIM-3, anti-LAG3, anti-OX40, anti-4-lBB, anti-GITR, anti-PD-Ll, anti-PD-L2, anti-B7.1, anti- B7.2, anti-VTCNl, anti-ICOS ligand, anti-MHC class II, anti HVEM, anti-CD155, anti galectin-9, or anti-TIM-3 ligand antibody. The method may further comprise administering to the patient atezolizumab, avelumab, durvalumab, GSK3174998, MEDI6383,

prezalizumab, urelumab, or utomilumab.

[0009] In some aspects, the present invention provides compositions that inhibit

HPKl and that may comprise a small molecule HPKl inhibitor. A small molecule HPKl inhibitor may bind HPKl with a dissociation constant (K_d) of less than about 10 nM (e.g., less than about 5 nM, 4 nM 3 nM, 2 nM, or 1 nM). A small molecule HPKl inhibitor may selectively inhibit HPKl relative to 1, 2, 3, or all of lymphocyte-specific protein tyrosine kinase (LCK), zeta-chain-associated protein kinase 70 (ZAP70), protein kinase C theta (PKC- Θ), and Janus kinase 3 (JAK3). A small molecule HPKl inhibitor may bind HPKl with a K_d that is less than the K_d between the small molecule HPKl inhibitor and LCK, less than the K_d between the small molecule HPKl inhibitor and ZAP70, less than the K& between the small molecule HPKl inhibitor and PKC-Θ, and/or less than the K& between the small molecule HPKl inhibitor and JAK3. A small molecule HPKl inhibitor may have an IC₅₀ with HPKl that is less than the IC₅₀ between the small molecule HPKl inhibitor and LCK, less than the IC₅₀ between the small molecule HPKl inhibitor and ZAP70, less than the IC₅₀ between the small molecule HPKl inhibitor and PKC-Θ, and/or less than the IC₅₀ between the small molecule HPKl inhibitor and JAK3.

[0010] In some embodiments, the composition that inhibits HPKl comprises microRNA (miRNA) or small interfering RNA (siRNA) or a nucleic acid encoding miRNA or siRNA. The miRNA or siRNA may reduce the translation of mRNA (or pre-mRNA) encoding HPKl .

[0011] In some aspects, the present invention provides methods of treating a disease or condition in a human patient, comprising administering to the patient a small molecule inhibitor of HPKl . The small molecule inhibitor may bind HPKl with a K^ of less than about 10 nM (e.g., less than about 5 nM, 4 nM 3 nM, 2 nM, or 1 nM). The disease or condition may be cancer, an infection associated with a pathogen, or a disease or condition associated with extracellular protein aggregates. In some embodiments, the patient does not have a T- cell dysfunctional disorder. A method may further comprise comprising administering to the patient an anti-CTLA-4 antibody and/or an anti-PD-1 antibody.

[0012] In some embodiments, the patient does not have a colorectal tumor. A patient may not have a solid tumor. A patient may have previously had a malignant tumor, and the malignant tumor may have been excised or irradiated such that the patient no longer has a malignant tumor.

[0013] In some embodiments, the patient has cancer. For example, the patient may have bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, Merkel-cell carcinoma, mesothelioma, melanoma, non- small cell lung cancer, ovarian cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, transitional cell carcinoma, or urothelial cancer. In some embodiments, the cancer is associated with a virus.

[0014] In some embodiments, the patient does not have cancer.

[0015] A patient may be in remission from cancer.

[0016] In some embodiments, the patient has an infection associated with a pathogen.

An infection may be a viral infection. The infection may be an HIV infection. [0017] In some embodiments, the patient has a disease or condition associated with extracellular protein aggregates. A patient may have Alzheimer's disease.

[0018] Various embodiments relate to a method of treating an infection in a human patient, comprising administering to the patient a composition that inhibits HPKl . The method may further comprise administering an anti-CTLA-4 antibody and/or an anti-PD-1 to the patient.

[0019] Various embodiments relate to a method of treating a disease or condition associated with extracellular protein aggregates in a human patient. The method may further comprise administering an anti-CTLA-4 antibody and/or an anti-PD-1 to the patient.

[0020] Various embodiments relate to a composition that inhibits HPKl for use in a method of increasing T-cell activation in a human patient. The method may further comprise administering an anti-CTLA-4 antibody and/or an anti-PD-1 to the patient.

[0021] Various embodiments relate to a composition that inhibits HPKl for use treating cancer. The treatment may further comprise administering an anti-CTLA-4 antibody and/or an anti-PD-1 to the patient.

[0022] Various embodiments relate to a composition that inhibits HPKl for use treating an infection. The treatment may further comprise administering an anti-CTLA-4 antibody and/or an anti-PD-1 to the patient.

[0023] Various embodiments relate to a composition that inhibits HPKl for use treating a disease or condition associated with extracellular protein aggregates. The treatment may further comprise administering an anti-CTLA-4 antibody and/or an anti-PD-1 to the patient.

[0024] Various embodiments relate to a kit comprising a composition that inhibits

HPKl and a package insert comprising instructions for use. A kit may further comprise an anti-PD-1 antibody and/or an anti-CTLA-4 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings are for illustration purposes only, not for limitation.

[0026] Figure 1A depicts an inducible T-cell system. The drawing depicts a T-cell that inducibly expresses inhibitory checkpoint receptors PD-1 and CTLA-4. The T-cell also expresses the costimulatory receptor CD28 and the T-cell receptor (TCR). The drawing depicts a B-cell that expresses (a) either PD-L1, PD-L2, or both and (b) either B7.1, B7.2, or both. The drawing also depicts beads coated with anti-CD3 antibodies.

[0027] Figure IB illustrates two states of an inducible T-cell system. In a first state

(IB, left), the transcription inducer doxycycline (dox) is not present, and the T-cell does not express inducible PD-1 or CTLA-4 constructs. The T-cell may receive costimulatory signals from B7.1 or B7.2 (which interact with CD28) and anti-CD3 antibodies (which interact with the T-cell receptor). The costimulatory signals result in interleukin-2 (IL2) production by the T-cell in addition to other cytokines. In a second state (IB, right), the transcription inducer doxycycline (dox) is added, and the T-cell may express inducible PD-1 and CTLA-4 constructs. The T-cell may receive costimulatory signals from B7.1 or B7.2 and the anti- CD3 antibodies. The T-cell may also receive inhibitory signals from PD-L1 or PD-L2 (which interact with PD-1) and B7.1 or B7.2 (which interact with CTLA-4). The inhibitory signals result in decreased IL2 production by the T-cell relative to the first state.

[0028] Figure 1C illustrates a T-cell system that includes a knockout of the hpkl gene that encodes hematopoietic progenitor kinase 1 (HPKl).

[0029] Figure 2A shows exemplary data that demonstrate that hematopoietic progenitor kinase 1 (HPKl) expression is decreased and the percentage of interleukin-2 (IL2) positive cells is increased when HPKl is knocked down via shRNA.

[0030] Figure 2B shows exemplary data that demonstrate that hematopoietic progenitor kinase 1 (HPKl) expression is decreased and the percentage of interleukin-2 (IL2) positive cells is increased when HPKl is knocked down via CRISPR.

[0031] Figure 3A contains bar graphs that show the interleukin-2 (IL2) release of T- cells of inducible T-cell systems with or without an hpkl gene knockout. The hpkl knockout resulted in higher IL2 release for both systems comprising un-induced T-cells, which do not express transgenic PD-1 and CTLA-4, and systems comprising induced T-cells, which express the transgenic inhibitory checkpoint receptors PD-1 and CTLA-4.

[0032] Figure 3B contains bar graphs that show the interleukin-2 (IL2) release of T- cells of un-induced T-cell systems with or without an hpkl gene knockout in which the T- cells of the system were contacted with ipilimumab (Ipi) and nivolumab (Nivo) or a control anti-IgG antibody. The hpkl knockout resulted in >10 fold increased activation relative to the combination of ipilimumab and nivolumab. [0033] Figure 4 illustrates an inducible T-cell system in which the T-cells are contacted with small molecule compounds that target HPK1.

[0034] Figure 5 is a graph that depicts the effects of four small molecule compounds that target HPK1 relative to DMSO controls in inducible T-cell systems comprising doxycycline-induced T-cells (dox+) and un-induced T-cells (dox-). Each compound increased interleukin-2 (IL2) release by T-cells relative to DMSO controls in both induced T- cells expressing PD-1 and CTLA-4 transgenes and un-induced T-cells that do not express the transgenes.

[0035] Figure 6 illustrates five experiments that were used to assess the effect of anti-

PD-1 antibody nivolumab (Nivo) and/or anti-CTLA-4 antibody ipilimumab (Ipi) on T-cell activation. Experiment 1 depicts an inducible T-cell system in which T-cells are not induced to express the inhibitory checkpoint receptor transgenes for PD-1 and CTLA-4. The T-cells of the system nevertheless endogenously express CD28 and the T-cell receptor (TCR). The B-cells of the system constitutively express a PD-L1 or PD-L2 transgene and endogenously express B7.1 and/or B7.2. The T-cells of the system of experiment 1 may be incubated with a control antibody, such as an anti-IgG antibody. Experiment 3 depicts an inducible T-cell system in which T-cells are induced to express PD-1 and CTLA-4 transgenes. The T-cells of the system may be incubated with a control antibody, such as an anti-IgG antibody.

Experiment 4 depicts an inducible T-cell system in which (a) T-cells are induced to express PD-1 and CTLA-4 transgenes and (b) the T-cells are contacted with nivolumab. Experiment 5 depicts an inducible T-cell system in which (a) T-cells are induced to express PD-1 and CTLA-4 transgenes and (b) the T-cells are contacted with ipilimumab. Experiment 6 depicts an inducible T-cell system in which (a) T-cells are induced to express PD-1 and CTLA-4 transgenes and (b) the T-cells are contacted with the both nivolumab and ipilimumab.

[0036] Figure 7 is a graph that depicts relative interleukin-2 (IL2) release by T-cells of different inducible T-cell systems after incubating the T-cells of a system with nivolumab (nivo), ipilimumab (ipi), both nivolumab and ipilimumab (nivo+ipi), or a control antibody (IgG). T-cells treated with nivolumab or ipilimumab displayed higher IL2 release than the IgG control, and the combination of nivolumab and ipilimumab displayed an additive effect.

[0037] Figure 8 illustrates an inducible T-cell system in which the T-cells of the system are induced to express inhibitory checkpoint receptor PD-1 and CTLA-4 transgenes. The T-cells are also contacted with nivolumab, ipilimumab, and a small molecule compound that binds HPK1.

[0038] Figures 9A-9B are graphs that depict relative interleukin-2 (TL2) release by T- cells of different inducible T-cell systems after incubating the T-cells of a system with nivolumab (nivo), ipilimumab (ipi), both nivolumab and ipilimumab (nivo+ipi), or an IgG control antibody. The experiments were repeated after contacting the T-cells of the system with 1250 nM of the small molecule compound "compound 1" or 5000 nM of the small molecule compound "compound 2," each of which inhibit HPK1. Both compound 1 and compound 2 displayed an additive effect on T-cell activation in combination with nivolumab, ipilimumab, or both nivolumab and ipilimumab. The combination of compound 1, nivolumab, and ipilimumab appeared to display a synergistic effect. The combination of compound 2 with either nivolumab, ipilimumab, or both nivolumab and ipilimumab appeared to display a synergistic effect.

[0039] Figure 10 is a graph that depicts relative interferon γ (INFy) positive cell counts in GFP⁺ and GFP^" cell populations. Human primary T-cells were transfected with shRNA designed to knockdown HPK1 expression (shKinase#l) along with a green fluorescent protein (GFP) marker protein. Transfected cells (GFP+) were >3 times more likely to be INFy positive relative to untransfected cells (GFP-) or cells transfected with control shRNA (shControl).

[0040] Figure 11 contains a graph that depicts the relative release of interferon γ

(INFy) from activated human primary T-cells incubated with four different compounds (Cmpdl, Cmpd2, Cmpd3, Cmpd4). Figure 11 also shows the half maximal inhibitory concentration (IC₅₀ in nM) of the four compounds and FIPK1 (Kinase#l) as well as three other kinases (counter Target#l, counter Target#2, and counter Target#3). Counter Targets #1, #2 and #3 were lymphocyte-specific protein tyrosine kinase (Lck), zeta-chain-associated protein kinase 70 (ZAP70) and Janus kinase 3 (Jak3), respectively. Compound 2 (Cmpd2) and compound 3 (Cmpd3) had the greatest inhibition of FD P1 (i.e., lowest IC₅₀'s) and resulted in the highest levels of INFy release.

[0041] Figure 12 contains a graph that depicts the relative release of interferon γ

(INFy) from human peripheral blood mononuclear cells (PBMCs). PBMCs were activated with cytomegalovirus (CMV)-peptide and incubated with either 1 μg/mL nivolumab or 156 nM, 312 nM, 625 nM, or 1250 nM of small molecule HPK1 inhibitor compound 5 (Cmpd 5). [0042] Figure 13 contains a graph that depicts the relative release of interleukin 2

(TL2) from T-cells. The T-cells were contacted with an HPK1 inhibitor ("AP compound") or DMSO vehicle and either 10 μg/mL ipilimumab and 10 μg/mL nivolumab (Ipi + Nivo) or 10 μg/mL of an anti-IgG control antibody (IgG).

[0043] Figure 14 contains an exemplary bar graph demonstrating that HPK1 inhibits

T-cell signaling in Jurkat cells via an inducible HPK1 rescue construct.

DEFINITIONS

[0044] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. As used in the specification and claims, the singular form "a," "an," and "the" includes plural references unless the context clearly dictates otherwise.

[0045] As used herein, "agent" or "pharmaceutical agent" refers to a biological, pharmaceutical, or chemical compound or other moiety. Non-limiting examples include simple or complex organic or inorganic molecules, a peptide, a protein, a nucleic acid, an antibody, an antibody derivative, an antibody fragment, a cytokine, a vitamin, a vitamin derivative, a carbohydrate, a toxin, or a chemotherapeutic compound, and metabolites thereof. Various compounds can be synthesized including, for example, small molecules and oligomers (e.g., polypeptides and nucleic acid), and synthetic organic compounds based on various core structures. In addition, various natural sources may provide active compounds, such as plant or animal extracts, and the like. A skilled artisan will readily recognize that there is no limit to the structural nature of the agents of this disclosure.

[0046] The terms "antagonist" and "inhibitor" are used interchangeably, and they refer to a compound or agent having the ability to inhibit a biological function of a target protein or polypeptide, such as by inhibiting the activity or expression of the target protein (e.g., HPK1) or polypeptide. Accordingly, the terms "antagonist" and "inhibitor" are defined in the context of the biological role of the target protein or polypeptide. An antagonist or inhibitor may be a small molecule HPK1 inhibitor. While some antagonists herein specifically interact with (e.g., bind to) the target (e.g., HPK1), compounds that inhibit a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of that target protein or polypeptide are also specifically included within this definition. Non-limiting examples of biological activity inhibited by an antagonist include those associated with the development, growth, or spread of a tumor, or the progression of an infection or disease or condition associated with extracellular protein aggregates.

[0047] "Administration" encompasses the delivery to a subject of a composition or compound as described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, using any suitable formulation or route of administration described herein.

[0048] The term "co-administration," "administered in combination with," and their grammatical equivalents, as used herein, encompasses administration of two or more agents to a subject so that both agents and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0049] The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound or pharmaceutical composition described herein that is sufficient to affect the intended application including, but not limited to, the treatment of a disease or condition. In some embodiments, the amount is effective for detectable killing or inhibition of the growth or spread of cancer cells; the size or number of tumors; or other measure of the level, stage, progression or severity of the cancer. The amount may be effective for slowing the progression or reversing the course of an infection or a disease or condition associated with extracellular protein aggregates. A therapeutically effective amount may vary depending upon the intended indication, subject, and/or disease or condition being treated, e.g., the weight and age of the subject, the severity of the disease or condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in a target cell, e.g., increased activation of T-cells. The specific dose will vary depending on, for example, the particular compounds chosen, the species of subject, the age of the subject, existing health conditions or risk for health conditions, the dosing regimen to be followed, the severity of the disease or condition, whether a composition is administered in combination with other agents, the timing of administration, the tissue to which a composition is administered, and the physical delivery system in which it is carried. [0050] The term "protein aggregate" refers to the accumulation of misfolded proteins.

A protein aggregate is typically present as a precipitate in vivo {e.g., the aggregate is insoluble or sparingly soluble in the extracellular fluid). A protein aggregate may be characterized, for example, by β-sheet tertiary and/or quaternary structure, although the term protein aggregate is not limited to aggregates comprising β-sheets. For example, a protein aggregate may be an amorphous aggregate or an oligomeric aggregate. Examples of proteins that may form misfolded protein aggregates include a-synuclein, apolipoprotein AI, atrial natriuretic factor, β amyloid, β-2 microglobulin, calcitonin, cystatin, gelsolin, huntingtin protein, amylin, immunoglobulin light chain AL, keratoepithelin, lysozyme, medin, prolactin, PrP^Sc, serum amyloid A, tau, and transthyretin. The skilled artisan will recognize that some of these proteins primarily form extracellular protein aggregates {e.g., β amyloid) or and others will primarily form intracellular protein aggregates {e.g., huntingtin protein). In some embodiments, a protein aggregate is amyloid.

[0051] "Signal transduction" is a process during which stimulatory or inhibitory signals are transmitted into and within a cell to elicit an intracellular response. A

"modulator" of a signal transduction pathway refers to a compound which modulates the activity of one or more cellular proteins mapped to the same specific signal transduction pathway. A modulator can augment (agonist) or suppress (antagonist) the activity of a signaling molecule.

[0052] The terms "selective inhibition," "selectively inhibit," and "selective inhibitor" refer to the ability of a small molecule HPKl inhibitor to selectively reduce target signaling activity relative to off-target signaling activity, via direct or interact interaction with the target. For example, a small molecule FIPK1 inhibitor that selectively inhibits FIPK1 may have an activity of at least about 2x relative to another kinase {e.g., at least about 3x, about 5x, about lOx, about 20x, about 50x, or about lOOx). A small molecule FIPK1 inhibitor may be a selective inhibitor relative to a specific {i.e., single) serine/threonine kinase, or a small molecule FIPK1 inhibitor may be a selective inhibitor relative to a genus of kinases {e.g., all human serine/threonine kinases).

[0053] A "subject" to which administration is contemplated includes, but is not limited to, humans {i.e., a male or female of any age group, e.g., a pediatric subject {e.g., infant, child, adolescent) or adult subject {e.g., young adult, middle-aged adult, or senior adult)) and/or other primates {e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and/or turkeys. The terms "subject" and "patient" are used synonymously herein.

[0054] As used herein, the term "treating" refers to an approach for obtaining a beneficial or desired result including, but not limited to, therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit may refer to the eradication or amelioration of an underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with an underlying disorder such that an improvement is observed in a subject, notwithstanding that the subject may still be afflicted with the underlying disorder. For prophylactic benefit, a pharmaceutical compound or compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0055] A "therapeutic effect," as the term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described above. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

DETAILED DESCRIPTION

[0056] The present invention provides, among other things, methods of increasing T- cell activation, comprising administering to a subject a composition that inhibits

hematopoietic progenitor kinase 1 (HPKl). HPKl is a protein expressed by T-cells, which play a central role in cell-mediated immunity. T-cells may be activated or inhibited in various disease states, and increased T-cell activation may be beneficial in treating various diseases and conditions including cancer, infection, and conditions associated with extracellular protein aggregates.

[0057] A knockout of the hpkl gene that encodes HPKl in human T-cells was found to increase T-cell activation. Accordingly, compositions that knockout or knockdown HPKl in T-cells may be useful to increase T-cell activation in vivo. Similarly, compositions that inhibit HPKl activity in T-cells may be useful to increase T-cell activation in vivo. [0058] Compositions that knockout, knockdown, or inhibit HPKl may be particularly useful when used in combination with other pharmaceutical agents that activate T-cells. For example, a composition that knocks-out, knocks-down, or inhibits FIPK1 may be combined with an anti-CTLA-4 antibody and/or an anti-PD-1 antibody to further-increase T-cell activation.

[0059] Various embodiments relate to a composition for use in a method disclosed herein. For example, the composition may be a composition comprising a small molecule FIPK1 inhibitor and the method may be a method of increasing T-cell activation in a human patient. Similarly, the composition may be a composition comprising a small molecule FIPK1 for use in the treatment of cancer.

HPKl Protein and Signaling

[0060] FIPK1, also referred to as mitogen-activated protein kinase kinase kinase kinase 1 or MAP4K1, is a member of the germinal center kinase subfamily of the Sterile-20 (Ste20) related serine/threonine kinase family. FIPK1 functions as a MAP4K by

phosphorylating and activating MAP3K proteins, including MEKK1, MLK3, and TAK1, leading to the activation of the mitogen-activated protein kinase Jnk.

[0061] A non-limiting example of a nucleotide sequence encoding FIPK1 includes various human HPKl mRNA transcripts {e.g., NCBI Reference Sequence: M_007181.5). A non-limiting example of an HPKl amino acid sequence includes various human HPKl amino acid sequences {e.g., NCBI Reference Sequence: NP 009112.1; 833 amino acids) which may be defined by different mRNA transcripts.

[0062] The HPKl protein comprises a variety of conserved structural motifs, which are described in relation to NCBI Reference Sequence: NP 009112.1 herein. HPKl comprises an amino-terminal Ste20-like kinase domain that spans amino acid residues 17- 293, which includes an ATP-binding site from amino acid residues 23-46. The kinase domain is followed by four proline-rich (PR) motifs that serve as binding sites for SH3- containing proteins, such as CrkL, Grb2, HIP-55, Gads, Nek, and Crk. The four PR motifs span amino acid residues 308-407, 394-402, 432-443, and 468-477, respectively.

[0063] Postnatal expression of HPKl is primarily restricted to hematopoietic organs and cells. HPKl becomes phosphorylated and activated in response to T-cell receptor

("TCR") stimulation or B-cell receptor ("BCR") stimulation. TCR- and BCR-induced phosphorylation of the tyrosine at position 381, located between PR1 and PR2, mediates binding to SLP-76 in T-cells (or BLNK in B-cells) via a SLP-76 (or BL K) SH2 domain, and binding is required for activation of the kinase. A citron homology domain (C H) of the C-terminus of HPKl, approximately spanning residues 495-800, may act as a regulatory domain and may be involved in macromolecular interactions.

[0064] HPKl can also become activated in response to prostaglandin E2, which is often secreted by tumors, thereby contributing to the escape of tumor cells from the immune system.

[0065] HPKl is a negative regulator of T- and B-cell responses. In T-cells, it is believed that HPKl negatively regulates T-cell activation by reducing the persistence of signaling microclusters by phosphorylating SLP76 at Ser376 (Di Bartolo, V., et al. J.

Experimental Medicine 204:681-691 (2007)) and Gads at Thr254, which leads to the recruitment of 14-3-3 proteins that bind to the phosphorylated SLP76 and Gads, releasing the SLP76-Gads-14-3-3 complex from LAT-containing microclusters (Lasserre, R. et al. J. Cell Biology 195(5):839-853 (2011)).

Small Molecule Inhibitors of HPKl

[0066] Various embodiments relate to a composition that inhibits HPKl . The composition that inhibits HPKl may comprise a small molecule HPKl inhibitor. A small molecule HPKl inhibitor may be an organic or inorganic compound {e.g., including heteroorganic and organometallic compounds).

[0067] A small molecule HPKl inhibitor may inhibit the serine/threonine kinase activity of HPKl . A small molecule HPKl inhibitor may be a competitive inhibitor of HPKl . A competitive inhibitor may inhibit {e.g., block or sterically inhibit) the binding of a substrate {e.g., ATP or a protein substrate) to HPKl . A small molecule HPKl inhibitor may be a non-competitive inhibitor of HPKl . A non-competitive inhibitor may bind to HPKl regardless of whether substrate {e.g., ATP or a protein substrate) is also bound to HPKl . A small molecule HPKl inhibitor may be an un-competitive inhibitor of HPKl. An uncompetitive inhibitor may only bind to HPKl after HPKl is bound to a substrate {e.g., ATP and a protein substrate). A small molecule HPKl inhibitor may be an allosteric inhibitor of HPKl . An allosteric inhibitor may bind to a site on HPKl other than the active site. [0068] A small molecule HPKl inhibitor may function as a competitive inhibitor by binding within the substrate-binding domain (e.g., ATP -binding domain or protein substrate- binding domain), thereby blocking the binding of a substrate (e.g., ATP or protein substrate). Competitive inhibitors may also function as allostenc inhibitors and bind to sites outside of the substrate binding site of the free enzyme, thereby blocking the binding of a substrate (e.g., ATP or a protein substrate) to HPKl .

[0069] A small molecule HPKl inhibitor may be a competitive inhibitor that binds to an ATP binding site of HPKl when HPKl is in an active conformation, e.g., thereby functioning as an ATP mimic and inhibiting the binding of ATP to HPKl. In some embodiments, a small molecule HPKl inhibitor may bind to an inactive conformation of HPKl . In some embodiments a small molecule HPKl inhibitor may be an allostenc inhibitor that does not bind to the active site of HPKl .

[0070] A small molecule HPKl inhibitor may have a dissociation constant (K_d) with

HPKl of less than 100 nM, such as less than 50nM, 40 nM, 30nM, 25nM, 20nM, 15nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2, nM, or 0.1 nM. For example, nintedanib (BIBF-1120) has a K_d of 35 nM with HPKl, lestaurtinib (CEP-701) has a K_d of 38 nM, dovitinib (TKI-258, CHIR-258; RAF-265) has a K_d of 44 nM, crizotinib has a K_d of 39 nM, foretinib (GSK1363089; EXEL-2880) has a K_d of 44 nM, neratinib (HKI-272) has a K_d of 93 nM, KW-2449 has a K_d of 63 nM, bosutinib (SKI-606) has a K_d of 15 nM, staurosporine has a K_d of 4.3 nM, sunitinib has a K_d of 16 nM, SU-14813 has a K_d of 15 nM, tozasertib (VX-680; MK-0457) has a K_d of 72 nM (see Davis, M.I., et al., Nature Biotechnology 29: 1046-51 (2011); Karaman, M.W., et al., Nature

Biotechnology 26: 127-32 (2008)). In some embodiments, a small molecule HPKl inhibitor is selected from bosutinib, crizotinib, dovitinib, foretinib, lestaurtinib, neratinib, nintedanib, KW-2449, staurosporine, sunitinib, SU-14813, and tozasertib.

[0071] A small molecule HPKl inhibitor may or may not be a multi-kinase inhibitor.

[0072] A small molecule HPKl inhibitor may or may not be a specific HPKl inhibitor. A specific HPKl inhibitor reduces the biological activity of HPKl by an amount that is greater than the inhibitory effect of the inhibitor on any other human kinase. In certain embodiments, a small molecule HPKl inhibitor specifically inhibits the serine/threonine kinase activity of HPKl . In some of these embodiments, the IC₅₀ of the small molecule HPKl inhibitor for HPKl is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 0.1%, 0.01%, 0.001%, or less of the IC₅₀ of the small molecule HPKl inhibitor for another serine/threonine kinase.

[0073] A small molecule HPKl inhibitor may or may not be a selective HPKl inhibitor as defined herein. A small molecule HPKl inhibitor may selectively inhibit HPKl relative to one or more of lymphocyte-specific protein tyrosine kinase (LCK), zeta-chain- associated protein kinase 70 (ZAP70), protein kinase C theta (PKC-Θ), and Janus kinase 3 (JAK3). A small molecule HPKl inhibitor may bind HPKl with a that is less than the between the small molecule HPKl inhibitor and 1, 2, 3, or all of LCK, ZAP70, PKC-Θ, and JAK3. For example, the between the small molecule HPKl inhibitor and HPKl may be less than: the K_& between the small molecule HPKl inhibitor and LCK; the K_& between the small molecule HPKl inhibitor and ZAP70; the between the small molecule HPKl inhibitor and PKC-Θ; and/or the between the small molecule HPKl inhibitor and JAK3. The small molecule HPKl inhibitor may have an IC₅₀ with HPKl that is lower than the IC₅₀ between the small molecule HPKl inhibitor and 1, 2, 3, or all of LCK, ZAP70, PKC-Θ, and JAK3. For example, the IC₅₀ of the small molecule HPKl inhibitor and HPKl may be less than: the IC₅₀ of the small molecule HPKl inhibitor and LCK; the IC₅₀ of the small molecule HPKl inhibitor and ZAP70; the IC₅₀ of the small molecule HPKl inhibitor and PKC-Θ; and/or the IC₅₀ of the small molecule HPKl inhibitor and JAK3.

[0074] In some embodiments, the small molecule HPKl inhibitor is not

staurosporine, bosutinib, sunitinib, lestaurtinib, crizotinib, foretinib, dovitinib, or KW-2449. A small molecule HPKl inhibitor may be, for example, an analogue of staurosporine, bosutinib, sunitinib, lestaurtinib, crizotinib, foretinib, dovitinib, or KW-2449. An analogue may be generated by organic synthesis using known methods.

[0075] Small molecule HPKl inhibitors may be identified using a high-throughput screen followed by lead optimization using methods known in the art. An assay of a high- throughput screen may be, for example, a binding assay, a kinase-activity assay, or a cellular assay. Cellular assays include systems in which T-cell activation is measured in T-cells either comprising or lacking an HPKl knockout/knockdown, each in the presence of a candidate HPKl inhibitor.

[0076] Small molecule HPKl inhibitors may be rationally designed based on a 3D model of HPKl . A 3D structure of HPKl is not currently publicly available. Structures of the kinase domains of related proteins are available, including MSTl (e.g., PDB ID: 3COM), MST3 (e.g., PDB ID: 3A7J; 3CKX; 3CKW; 3A7I; 3A7H; 3A7G; 3A7F; 4Q09; 4QML; 4QMM; 4QMM; 4QMO; 4QMP; 4QMQ; 4QMR; 4QMS; 4QMT; 4QMU; 4QMV; 4QMW; 4QMX; 4QMY; 4QMZ; 4QNA; 4U8Z; 4W8E; 4W8D), MST4 (e.g., PDB ID: 3GGF), PAK6 (e.g., PDB ID: 2C30; 4KS7; 4KS8), and TA02 (PDB ID: 1U5R; 2GCD). These structures display striking similarity, and some include a bound small molecule kinase inhibitor (see, e.g., Record, C.J., et al., PLoS ONE 5(8): el l905. doi: 10.1371/journal.pone.001 1905 (2010)). A crystal structure of HPK1 may be solved with or without a small molecule kinase inhibitor according to the methods used to arrive at one or more of the foregoing structures.

Additionally, 3D models of the HPK1 kinase domain may be generated by homology modeling using a known structure as a template. Such modeling may be especially useful for the rational design of staurosporine analogues (e.g., based on PDB ID: 4QMY or 3CKX, "MST3 in complex with staurosporine"; 2GCD, "TA02 kinase domain-staurosporine structure"), sunitinib analogues (e.g., based on PDB ID: 4QMZ, "MST3 in complex with sunitinib"; 4KS8, "PAK6 kinase domain in complex with sunitinib"), and bosutinib analogues (e.g., based on PDB ID: 4QMN, "MST3 in complex with bosutinib").

[0077] A small molecule HPK1 inhibitor may have a molecular weight of less than about 1000 amu, such as less than about 900, 800, 700, 600, or 500 amu. A small molecule HPK1 inhibitor may have a molecular weight of about 3 amu to about 1000 amu, such as about 100 to about 1000 amu, about 100 to about 900 amu, about 100 to about 800 amu, about 100 to about 700 amu, about 100 to about 600 amu, about 100 to about 500 amu, about 200 to about 1000 amu, about 200 to about 900 amu, about 200 to about 800 amu, about 200 to about 700 amu, about 200 to about 600 amu, about 200 to about 500 amu, about 300 to about 1000 amu, about 300 to about 900 amu, about 300 to about 800 amu, about 300 to about 700 amu, about 300 to about 600 amu, about 300 to about 500 amu, about 400 to about 1000 amu, about 400 to about 900 amu, about 400 to about 800 amu, about 400 to about 700 amu, about 400 to about 600 amu, about 400 to about 500 amu, about 500 to about 1000 amu, about 500 to about 900 amu, about 500 to about 800 amu, about 500 to about 700 amu, about 500 to about 600 amu, about 600 to about 1000 amu, about 600 to about 900 amu, about 600 to about 800 amu, or about 600 to about 700 amu. In certain embodiments, the small molecule HPK1 inhibitor has a molecular weight of about 400 amu to about 700 amu.

[0078] A small molecule HPK1 inhibitor may have a net charge (positive or negative charge) or neutral charge at physiological pH (e.g., 7.4). A small molecule HPK1 inhibitor may have a net charge or neutral charge at the pH of a tumor microenvironment (e.g., pH 6.5- 6.9).

[0079] About 0.0001 mg to about 5 g of a small molecule HPK1 inhibitor may be administered to a subject per day, such as about 0.001 mg to about 2 g per day, about 0.01 mg to about 1000 mg per day, about 0.1 mg to about 800 mg per day, about 0.1 mg to about 250 mg per day, about 0.0001 mg to about 500 mg per day, about 0.001 mg to about 500 mg per day, about 0.01 mg to about 800 mg per day, about 0.01 mg to about 500 mg per day, about 0.1 mg to about 500 mg per day, about 1 mg to about 250 mg per day, about 1 mg to about 150 mg per day, or about 5 mg to about 100 mg per day. An exemplary dosage may be about 5 mg to about 100 mg per day. In some embodiments, for a 70 kg human patient, a suitable dose may be about 1 mg to about 7 g per day, such as about 5 mg to about 2 g per day.

[0080] Administering a composition that inhibits HPKl to a subject (e.g., wherein the composition comprises a small molecule HPKl inhibitor) may comprise administering the composition to the subject at least once per day, such as 1, 2, 3, 4, 5, or 6 times per day, or at least once per week, such as 1, 2, 3, 4, 5, 6, or 7 times per week.

[0081] A small molecule HPKl inhibitor may be formulated in a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient, which includes any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and the like. A pharmaceutically acceptable carrier or excipient preferably does not destroy the pharmacological activity of a small molecule HPKl inhibitor and is preferably nontoxic when administered in doses sufficient to deliver a therapeutic amount of the inhibitor. The use of such media and agents for pharmaceutically active substances is well known. Except insofar as any conventional media or agent is incompatible with a specific small molecule HPKl inhibitor, its use in various therapeutic compositions is contemplated. Non-limiting examples of pharmaceutically acceptable carriers and excipients include sugars such as lactose, glucose, dextrose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as polyethylene glycol and propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; isotonic saline; Ringer's solution; ethyl alcohol;

phosphate buffer solutions; non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; releasing agents; coating agents; sweetening, flavoring, and perfuming agents; preservatives; antioxidants; ion exchangers; alumina; aluminum stearate; lecithin; self-emulsifying drug delivery systems (SEDDS) such as d-a tocopherol polyethyleneglycol 1000 succinate; surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices; serum proteins such as human serum albumin; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts;

colloidal silica; magnesium trisilicate; polyvinyl pyrrolidone; cellulose-based substances; polyacrylates; waxes; and polyethylene-polyoxypropylene-block polymers. Cyclodextrins such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives such as

hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl-cyclodextrins, or other solubilized derivatives may also be used to enhance delivery of a specific small molecule HPK1 inhibitor.

[0082] Pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), capsules, boluses, powders, granules, pastes for application to the tongue, and intraduodenal routes; parenteral administration, including intravenous, intraarterial, subcutaneous, intramuscular,

intravascular, intraperitoneal or infusion as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; intravaginally or intrarectally, for example, as a pessary, cream, stent, or foam; sublingually; ocularly; pulmonarily; local delivery by catheter or stent; intrathecally, or nasally.

[0083] Examples of suitable aqueous and nonaqueous carriers which can be employed in pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0084] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents, lubricants, and/or antioxidants. Prevention of the action of microorganisms upon a small molecule HPKl inhibitor may be ensured by the inclusion of various antibacterial and/or antifungal agents, for example, paraben,

chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0085] Methods of preparing various formulations or compositions may include the step of bringing into association a small molecule HPKl inhibitor with a carrier and, optionally, one or more accessory ingredients. In general, formulations may be prepared by uniformly and intimately bringing into association a small molecule HPKl inhibitor with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0086] Preparations for such pharmaceutical compositions are well-known. See, e.g.,

Anderson, Philip O.; Knoben, James E.; Troutman, William G., eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 2003; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remington's Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all of which are incorporated by reference herein in their entirety. Except insofar as any conventional excipient medium is incompatible with a specific small molecule HPKl inhibitor, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, an excipient' s use is contemplated to be within the scope of this disclosure.

Genetic Approaches to HPKl Inhibition

[0087] In some embodiments, the composition that inhibits HPKl comprises a nucleic acid. A nucleic acid may enable the knockdown or knockout of HPKl, thereby reducing the concentration of HPKl in a T-cell. A composition that inhibits HPKl may knockdown HPKl, for example, by depleting the intracellular mRNA encoding HPKl in a T- cell of a subject. A composition that inhibits HPKl may knockdown HPKl, for example, by decreasing the rate at which intracellular mRNA encoding HPKl is translated in a T-cell of a subject (e.g., by binding to a control region of the mRNA). A nucleic may be, for example, microRNA (miRNA) or a small interfering RNA (siRNA).

[0088] A nucleic acid may be capable of specifically binding either pre-mRNA encoding HPKlor mRNA encoding HPKl, e.g., the nucleic acid may comprise a nucleotide sequence that is the reverse complement of a nucleotide sequence of either pre-mRNA encoding HPKl or mRNA encoding HPKl . The reverse complement may be an antisense sequence (i.e., wherein the reverse complement is the reverse complement of a coding sequence). In some embodiments, the reverse complement is not an antisense sequence, e.g., wherein the reverse complement binds to an intron, a unique control region, or other unique untranslated region of an RNA molecule. The melting temperature of the complementary portion of the nucleic acid and the pre-mRNA or mRNA may be greater than body temperature (e.g., greater than 37°C) at intracellular ionic strength.

[0089] Interfering RNA (RNAi) utilizes a reverse complement of mRNA to knockdown protein expression. RNAi strategies include small RNA (sRNA), siRNA, miRNA, hairpin RNA, and short hairpin RNA (shRNA) (see, e.g., Meister and Tuschl, Nature 431 :343-349 (2004); Bonetta et al., Nature Methods 1 :79-86 (2004); Chuang and Meyerowitz, Proc. Natl. Acad. Sci. USA 97:4985-4990 (2000); McManus,et al., Nature Reviews Genetics 3 :737-47 (2002); Dykxhoorn et al., Nature Reviews Molecular Cell Biology 4:457-467 (2003)). A variety of software programs are available to assist with the selection of siRNA sequences (see, e.g., http://www.thermofisher.com/us/en/home/life- sci ence/ rnai . html) .

[0090] A nucleic acid may comprise DNA or RNA. For example, DNA comprising a reverse complement of an mRNA molecule may target the mRNA for RNAse H digestion. Similarly, either DNA or RNA may bind a unique nucleotide sequence of a translation initiation site of an mRNA to inhibit translation.

[0091] As non-limiting examples, antisense nucleic acids may be targeted to hybridize to the following regions: mRNA cap region, translation initiation site, translational termination site, transcription initiation site, transcription termination site, polyadenylation signal, 3' untranslated region, 5' untranslated region, 5' coding region, mid coding region, and 3' coding region. In some embodiments, a complementary nucleic acid is designed to hybridize to the most unique 5' sequence of a gene or mRNA (e.g., relative to other human DNA or RNA sequences), including any of about 15-35 nucleotides spanning the 5' coding sequence. Antisense nucleic acids may be produced by standard techniques (see, e.g., U.S. Pat. No. 5,107,065). Appropriate nucleic acids may be designed using OLIGO software (Molecular Biology Insights, Inc., Cascade, Colo.; http://www.oligo.net).

[0092] In some embodiments, a nucleic acid may bind to double-stranded DNA, thereby forming a triplex nucleic acid, which inhibits the transcription of the hpkl gene. Triple helix pairing inhibits the double helix from opening sufficiently to allow the binding of polymerases, transcription factors, or regulatory molecules. Such nucleic acids can be constructed using the base-pairing rules of triple helix formation and the nucleotide sequences of the target hpkl gene.

[0093] In some embodiments, a composition that inhibits HPK1 comprises a nucleic acid, the nucleic acid is DNA, and the DNA encodes RNA that is capable of specifically binding either pre-mRNA encoding HPK1 or mRNA encoding HPK1. For example, the DNA may encode one or more of the RNAs described herein, supra, such as a miRNA or a siRNA. The composition may knock down HPK1 by depleting the intracellular mRNA encoding HPK1 in a T-cell of a subject. The composition may knockdown HPK1 by decreasing the rate at which intracellular mRNA encoding HPK1 is translated in a T-cell of a subject {e.g., by binding to a control region of the mRNA).

[0094] A DNA nucleic acid may comprise a template for miRNA, siRNA, dsRNA, shRNA, or antisense RNA. The DNA nucleic acid may comprise or consist of an expression cassette.

[0095] An expression cassette may comprise one or more regulatory sequences that are operably-linked to the nucleotide sequence encoding an RNA nucleotide sequence.

"Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing, or stability of the nucleotide sequence encoding the RNA nucleotide sequence {see, e.g., Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.)). Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. For example, a DNA nucleic acid may comprise a promoter that is operably-linked to a nucleotide sequence encoding a miRNA, siRNA, dsRNA, shRNA, or antisense RNA. [0096] Regulatory sequences may be operably-linked with a coding sequence to allow for expression of an RNA encoded by the coding sequence. "Operably-linked" refers to a coding sequence that is functionally linked to one or more regulatory sequences. Operably- linked elements may be contiguous or non-contiguous. Coding sequences may be operably- linked to regulatory sequences in a sense or antisense orientation.

[0097] Regulatory regions (e.g., promoters or transcriptional regulatory regions) and/or coding nucleotide sequences may be native/analogous to the cell to which a nucleic acid is being introduced (e.g., they may be human, such as a human elongation factor 1 alpha (EFla) promoter, phosphoglycerate kinase gene (PGK1) promoter, ubiquitin C (Ubc) promoter, or human beta actin promoter). Regulatory regions and/or coding nucleotide sequences may be native/analogous to each other. Regulatory regions and/or coding nucleotide sequences may be heterologous to a cell to which a nucleic acid is being introduced (e.g., a promoter may be a cytomegalovirus (CMV) promoter or a simian vacuolating virus 40 (SV40) promoter). Regulatory regions and/or coding nucleotide sequences may be heterologous to each other.

[0098] The term "heterologous" in reference to a nucleotide sequence is a nucleotide sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form by deliberate human intervention. For example, a promoter operably-linked to a heterologous nucleotide sequence may be of a species different than the species from which the heterologous nucleotide sequence was derived. Similarly, a nucleotide sequence that is heterologous to a cell may be a nucleotide sequence that originates from a foreign species of cell.

[0099] A DNA nucleic acid may be a plasmid.

[0100] A nucleic acid may be administered naked, i.e., in the absence of a carrier that facilitates the transport of a nucleic acid across a T-cell membrane. For example, a composition that inhibits FIPK1 may comprise a nucleic acid and saline. A composition may comprise a nucleic acid associated with a carrier, which may protect the nucleic acid from degradation in the blood or extracellular fluid of a subject and/or facilitate the transport of the nucleic acid across a T-cell membrane. A composition may comprise lipid nanoparticles (L Ps), polymer nanoparticles, a stable nucleic acid lipid particle (SNALP), cyclodextrin, a cholesterol-RNA conjugate, a Local Drug EluteR (LODER) polymer, a dynamic

polyconjugate (DPC), or a GalNAc conjugate (see, e.g., Khvalevsky, E.Z., et al., Proc. Nat'l Acad. Sci. USA, 110(51):20723-28 (2013); Rozema, D.B., et al., J Controlled Release, 209:57-66 (2015)).

[0101] A DNA nucleic acid may be the genome of a recombinant virus, e.g., wherein the genome encodes a miRNA, siRNA, dsRNA, shRNA, or antisense RNA as described herein. A recombinant virus may be a replication defective virus. A recombinant virus may be, for example, an adenovirus or an adeno-associated virus.

[0102] In some embodiments, a nucleic acid is the RNA of a retrovirus, such as a lentivirus (e.g., a replication defective retrovirus). An RNA nucleic acid of a retrovirus may encode DNA, which in turn encodes a miRNA, siRNA, dsRNA, shRNA, or antisense RNA as described herein.

[0103] A recombinant virus may have a tropism for T-cells.

[0104] In some embodiments, a composition comprising a nucleic acid is

administered ex vivo. For example, a sample comprising T-cells (or other lymphoid lineage cell, such as a small lymphocyte, common lymphoid progenitor, or multipotential hematopoietic stem cell) may be obtained from a subject, the cells of the sample may be transfected with the composition comprising the nucleic acid, and the cells may be re- administered to the subject. Such adoptive cell transfer (ACT) therapies are well-known, including methods of transfecting and culturing cells ex vivo (see, e.g., PCT Patent

Publication No. WO 2016/126608, hereby incorporated by reference).

[0105] Exemplary techniques for introducing foreign nucleic acids into cells, include, but are not limited to calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and viral vectors. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art (see, e.g., Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A nucleic acid may be stably incorporated into the genome of the cell or presented transiently in the cell. Viral vector delivery systems include DNA and RNA viruses, which may result in either episomal or integrated genomes after delivery to the cell. Conventional viral based systems for the delivery of nucleic acids into T-cells include retroviral (e.g., lentivirus), adenoviral, adeno-associated virus, and herpes virus vectors. Integration into a host genome is possible with retrovirus, lentivirus, and adeno-associated virus transfection methods, which may result in long term expression of an inserted expression construct. [0106] In some embodiments, the composition that inhibits HPK1 comprises a single guide RNA (sgRNA) and Cas9 (or an expression cassette encoding Cas9). Methods for designing sgRNAs that target a specific nucleotide sequence are known (see, e.g., Pennisi, Science 341 :833-836 (2013); U.S. Patent Application Publication No. 2014/0068797 and U.S. Patent Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359, each of which is hereby incorporated by reference). These methods include the automated design and/or selection of sgRNA sequences (e.g., CRISPR design, http://crispr.mit.edu/; CasFinder, http://arep.med.harvard.edu/CasFinder/; E-CRISP, http://www.e-crisp.org/E- CRISP/designcrispr.html; CRISPR-ERA http://crispr-era.stanford.edu/index.jsp). A Cas9 protein (or expression cassette encoding a Cas9 protein) and sgRNA (or expression cassette encoding a sgRNA) may be introduced into a T-cell using various methods described herein for introducing a nucleic acid into a T-cell as known in the art. For example, expression cassettes encoding a Cas9 protein and a sgRNA may be introduced into a cell using a viral vector, such as a retroviral vector (e.g., a lentivirus).

[0107] In some embodiments, the composition that inhibits HPK1 comprises a transcription activator-like effector nuclease (TALEN) engineered to target a gene encoding HPK1. In some embodiments, the composition that inhibits HPK1 comprises an expression cassette encoding a TALEN engineered to target a gene encoding HPK1.

[0108] TALENs are produced artificially by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effects (TALEs) may be engineered to bind any desired DNA sequence, including a portion of the hpkl gene. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme may be produced which is specific to any desired DNA sequence, including an hpkl sequence. These can then be introduced into a cell for genome editing (see, e.g., Boch Nature Biotech. 29: 135- 6 (2011); Boch et al. Science 326: 1509-12 (2009); Moscou et al. Science 326:3501 (2009)).

[0109] TALEs are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence.

[0110] To produce a TALEN, a TALE protein is fused to a nuclease (N), which may be a wild-type or mutated Fokl endonuclease. Several mutations to Fokl have been made for use in TALENs, which improve cleavage specificity or activity (see, e.g., Cermak et al. Nucl. Acids Res. 39:e82 (2011); Miller et al. Nature Biotech. 29: 143-8 (2011); Hockemeyer et al. Nature Biotech. 29:731-734 (2011); Wood et al. Science 333 :307 (2011); Doyon et al. Nature Methods 8:74-79 (2010); Szczepek et al. Nature Biotech. 25:786-793 (2007); and Guo et al. J. Mol. Biol. 200:96 (2010)).

[0111] The Fokl domain functions as a dimer, requiring two constructs with unique

DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the Fokl cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity (see, e.g., Miller et al. Nature Biotech. 29: 143-8 (2011)).

[0112] An hpkl TALEN may be used inside a cell to produce a double-stranded break in an hpkl gene. A mutation may be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining. For example, improper repair may introduce a frame shift mutation. Alternatively, foreign DNA can be introduced into the cell along with the TALEN; depending on the sequences of the foreign DNA and

chromosomal sequence, this process may be used to knockout an hpkl gene, mutate an hpkl gene, or to introduce a defect that decreases hpkl expression. Mutation of lysine 46 to glutamate (K46E), for example, results in a kinase dead FIPK1 protein.

[0113] TALENs specific to nucleotide sequences of the hpkl gene may be

constructed using any method known in the art (see, e.g., Zhang et al. Nature Biotech.

29: 149-53 (2011); Geibler et al. PLoS ONE 6:el9509 (2011)).

[0114] In some embodiments, a composition that inhibits FIPK1 comprises a zinc- finger nuclease (ZFN) engineered to target a gene encoding FIPK1. In some embodiments, the composition that inhibits HPK1 comprises an expression cassette encoding a ZFN engineered to target a gene encoding HPKL

[0115] Like a TALEN, a ZFN comprises a Fokl nuclease domain (or derivative thereof) fused to a DNA-binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zinc fingers (see, e.g., Carroll et al. Genetics Society of America 188:773-782 (2011); and Kim et al. Proc. Natl. Acad. Sci. USA 93 : 1156-1160 (1996)).

[0116] A ZFN protein or expression vector encoding a ZFN protein may be introduced into a T-cell using various methods described herein for introducing a nucleic acid into a T-cell as known in the art. For example, expression cassettes encoding a ZFN protein may be introduced into a cell using a viral vector, such as a retroviral vector (e.g., a lentivirus).

[0117] A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger may comprise, for example, CyS₂His₂, and can recognize an

approximately 3-bp sequence. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize about 6, 9, 12, 15 or 18-bp sequences. Various selection and modular assembly techniques are available to generate zinc fingers recognizing specific sequences, including phage display, yeast one-hybrid and two-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian screening systems.

[0118] A ZFN must dimerize to cleave DNA. Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart (see, e.g., Bitinaite et al. Proc. Natl. Acad. Sci. USA 95: 10570-5 (1998)).

[0119] A ZFN can create a double-stranded break in DNA, which can create a frame- shift mutation if improperly repaired, thereby leading to a decrease in the expression and amount of FIPK1 in a cell. ZFNs may also be used with homologous recombination to mutate the hpkl gene. Mutation of lysine 46 to glutamate (K46E), for example, results in a kinase dead FIPK1 protein.

[0120] ZFNs specific to a sequence of an hpkl gene may be constructed using any method known in the art (see, e.g., Provasi, Nature Med. 18:807-815 (2011); Torikai, Blood 122: 1341-1349 (2013); Cathomen et al., Mol. Ther. 16: 1200-7 (2008); Quo et al., J. Mol. Biol. 400:96 (2010); U.S. Patent Nos. 8,956,828 and 8,945,868, each of which is hereby incorporated by reference).

[0121] A ZFN protein or expression vector encoding a ZFN protein may be introduced into a T-cell using various methods described herein for introducing a nucleic acid into a T-cell as known in the art. For example, expression cassettes encoding a ZFN protein may be introduced into a cell using a viral vector, such as a retroviral vector (e.g., a lentivirus).

Therapeutic Antibodies [0122] In some embodiments, a method comprises administering to a subject (e.g., a human patient) one or more antibodies. A method may comprise administering to a subject an anti-CTLA-4 antibody, e.g., ipilimumab, tremelimumab, or an antigen-binding portion of either one of the foregoing. A method may comprise administering to a subject an antibody that binds to an epitope capable of binding ipilimumab or tremelimumab. A method may comprise administering to a subject an antibody that competes with ipilimumab or tremelimumab for binding to CTLA-4 in a competitive binding assay. As shown in the examples, infra, the combination of an anti-CTLA-4 antibody and a composition that inhibits HPK1 displays synergy as assessed by T-cell interleukin-2 production.

[0123] A method may comprise administering to a subject an anti-PD-1 antibody, e.g., BGB-A317, nivolumab, pembrolizumab, pidilizumab, or an antigen-binding portion of any one of the foregoing. A method may comprise administering to a subject an antibody that binds to an epitope capable of binding BGB-A317, nivolumab, pembrolizumab, or pidilizumab. A method may comprise administering to a subject an antibody that competes with BGB-A317, nivolumab, pembrolizumab, or pidilizumab for binding to PD-1 in a competitive binding assay. Various anti-PD-1 antibodies are described in US Patent

Application Publication No. 2016/0158360, hereby incorporated by reference). As shown in the examples, infra, the combination of an anti-PD-1 antibody and a composition that inhibits HPK1 displays synergy as assessed by T-cell interleukin-2 production.

[0124] A method may comprise administering to a subject an anti-CTLA-4 antibody and an anti-PD-1 antibody.

[0125] An antibody may be administered to a subject {e.g., human patient) at a dose of about 0.001 mg/kg to about 1000 mg/kg, such as about 0.01 mg/kg to about 100 mg/kg, about 0.1 mg/kg to about 100 mg/kg, about 0.5 mg/kg to about 20 mg/kg, or about 1 mg/kg to about 10 mg/kg {i.e., mg antibody per kg bodyweight of the subject). For example, an antibody may be administered to a subject {e.g., human patient) at a dose of about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.3 mg/kg, 3.7 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg. [0126] An antibody may be administered to a subject by intravenous injection, intravenous infusion, subcutaneous injection, intramuscular injection, intratumoral injection, peritumoral injection, intraperitoneal injection, or intrathecal injection. For example, an antibody may be administered by intravenous infusion over the course of about 10 minutes to about 300 minutes, such as about 15 minutes to about 240 minutes, or about 20 minutes to about 200 minutes. An antibody may be administered by intravenous infusion over about 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, or 180 minutes.

[0127] An antibody may be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. For example, an antibody may be administered 1, 2, 3, 4, 5, 6, or 7 times per week, once every 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, once every 1, 2, 3, or 4 weeks, or 1, 2, 3, or 4 times per month. In some embodiments, an antibody is administered once every 1, 2, or 3 weeks.

[0128] An antibody may be formulated in sterile water. A composition comprising an antibody may further comprise a metal chelator (e.g., ethyl enediaminetetraacetic acid or diethyl en etriamine pentaacetic acid), a sugar or sugar alcohol (e.g., glucose, sucrose, dextrose, or mannitol), an emulsifier (e.g., polysorbate such as polysorbate 80), a toni city- adjusting agent (e.g., sodium chloride), and/or a buffer (e.g., phosphate, citrate,

tris(hydroxymethyl)aminomethane, or histidine). An antibody may be formulated at a concentration of about 0.1 mg/mL to about 100 mg/mL, such as about 1 mg/mL to about 10 mg/mL. An antibody may be formulated at a concentration of about 1 mg/mL to about 50 mg/mL, such as about 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, about 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, about 10 mg/mL, 1 1 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL, or about 25 mg/mL.

[0129] The term "antibody" includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies), and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab')₂, and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0130] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (V_H) followed by three constant domains (C_H) for each of the a and γ chains and four C_H domains for μ and ε isotypes. Each L chain has at the N- terminus, a variable domain (V_L) followed by a constant domain at its other end. The V_L is aligned with the V_H and the C_L is aligned with the first constant domain of the heavy chain (C_HI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a V_H and V_L together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Ten and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called κ and λ, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C_H), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and a classes are further divided into subclasses on the basis of relatively minor differences in the C_H sequence and function, e.g., humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl and IgA2.

[0131] The "variable region" or "variable domain" of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as "V_H" and "V_L," respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.

[0132] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.

However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of

Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0133] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be produced by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies of the presently disclosed compositions and methods may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal

Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N. Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004)), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO

1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625, 126;

5,633,425; and 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 :65-93 (1995)).

[0134] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel. An anti-CTLA antibody and/or an anti-PD-1 antibody may be a naked antibody.

[0135] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

[0136] An "antibody fragment" comprises a portion of an intact antibody, and in most cases, the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')₂ and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062(1995)); single- chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V_H), and the first constant domain of one heavy chain (C_H1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen- binding site. Pepsin treatment of an antibody yields a single large F(ab')₂ fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the C_H1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0137] The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region. The Fc region is also recognized by Fc receptors (FcR) found on certain types of cells.

[0138] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0139] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the V_H and V_L antibody domains connected into a single polypeptide chain. In some cases, the sFv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

[0140] "Functional fragments" of the antibodies useful in the presently disclosed compositions and methods comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fc region of an antibody which retains or has modified FcR binding capability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0141] The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments with short linkers (about 5-10 residues) between the V_H and V_L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the V_H and V_L domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, in EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0142] Monoclonal antibodies useful in the presently disclosed compositions and methods specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567, hereby incorporated by reference; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies, e.g., wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with an antigen of interest. As used herein, "humanized antibody" is used as a subset of "chimeric antibodies."

[0143] "Humanized" forms of non-human {e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an hypervariable region ("HVR") of the recipient antibody is replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having a desired specificity, affinity, and/or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, and/or immunogenicity. The number of amino acid substitutions in the FR includes typically no more than 6 in the H chain and no more than 3 in the L chain. A humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23 : 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0144] A "human antibody" is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries {see, e.g., Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice {see, e.g., U.S. Pat. Nos. 6,075, 181 and 6,150,584 regarding XENOMOUSE® technology). See also Li et al., Proc. Natl. Acad. Sci. USA, 103 :3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0145] The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs, e.g., three in the V_H (HI, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies {see, e.g., Xu et al., Immunity 13 :37-45 (2000);

Johnson and Wu, in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, N. J., 2003)). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of a light chain (see, e.g., Hamers-Casterman et al., Nature 363 :446-448 (1993); Sheriff et al., Nature Struct. Biol. 3 :733-736 (1996)).

Additional Pharmaceutical Agents

[0146] A method may further comprises administering to a subject one or more additional pharmaceutical agents. The one or more additional pharmaceutical agents may comprise a small molecule compound or a large molecule.

[0147] The one or more additional pharmaceutical agents may comprise a BRAF inhibitor. The one or more additional pharmaceutical agents may comprise dabrafenib, encorafenib, GDC-0879, PLX-4720, regorafenib, sorafenib, or vemurafenib.

[0148] The one or more additional pharmaceutical agents may comprise a MEK inhibitor. The one or more additional pharmaceutical agents may comprise binimetinib, cobimetinib, PD-0325901, PD-184352, PD-325901, selumetinib, TAK-733, or trametinib.

[0149] The one or more additional pharmaceutical agents may comprise a MEK inhibitor and a BRAF inhibitor, such as cobimetinib and vemurafenib or dabrafenib and trametinib.

[0150] The one or more additional pharmaceutical agents may comprise a c-KIT inhibitor. The one or more additional pharmaceutical agents may comprise cediranib, crenolanib, imatinib, linifanib, masitinib, nilotinib, pazopanib, semaxanib, sorafenib, or sunitinib.

[0151] The one or more additional pharmaceutical agents may comprise an EGFR inhibitor. The one or more additional pharmaceutical agents may comprise afatinib, brigatinib, canertinib, dacomitinib, erlotinib, gefitinib, icotinib, lapatinib, neratinib, vandetanib, or osimertinib. The one or more additional pharmaceutical agents may comprise cetuximab, nimotuzumab, matuzumab, panitumumab, or zalutumumab.

[0152] The one or more additional pharmaceutical agents may comprise a platinum- based antineoplastic drug, e.g., carboplatin, cisplatin, nedaplatin, oxaliplatin, picoplatin, satraplatin, or triplatin tetranitrate.

[0153] The one or more additional pharmaceutical agents my comprise an indoleamine 2,3-dioxygenase (IDO) inhibitor. The one or more pharmaceutical agents may comprise epacadostat, 1-methyltryptophan (e.g., indoximod), PF-06840003, GDC-0919, or imatinib mesylate.

[0154] The one or more additional pharmaceutical agents may comprise adriamycin, dacarbazine, fotemustine, gemcitabine, methotrexate, temozolomide, and/or vinblastine.

[0155] The one or more additional pharmaceutical agents may comprise a

corticosteroid (e.g., an oral corticosteroid), such as prednisone or methylprednisolone.

[0156] The one or more additional pharmaceutical agents may comprise a mitomycin.

[0157] The one or more additional pharmaceutical agents may comprise a cytokine, such as interleukin-2 (e.g., aldesleukin).

[0158] The one or more additional pharmaceutical agents may comprise AMP -224, which is a PD-L2/Fc fusion protein (see U.S. Patent Application Publication Nos.

2013/0017199 and 2014/0227262, each of which is hereby incorporated by reference in its entirety).

[0159] The one or more additional pharmaceutical agents may comprise an anti-PD-

Ll antibody such as atezolizumab, avelumab, MDX-1105 (see US Patent No. 7,943,743, hereby incorporated by reference), durvalumab, or YW243.55.S70 (see US Patent No.

8,217, 149, hereby incorporated by reference). Anti-PD-Ll antibodies are described in US Patent Application Publication No. 2016/0158360 (hereby incorporated by reference).

[0160] The one or more additional pharmaceutical agents may comprise an anti-TIM-

3 antibody, anti-LAG3 antibody, anti-OX40 antibody, anti-4-lBB antibody, anti-GITR antibody, anti-PD-Ll antibody, anti-PD-L2 antibody, anti-B7.1 antibody, anti-B7.2 antibody, anti-VTCNl antibody, anti-ICOS ligand antibody, anti-MHC class II antibody, anti HVEM antibody, anti-CD 155 antibody, anti galectin-9 antibody, or anti-TIM-3 ligand antibody.

[0161] The one or more additional pharmaceutical agents may comprise

atezolizumab, avelumab, durvalumab, GSK3174998, MEDI6383, prezalizumab, urelumab, or utomilumab.

[0162] The one or more additional pharmaceutical agents may comprise one or more alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines, and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan, innotecan, acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed; callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide;

cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; TLK-286; CDP323, an oral alpha-4 integrin inhibitor; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and

calicheamicin omegall (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33 : 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin (including, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®) and

deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine, tegafur,

capecitabine, an epothilone, and 5-fluorouracil; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea;

lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; thiotepa; taxoids, e.g., paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE®), and doxetaxel; chloranbucil; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; oxaliplatin; leucovovin; vinorelbine; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine; retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN®) combined with 5-FU and leucovovin.

[0163] Additional examples of pharmaceutical agents include anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that promote the growth of cancer. Anti-hormonal agents may be hormones themselves. Examples include anti- estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; anti-progesterones; estrogen receptor down-regulators (ERDs); estrogen receptor antagonists such as fulvestrant; agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole. In addition, such definition of

chemotherapeutic agents includes bisphosphonates such as, etidronate, NE-58095, zoledronic acid/zoledronate, alendronate, pamidronate, tiludronate, or risedronate; as well as

troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); anti-sense nucleic acids, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); an anti-estrogen such as fulvestrant; EGFR inhibitor such as erlotinib or cetuximab; an anti-VEGF inhibitor such as bevacizumab; arinotecan; rmRH (e.g., ABARELIX®); tanespimycin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

Applications and Indications

[0164] Various embodiments relate to a method of increasing T-cell activation in a subject comprising administering to the subject a composition that inhibits hematopoietic progenitor kinase 1 (HPK1). A method may be a method of treating cancer, a method of treating an infection, or a method of treating a disease or condition associated with extracellular protein aggregates.

[0165] The method may further comprise administering an anti-CTLA-4 (cytotoxic

T-lymphocyte-associated protein 4) antibody to the subject. Anti-CTLA-4 antibodies include ipilimumab and tremelimumab. The method may further comprise administering an anti-PD- 1 (programmed cell death protein 1) antibody to the subject. Anti-PD-1 antibodies include BGB-A317, nivolumab, pembrolizumab, and pidilizumab. The method may comprise administering to a subject a composition that inhibits hematopoietic progenitor kinase 1 (HPK1), an anti-CTLA-4 antibody, and an anti-PD-1 antibody.

[0166] Various embodiments relate to a method of increasing T-cell activation in a subject. In some embodiments, the subject does not have a T-cell dysfunction. For example, the T-cell may have unimpaired capacity to translate antigen recognition into down-stream T- cell effector functions, such as proliferation, cytokine production, and/or target cell killing. In some embodiments, the subject lacks T-cell anergy, e.g., the T-cells of the subject are responsive to antigen stimulation. In some embodiments, the subject lacks T-cell exhaustion, e.g., the T-cells of the subject may have effector function within a normal range. A subject may, however, have T-cell dysfunction, anergy, or exhaustion.

[0167] Various embodiments relate to a method of treating cancer in a subject. The cancer may be selected from colorectal cancer, melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, pancreatic cancer, a hematological malignancy, and a renal cell carcinoma. The cancer may be selected from carcinoma, lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumors, carcinoid tumors, gastrinoma, islet cell cancer, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancies, squamous cell cancer, epithelial squamous cell cancer, lung cancer, small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, Merkel cell cancer, mycosis fungoides, testicular cancer, esophageal cancer, tumors of the biliary tract, head and neck cancer, and hematological malignancies.

[0168] The cancer may be a hematologic cancer, such as chronic lymphocytic leukemia (CLL), an acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell-follicular lymphoma, a malignant

lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non- Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia.

[0169] The cancer may be a solid cancer selected from colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non- Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, and environmentally induced cancers.

[0170] The cancer may be selected from acute lymphoid leukemia (ALL), an acute leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoid leukemia (B-ALL), blastic plasmacytoid dendritic cell neoplasm, bone cancer, brain stem glioma, Burkitt lymphoma, cancer of the adrenal gland, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the small intestine, cancer of the urethra, carcinoma of the renal pelvis, carcinoma of the vagina, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), cutaneous malignant melanoma, diffuse large B-cell lymphoma, epidermoid cancer, follicular lymphoma, hairy cell leukemia, Hodgkin's Disease, Hodgkin's lymphoma, intraocular malignant melanoma, Kaposi's sarcoma, large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, neoplasm of the central nervous system (CNS), non-Hodgkin's lymphoma, pituitary adenoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, preleukemia, primary CNS lymphoma, small cell-follicular lymphoma, a solid tumor of childhood, a spinal axis tumor, T-cell acute lymphoid leukemia (T-ALL), T-cell lymphoma, uterine cancer, and Waldenstrom macroglobulinemia.

[0171] In some embodiments, the cancer is not a solid cancer. In some embodiments, the cancer is not colorectal cancer. For example, the cancer may be a solid cancer, wherein the solid cancer is not a colorectal cancer. In some embodiments, the subject does not have a solid tumor. For example, the cancer may be a solid cancer, and the subject may not have a solid tumor, e.g., because any tumor has been excised such that the subject no longer has a solid tumor. In some embodiments, the cancer is a solid cancer, and the subject does not have a colorectal tumor. In some embodiments, the subject does not have a malignant tumor. For example, the cancer may be a solid cancer, and the subject may not have a malignant tumor, e.g., because any malignant tumor has been excised or irradiated such that the subject no longer has a malignant tumor. In some embodiments, a subject previously had a malignant tumor, and the malignant tumor has been excised or irradiated such that the subject no longer has a malignant tumor. In some embodiments, the subject does not have a primary tumor. For example, the cancer may be a solid cancer, and the subject may not have a primary tumor, e.g., because any primary tumor has been excised or irradiated such that the subject no longer has a primary tumor. In some embodiments, a subject previously had a primary tumor, and the primary tumor has been excised or irradiated such that the subject no longer has a primary tumor. In some embodiments, the subject does not have a secondary tumor. A subject may have had a lymphadenectomy (e.g., a total lymphadenectomy). In some embodiments, the cancer is associated with a virus (e.g., HPV).

[0172] In some embodiments, a subject does not have cancer. A subject may be in remission from cancer. A subject may be in partial remission or complete remission.

[0173] Various embodiments relate to a method of preventing a recurrence of cancer in a subject, e.g., wherein the subject is in remission. The term "prevent" as used herein refers to reducing the likelihood that a subject will develop a condition (such as a recurrence of cancer), delaying the onset of the condition, and/or delaying the severity of the condition should it occur in a subject relative to an untreated control subject. The term "prevent" may also refer to reducing the likelihood that any subject of a group of treated subjects will develop a condition (such as a recurrence of cancer) relative to an untreated group of control subjects, delaying the onset of the condition on average in the group of treated subjects relative to an untreated group of control subjects, and/or delaying the severity of the condition (should it occur) on average in the group of treated subjects relative to an untreated group of control subjects. A method of preventing may be a prophylactic method.

[0174] Various embodiments relate to a method of treating an infection in a subject.

The infection may be an infection associated with a pathogen such as a bacterium, protist, virus, viroid, or prion. The infection may be a latent infection or an active infection. The infection may be, for example, HIV-1 or HIV-2.

[0175] Various embodiments relate to a method of treating a disease or condition associated with extracellular protein aggregates. Diseases associated with misfolded protein aggregates include Alzheimer's disease (amyloid β), aortic medial amyloid (medin), atherosclerosis (apolipoprotein AI), isolated atrial amyloidosis (atrial natriuretic factor), cerebral amyloid angiopathy (amyloid β), Icelandic cerebral amyloid angiopathy (cystatin), diabetes mellitus type 2 (amylin), dialysis related amyloidosis (β-2 microglobulin), familial amyloid polyneuropathy (transthyretin), fatal familial insomnia (PrP^Sc), Finnish amyloidosis (gelsolin), hereditary non-neuropathic systemic amyloidosis (lysozyme), Huntington's disease (Huntingtin), lattice corneal dystrophy (keratoepithelin), medullary carcinoma of the thyroid (calcitonin), Parkinson's disease (a-synuclein), prolactinoma (prolactin), rheumatoid arthritis (serum amyloid A), sporadic inclusion body myositis, systemic amyloid light-chain amyloidosis (immunoglobulin light chain AL), and transmissible spongiform encephalopathy (PrP^Sc).

Subjects

[0176] A subject may have cancer, including any one of the cancers described herein.

A subject may be in remission from cancer, e.g., complete remission.

[0177] A subject may have bladder cancer, breast cancer {e.g., metastatic triple- negative breast cancer), colorectal cancer, gastric cancer, head and neck squamous cell carcinoma (HNSCC, e.g., metastatic HNSCC), Hodgkin lymphoma, Merkel-cell carcinoma {e.g., virus positive Merkel-cell carcinoma or virus-negative Merkel-cell carcinoma), mesothelioma {e.g., unresectable malignant mesothelioma), melanoma {e.g., cutaneous melanoma, metastatic melanoma, unresectable melanoma, ocular melanoma), non-small cell lung cancer (NSCLC, e.g., metastatic NSCLC, squamous NSCLC), ovarian cancer, prostate cancer {e.g., metastatic castration-resistant prostate cancer or metastatic hormone-refractory prostate cancer), renal cell carcinoma (RCC), small cell lung cancer (SCLC), transitional cell carcinoma, or urothelial cancer.

[0178] A subject may have a cancer comprising BRAF mutation {e.g., V600E, R461I,

I462S, G463E, G463 V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K, or A727V). For example, a subject may have a cancer selected from colorectal cancer, craniopharyngioma, hairy cell leukemia, Langerhans cell histiocytosis, lung cancer {e.g., NSCLC), melanoma, or thyroid cancer {e.g., papillary thyroid cancer), wherein the cancer comprises a BRAF mutation {e.g., V600E).

[0179] A subject may have an EGFR genomic tumor aberration. In some

embodiments, a subject does not have an EGFR genomic tumor aberration. A subject may have an ALK genomic tumor aberration. In some embodiments, a subject does not have an ALK genomic tumor aberration.

[0180] A subject may have positive PD-L1 expression. In some embodiments, a subject does not have positive PD-L1 expression. [0181] In some embodiments, the subject does not have a solid cancer. In some embodiments, the subject does not have colorectal cancer. For example, the subject may have a solid cancer, wherein the solid cancer is not a colorectal cancer. In some

embodiments, the subject does not have a solid tumor. For example, the subject may have a solid cancer, and the subject may not have a solid tumor, e.g., because any tumor has been excised such that the subject no longer has a solid tumor. In some embodiments, the cancer is a solid cancer, and the subject does not have a colorectal tumor. In some embodiments, the subject does not have a malignant tumor. For example, the cancer may be a solid cancer, and the subject may not have a malignant tumor, e.g., because any malignant tumor has been excised or irradiated such that the subject no longer has a malignant tumor. In some embodiments, a subject previously had a malignant tumor, and the malignant tumor has been excised or irradiated such that the subject no longer has a malignant tumor. A subject may have a metastatic cancer wherein any primary tumor of has been excised or irradiated such that any primary tumor is no longer malignant. In some embodiments, the subject has a cancer that is associated with a virus (e.g., UPV).

[0182] In some embodiments, a patient has undergone adoptive cell transfer (ACT) therapy. For example, a patient may have undergone ACT within 24 months, 18 months, 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week prior to administering a composition that inhibits HPK1. A subject may have undergone hematopoietic stem cell transplantation (HSCT; e.g., autologous HSCT). For example, a patient may have undergone HSCT within 24 months, 18 months, 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week prior to administering a composition that inhibits HPK1.

[0183] In some embodiments, a patient has undergone treatment with a platinum- based antineoplastic drug. For example, a patient may have undergone treatment with a platinum-based antineoplastic drug within 24 months, 18 months, 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week prior to administering a composition that inhibits HPK1.

[0184] In some embodiments, a patient has undergone treatment with a programmed cell death protein 1 (PDl) monoclonal antibody (mAb). For example, a patient may have undergone treatment with a PDl mAb within 24 months, 18 months, 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week prior to administering a composition that inhibits HPK1. [0185] In some embodiments, a patient has undergone treatment with an epidermal growth factor receptor (EGFR) inhibitor. For example, a patient may have undergone treatment with a EGFR inhibitor within 24 months, 18 months, 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week prior to administering a composition that inhibits FIPK1.

[0186] In some embodiments, a patient has undergone treatment with a Anaplastic lymphoma kinase (ALK) inhibitor. For example, a patient may have undergone treatment with a ALK inhibitor within 24 months, 18 months, 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week prior to administering a composition that inhibits HPK1.

[0187] In some embodiments, a patient has undergone treatment with an anti-CTLA-4 antibody (e.g., ipilimumab). For example, a patient may have undergone treatment with an anti-CTLA-4 antibody within 24 months, 18 months, 12 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week prior to administering a composition that inhibits HPK1.

[0188] In some embodiments, the subject has a partial insufficiency of T-cell function, e.g., caused by acquired immune deficiency syndrome (AIDS), DiGeorge syndrome, chromosomal breakage syndrome (CBS), ataxia telangiectasia, or Wiskott-Aldrich syndrome.

[0189] In some embodiments, a subject does not have a T-cell dysfunction. For example, the T-cells of a subject may have unimpaired capacity to translate antigen recognition into down-stream T-cell effector functions, such as proliferation, cytokine production, and/or target cell killing. In some embodiments, a subject lacks T-cell anergy, e.g., the T-cells of the subject are responsive to antigen stimulation. In some embodiments, the subject lacks T-cell exhaustion, e.g., the T-cells of the subject may have effector function within a normal range. A subject may, however, have T-cell dysfunction, anergy, or exhaustion.

[0190] A subject may have an infection. For example, a subject may have an infection caused by a bacterium, protist, virus, viroid, or prion. A subject may have human immunodeficiency virus (e.g., HIV-1 or HIV-2). A subject may have herpes simplex virus (e.g., HSV-1 or HSV-2). A subject may have human papilloma virus (e.g., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, or 82). A subject may have hepatitis, such as hepatitis caused by a vims, bacteria, or parasite. A subject may have hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E. A patient may have tuberculosis.

Kits

[0191] Various embodiments relate to a kit comprising a composition that inhibits

HPKl as described herein. A kit may further comprise an anti-PD-1 antibody and/or an anti- CTLA-4 antibody as described herein. A kit may be used in any of the methods described herein. A kit may be used for the treatment of any of the diseases or conditions described here. For example, a kit may be used for the treatment of any of the diseases or conditions described herein in a subject described herein (e.g., a human patient).

[0192] A kit may further comprise a package insert with instructions for use. A

"package insert" refers to instructions customarily included in commercial packages of medicaments that contain information about the indications customarily included in commercial packages of medicaments that contain information about the indications, usage, dosage, administration, contraindications, other medicaments to be combined with the packaged product, and/or warnings concerning the use of such medicaments. For example, a package insert may comprise instructions for using a composition that inhibits FIPK1, e.g., in combination with an anti-PD-1 antibody and/or an anti-CTLA-4 antibody. A package insert may comprise instructions for using a composition that inhibits FIPK1 according to any of the methods disclosed herein.

[0193] In some embodiments, a kit does not comprise an anti-PD-1 antibody or an anti-CTLA-4 antibody, and yet, the kit comprises a package insert having instructions for using a composition that inhibits FIPK1 in combination with an anti-PD-1 antibody and/or an anti-CTLA-4 antibody. For example, the kit may comprise a composition comprising a small molecule FIPK1 inhibitor for self-administration (e.g., oral administration), and the kit may comprise a package insert having instructions for administering the small molecule FIPK1 inhibitor in combination with an anti-PD-1 antibody and/or an anti-CTLA-4 antibody (e.g., wherein the anti-PD-1 antibody and/or an anti-CTLA-4 antibody are administered by intravenous infusion).

[0194] In some embodiments, a kit comprises an antibody (e.g., an anti-PD-1 or anti-

CTLA antibody). An antibody may be formulated as described herein, e.g., the antibody may be formulated with a metal chelator (e.g., ethyl enediaminetetraacetic acid or diethyl en etriamine pentaacetic acid), a sugar or sugar alcohol (e.g., glucose, sucrose, dextrose, or mannitol), an emulsifier (e.g., polysorbate such as polysorbate 80), a toni city- adjusting agent (e.g., sodium chloride), and/or a buffer (e.g., phosphate, citrate,

tris(hydroxymethyl)aminomethane, or histidine). An antibody may be formulated at a concentration of about 0.1 mg/mL to about 100 mg/mL, such as about 1 mg/mL to about 10 mg/mL. An antibody may be formulated at a concentration of about 1 mg/mL to about 50 mg/mL, such as about 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, about 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, about 10 mg/mL, 1 1 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL, or about 25 mg/mL. In some embodiments, an antibody of a kit is present as a lyophilisate, e.g., for reconstitution. A kit may comprise a lyophilized antibody, such as about 5 mg to about 10 g of the lyophilized antibody. A kit may further comprise sterile water, e.g., a vial of sterile water, which may be used to dissolve a lyophilized antibody. The sterile water may be distilled water or deionized water.

EXAMPLES

[0195] While certain compounds, compositions, and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.

Example 1. Inducible T-cell system for assessing drug targets and combination therapies

[0196] A T-cell assay was developed and utilized to probe the effects of an HPKl knockout, HPKl inhibitors, and combination therapies on T-cell activation. The T-cell assay comprises genetically-modified Jurkat cells (T-cells), genetically-modified Raji cells (B- cells), and anti-CD3 coated beads (Figure 1 A).

[0197] The Jurkat cells were genetically modified to inducibly express inhibitory checkpoint receptors PD-1 and CTLA-4. The Raji cells were genetically modified to constitutively express either PD-Ll or PD-L2, which are ligands that bind PD-1. Raji cells endogenously express B7.1 and B7.2, which are ligands that bind CTLA-4. Jurkat cells endogenously express costimulatory receptor CD28, and both B7.1 and B7.2 are ligands that bind CD28. The anti-CD3 coated beads can bind the T-cell receptor (TCR) of the Jurkat cells, thereby providing a costimulatory signal.

[0198] In the absence of the transcription inducer doxycycline (- dox), the T-cells of the inducible T-cell system express signaling molecules, such as secreted interleukin-2 (TL2), which correlate with T-cell activation. In the presence of the transcription inducer (+ dox), the T-cells of the inducible T-cell system express the inhibitory checkpoint receptor constructs for PD-1 and CTLA-4 (Figure IB). The T-cells of the induced system express less IL2 than T-cells of non-induced systems, which is consistent with checkpoint receptor- mediated T-cell inhibition.

[0199] For some experiments, the Jurkat cells further comprised a knockout of the hpkl gene that encodes HPK1 (Figure 1C). The hpkl gene was knocked out using 3 different single guide RNAs (sgRNAs) designed and cloned by Cellecta. The vector expressed Cas9 enzyme and one of the sgRNAs that provided sequence specificity. The complementarity between the -20 nt region of the sgRNA and the target sequence {hpkl gene) determined the cutting position. The cut hpkl gene was imperfectly repaired in cells, leading to short insertions or deletions that destroyed the gene. The sgRNAl target sequence was

GCCAACATCGTGGCCTACCA (SEQ ID NO: 1); the sgRNA2 target sequence was CCTTTGCAAGGCTCGAGACA (SEQ ID NO: 2); and the sgRNA3 target sequence was AGGCCACGATGTTGGCGTGC (SEQ ID NO: 3). The control sgRNA target sequence was AAGATCGAGTGCCGCATCAC (SEQ ID NO: 4). The vector that expressed each of the sgRNA and Cas9 enzyme was pRSGC4-U6-sg-CMV-Cas9-2A-TagGFP2.

Example 2. The hpkl knockout increases T-cell activation

[0200] The percentage of interleukin-2 (IL2) positive cells was measured for Jurkat

T-cells transfected with short hairpin RNA (shRNA) against the hpkl gene to silence hpkl gene expression. As shown in Figure 2 A, shUPKl lead to a decrease in HPK1 relative to control and an increase in the percentage of IL2 positive cells compared to the control when the cells were stimulated. Additionally, HPK1 was knocked down using a CRISPR system with three different sgRNAs (Figure 2B). All three lead to a decrease in HPK1 relative to control sgRNA and an increase in the percentage of IL2 positive cells compared to the control when the cells were stimulated. Example 3. The hpkl knockout increases T-cell activation in the absence and presence of inhibitory checkpoint receptors PD-1 and CTLA-4

[0201] Interleukin-2 (IL2) secretion was measured for different inducible T-cell systems described in Example 1. One system had intact hpkl genes, and the system was assayed in the absence of transcription inducer doxycycline. One system had an hpkl knockout, and the system was assayed in the absence of transcription inducer doxycycline. One system had intact hpkl genes, and the system was assayed after incubating the T-cells of the system with doxycycline. One system had an hpkl knockout, and the system was assayed after incubating the T-cells of the system with doxycycline. IL2 expression was measured by ELISA.

[0202] Each T-cell system comprising a T-cell hpkl knockout expressed substantially more IL2 than its control. The un-induced knockout system expressed 13.6-fold more IL2 than the un-induced control, and the induced knockout system expressed 12.2-fold more IL2 than the induced control (Figure 3 A). These results suggest that the hpkl gene product HPK1 is a suitable drug target for modulating an immune response. Specifically, drugs that decrease HPK1 activity are likely to increase T-cell activation.

[0203] The T-cells of the un-induced hpkl knockout and control systems were incubated with 10 μg/mL ipilimumab and 10 μg/mL nivolumab or 10 μg/mL of a control anti-IgG antibody. The hpkl knockout system incubated with ipilimumab and nivolumab displayed a 2-fold increase in activation over the control system incubated with IgG and a >10 increase in activation over the wild-type system incubated with ipilimumab and nivolumab (Figure 3B).

Example 4. Small molecule compounds that bindHPKl increase T-cell activation

[0204] The effects of small molecule compounds that bind FIPK1 were assessed using the inducible T-cell system described in Example 1. Figure 4 depicts a cartoon of this system in which the T-cells have been contacted with the transcription inducer doxycycline and express the inhibitory checkpoint receptor constructs PD-1 and CTLA-4. The T-cells of this system lacked the hpkl gene knockout. [0205] Four compounds were assessed in the absence or presence of transcription inducer doxycycline. The T-cells of each un-induced system that was contacted with 1250 nM of a small molecule compound released more IL2 than an un-induced system contacted with the DMSO vehicle (Figure 5). The T-cells of each induced system that was contacted with a small molecule compound released more IL2 than an induced system contacted with the DMSO vehicle (Figure 5). These results suggest that FIPK1 inhibitors are suitable drug candidates to increase human T-cell activation.

Example 5. Small molecule compounds that bindHPKl display a synergistic effect on T- cell activation when combined with anti-PD-1 and anti-CTLA-4 antibodies in hpkl KO cells

[0206] The effects anti-PD-1 antibody nivolumab (nivo) and/or anti-CTLA-4 antibody ipilimumab (ipi) on T-cell activation were probed with an inducible T-cell system described in Example 1 wherein hpkl was knocked out. Nivolumab and ipilimumab were added to different T-cell systems either separately or together in systems in which the T-cells had been induced to express the PD-1 and CTLA-4 constructs. An anti-IgG antibody was added to induced and un-induced T-cell systems to serve as controls. Figure 6 depicts cartoons of an un-induced T-cell system contacted with the control antibody (experiment "1"), an induced T-cell system contacted with the control antibody (experiment "2"), an induced T-cell system contacted with a nivolumab (experiment "3"), an induced T-cell system contacted with ipilimumab (experiment "4"), and an induced T-cell system contacted with both nivolumab and ipilimumab (experiment "5").

[0207] Both nivolumab and ipilimumab increased interleukin-2 (IL2) secretion relative to the control anti-IgG antibody in systems induced to express the PD-1 and CTLA-4 constructs (Figure 7). The combination of nivolumab and ipilimumab with hpkl knockout displayed a synergistic effect (Figure 7) (p-value 0.00024, Wilcoxon signed rank test, Bliss Independence model).

[0208] The experiments with nivolumab, ipilimumab, and IgG antibodies were repeated with small molecule HPK1 inhibitors, "compound 1" and "compound 2." A cartoon depiction of this experiment is shown in Figure 8. T-cells were contacted with 1250 nM of compound 1 or 5000 nM of compound 2 after incubating the T-cells with doxycycline to induce PD-1 and CTLA-4 transgene expression. Both compound 1 and compound 2 displayed synergistic effects on JL2 secretion when combined with either nivolumab, ipilimumab, or both nivolumab and ipilimumab (p-value 0.002, Bliss Independence model) (Figure 9A and Figure 9B). These results suggest that HPK1 may be used in combination with anti-PD-1 and/or anti-CTLA-4 antibodies to increase T-cell activation.

Example 6. shRNA knockdown of HPK1 expression in human primary T-cells increases activation

[0209] shRNA against hpkl mRNA was used to knockdown HPK1 expression in human primary T-cells. A green fluorescent protein (GFP) construct was transfected into cells along with the shRNA as a transfection marker. Cells were stimulated with anti- CD3/anti-CD28 antibodies, and interferon γ (INFy) expression was monitored by flow cytometry. GFP⁺ cells were >3 times more likely to be INFy⁺ relative to GFP^" cells or cells transfected with a control shRNA (Figure 10).

Example 7. HPK1 inhibitors increase the activation of human primary T-cells

[0210] Human primary T-cells were activated with anti-CD3/anti-CD28 beads in the presence of four small molecule compounds including three compounds that specifically bind HPK1. T-cell activation was assessed by interferon γ (INFy) release. Compound 1 had an IC₅₀ of 44 nM with HPK1, compound 2 had a IC₅₀ of 12 nM with HPK1, compound 3 had a IC₅₀ of 7 nM with HPK1, and compound 4 had a IC₅₀ of 50,000 nM with HPK1.

Additionally, compounds 2 and 3 were selective for HPK1 relative to three other targets. (Compounds 1 and 2 of the instant example and Figure 11 are different from compounds 1 and 2 of Example 5 and Figures 9A and 9B). Counter Targets #1, #2 and #3 were lymphocyte-specific protein tyrosine kinase (Lck), zeta-chain-associated protein kinase 70 (ZAP70) and Janus Kinase 3 (Jak3), respectively.

[0211] Compound 2 and compound 3 produced the greatest inhibition of HPK1 of the four compounds and also lead to the highest interferon γ release (Figure 11). These results suggest that HPK1 inhibitors can increase T-cell activation in human patients. Example 8. HPK1 inhibitors increase the activation human PBMCs

[0212] Human peripheral blood mononuclear cells (PBMCs) were activated with cytomegalovirus (CMV)-peptide and incubated with 1 μg/mL nivolumab, 156 nM, 312 nM, 625 nM, or 1250 nM of small molecule HPK1 inhibitor compound 5, or DMSO vehicle. PBMC activation was monitored by interferon γ (INFy) release. Compound 5 displayed a similar magnitude of activation as nivolumab (Figure 12). These results suggest that FIPK1 inhibitors can increase PBMC activation in human patients.

Example 9. HPK1 inhibitors display synergy with anti-PD-1 and anti-CTLA-4 antibodies

[0213] T-cells were incubated with a small molecule FIPK1 inhibitor (AP Compound) or DMSO vehicle and either 10 μg/mL ipilimumab and 10 μg/mL nivolumab or 10 μg/mL of an anti-IgG control antibody in the CRISPR engineered platform described in Example 1. T- cell activation was monitored by ELISA for interleukin-2 (IL2). The combination of the small molecule FIPK1 inhibitor, ipilimumab, and nivolumab displayed ~5 fold increased activation over just the combination of ipilimumab and nivolumab (Figure 13). These results suggest that small molecule FIPK1 inhibiters act synergistically on T-cell activation in combination with anti-PD-1 and anti-CTLA-4 antibodies.

Example 10. HPKlinhibits T-cell signaling

[0214] The CRISPR engineered platform described in Example 1 was also used to incubate FIPK1. T-cell activation was monitored by ELISA for interleukin-2 (IL2). Figure 14 demonstrates that HPK1 inhibits T-cell signaling in Jurkat cells via an inducible HPK1 rescue construct.

EQUIVALENTS

[0215] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMS We claim:

1. A method of increasing T-cell activation in a human patient, comprising:

administering to the patient a composition that inhibits hematopoietic progenitor kinase 1 (HPK1); and

administering to the patient an anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibody.

2. The method of claim 1, further comprising administering to the patient an anti-PD-1 (programmed cell death protein 1) antibody.

3. A method of increasing T-cell activation in a human patient, comprising:

administering to the patient a composition that inhibits hematopoietic progenitor kinase 1 (HPK1); and

administering to the patient an anti-PD-1 (programmed cell death protein 1) antibody.

4. The method of any one of the preceding claims, wherein the anti-CTLA-4 antibody is selected from ipilimumab and tremelimumab.

5. The method of any one of claims 2 to 4, wherein the anti-PD-1 antibody is selected from nivolumab and pembrolizumab.

6. The method of any one of the preceding claims, further comprising administering to the patient an anti-TIM-3, anti-LAG3, anti-OX40, anti-4-lBB, anti-GITR, anti-PD-Ll, anti- PD-L2, anti-B7.1, anti-B7.2, anti-VTCNl, anti-ICOS ligand, anti-MHC class II, anti HVEM, anti-CD155, anti galectin-9, or anti-TIM-3 ligand antibody.

7. The method of any one of the preceding claims, further comprising administering to the patient atezolizumab, avelumab, durvalumab, GSK3174998, MEDI6383, prezalizumab, urelumab, or utomilumab.

8. The method of any one of the preceding claims, wherein the composition that inhibits HPKl comprises a small molecule HPKl inhibitor.

9. The method of claim 8, wherein the small molecule HPKl inhibitor binds HPKl with a dissociation constant (K_d) of less than about 10 nM.

10. The method of claim 8 or 9, wherein the small molecule HPKl inhibitor selectively inhibits HPKl relative to LCK, ZAP70, PKC-Θ, and/or JAK3.

1 1. The method of claim 10, wherein the small molecule HPKl inhibitor binds HPKl with a K_d that is less than the K& between the small molecule HPKl inhibitor and either LCK, ZAP70, PKC-Θ, or JAK3.

12. The method of claim 10 or 1 1, wherein the small molecule HPKl inhibitor has an IC₅₀ with HPKl that is less than the IC₅₀ between the small molecule HPKl inhibitor and either LCK, ZAP70, PKC-Θ, or JAK3.

13. The method of any one of the preceding claims, wherein the composition that inhibits HPKl comprises a microRNA (miRNA) or small interfering RNA (siRNA).

14. A method of treating a disease or condition in a human patient, comprising

administering to the patient a small molecule inhibitor of hematopoietic progenitor kinase 1 (HPKl), wherein:

the small molecule inhibitor binds HPKl with a dissociation constant (K_d) of less than about ΙΟ ηΜ;

the disease or condition is cancer, an infection associated with a pathogen, or a disease or condition associated with extracellular protein aggregates; and

the patient does not have a T-cell dysfunctional disorder.

15. The method of claim 14, further comprising administering to the patient an anti CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibody.

16. The method of claim 14 or 15, further comprising administering to the patient an anti- PD-1 (programmed cell death protein 1) antibody.

17. The method of any one of the preceding claims, wherein the patient does not have a colorectal tumor.

18. The method of any one of the preceding claims, wherein the patient does not have a solid tumor.

19. The method of any one of the preceding claims, wherein the patient previously had a malignant tumor, and the malignant tumor has been excised or irradiated such that the patient no longer has a malignant tumor.

20. The method of any one of the preceding claims, wherein the patient has cancer.

21. The method of claim 20, wherein the cancer is selected from bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, Merkel-cell carcinoma, mesothelioma, melanoma, non-small cell lung cancer, ovarian cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, transitional cell carcinoma, and urothelial cancer.

22. The method of claim 20 or 21, wherein the cancer is associated with a virus.

23. The method of any one of claims 1 to 19, wherein the patient does not have cancer.

24. The method of any one of the preceding claims, wherein the patient is in remission from cancer.

25. The method of any one of the preceding claims, wherein the patient has an infection associated with a pathogen.

26. The method of claim 25, wherein the infection is a viral infection.

27. The method of claim 26, wherein the infection is HIV.

28. The method of any one of the preceding claims, wherein the patient has a disease or condition associated with extracellular protein aggregates.

29. The method of claim 28, wherein the patient has Alzheimer's disease.

30. A method of treating an infection in a human patient, comprising:

administering to the patient a composition that inhibits hematopoietic progenitor kinase 1 (HPK1); and either

administering an anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibody to the patient;

administering an anti-PD-1 (programmed cell death protein 1) antibody to the patient; or

administering both an anti-CTLA-4 antibody and an anti-PD-1 antibody to the patient.

31. A method of treating a disease or condition associated with extracellular protein aggregates in a human patient, comprising:

administering to the patient a composition that inhibits hematopoietic progenitor kinase 1 (HPK1); and either

administering an anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibody to the patient;

administering an anti-PD-1 (programmed cell death protein 1) antibody to the patient; or

administering both an anti-CTLA-4 antibody and an anti-PD-1 antibody to the patient.

32. A composition that inhibits hematopoietic progenitor kinase 1 (HPK1) for use in a method of increasing T-cell activation in a human patient.

33. A composition that inhibits hematopoietic progenitor kinase 1 (HPK1) for use in a method of treating cancer.

34. A composition that inhibits hematopoietic progenitor kinase 1 (HPK1) for use in a method of treating an infection.

35. A composition that inhibits hematopoietic progenitor kinase 1 (HPK1) for use in a method of treating a disease or condition associated with extracellular protein aggregates.

36. The composition of any one of claims 32 to 35, wherein the method comprises:

administering the composition to the patient; and either

administering an anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibody to the patient;

administering an anti-PD-1 (programmed cell death protein 1) antibody to the patient; or

administering both an anti-CTLA-4 antibody and an anti-PD-1 antibody to the patient.

37. A kit comprising a composition that inhibits hematopoietic progenitor kinase 1 (HPK1) and a package insert comprising instructions for use.

38. The kit of claim 37, further comprising an anti-PD-1 antibody and/or an anti-CTLA-4 antibody.