WO2023168404A1

WO2023168404A1 - Methods of treating a tumor

Info

Publication number: WO2023168404A1
Application number: PCT/US2023/063680
Authority: WO
Inventors: Agnieszka Seyda; David Edward BALLI; Mark David STERN
Original assignee: Bristol-Myers Squibb Company
Priority date: 2022-03-04
Filing date: 2023-03-03
Publication date: 2023-09-07

Abstract

The disclosure provides a method of treating a tumor in a human subject in need thereof (e.g., a patient diagnosed with a NSCLC), comprising administering an effective amount of a checkpoint inhibitor to the subject, wherein the subject is identified as having decreased expression of one or more genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1.

Description

METHODS OF TREATING A TUMOR

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This PCT application claims the priority benefit of U.S. Provisional Application No. 63/316,843, filed March 4, 2022, which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure provides a method for treating a subject afflicted with a tumor using a checkpoint inhibitor, e.g, an immunotherapy.

BACKGROUND OF THE DISCLOSURE

[0003] In tumors, the rate of glucose uptake is known to be elevated together with lactate production, even in the presence of oxygen and fully functioning mitochondria. This process along with the resultant abnormal cell biology is known as the “Warburg effect”.

[0004] Elevated glucose metabolism is known to facilitate a reduction of pH in the tumor microenvironment (TME) due to lactate secretion. This in turn activates tumor associated fibroblasts which re-metabolize lactate and subsequently provide energy to tumor cells, in the form of glucose, as well as other energy sources such as fatty acids and proteins. The interaction between tumor cells and tumor-associated fibroblasts thus depletes various sources of energy from the TME which acts to “starve” other TME associated cells and causes direct competition for energy between tumor and tumor infiltrating lymphocytes (TIL). Without adequate sources of energy, tumor infiltrating immune cells are effectively prevented from functioning.

[0005] Discovering expression profiles that can detect relevant alterations in metabolism so as to predict response to IO therapies has proven extremely challenging. Many proteins involved in metabolic function (e.g., enzymes involved in pyruvate metabolism, electron transport chain enzymes, and glycolytic pathway enzymes) are not regulated based on increases or decreases in the amounts of protein or RNA but instead by metabolites via autoregulatory feedback loops (e.g., allosteric effects).

[0006] The present disclosure relates to uses of certain biomarkers auxiliary to the metabolic pathway that were identified as predictive of responses to IO therapy. Without wishing to be bound by theory, results provided herein are consistent with association of the identified biomarkers with depletion of energy in the TME and indicate that this energy depletion or “enhanced Warburg effect” can render immuno-oncology therapy ineffective.

SUMMARY OF THE DISCLOSURE

[0007] Some aspects of the present disclosure are directed to a method of treating a tumor in a human subject diagnosed with a non-small cell lung cancer (NSCLC), the method comprising administering an effective amount of a checkpoint inhibitor to the subject, wherein the subject is identified as suitable for the checkpoint inhibitor therapy by measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HBO1, and PDHA1; wherein the subject is identified as having a low expression profile of the panel of genes.

[0008] Some aspects of the present disclosure are directed to a method of treating a tumor in a human subject diagnosed with a non-small cell lung cancer (NSCLC), the method comprising: (a) measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HBO1, and PDHA1; and (b) administering an effective amount of a checkpoint inhibitor to the subject, wherein the subject is identified as having a low expression profile of the panel of genes.

[0009] Some aspects of the present disclosure are directed to a method of identifying a subject suitable for a checkpoint inhibitor therapy, wherein the subject has been diagnosed with non-small cell lung cancer (NSCLC), and the method comprises (i) measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HBO1, and PDHA1; and (ii) identifying a subject as suitable for an immune checkpoint inhibitor therapy if the subject is identified as having a low expression profile of the panel of genes. In some aspects, the method further comprises administering to the subject a checkpoint inhibitor.

[0010] Some aspects of the present disclosure are directed to a checkpoint inhibitor for treating a tumor in a human subject diagnosed with a non-small cell lung cancer (NSCLC), wherein the subject is identified as being suitable for a checkpoint inhibitor therapy by measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HBO1, and PDHA1; and wherein the subject is identified as having a low expression profile of the panel of genes.

[0011] In some aspects, the panel of genes comprises at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, or at least 9 genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDO A, GAPDH, LDHA, HBO1, and PDHA1. In some aspects, the panel of genes comprises SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HBO1, and PDHAL

[0012] In some aspects, the panel of genes comprises at least one housekeeping gene. In some aspects, the panel of genes comprises at least one control gene.

[0013] In some aspects, a high expression profile is characterized by measuring the composite expression of the genes in the gene panel in a tumor sample from the subject, and comparing the composite expression of the genes in the gene panel to the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with NSCLC. In some aspects, the average composite expression is determined by measuring the composite expression of the panel of genes in tumor samples obtained from the population of subjects. In some aspects, the expression of the genes in the panel of genes is determined by measuring the level of mRNA, the level of a protein encoded by the gene, or both. In some aspects, the level of mRNA is determined using reverse transcriptase PCR. In some aspects, the level of the protein encoded by the gene is determined using an IHC assay. In some aspects, the IHC assay is an automated IHC assay. In some aspects, the expression of the genes in the panel of genes is determined by whole transcriptome RNA-seq.

[0014] In some aspects, the checkpoint inhibitor comprises a polypeptide, a small molecule, a polynucleotide, or any combination thereof. In some aspects, the checkpoint inhibitor comprises an antibody or an antigen-binding fragment thereof. In some aspects, the antibody or antigenbinding fragment thereof specifically binds a target selected from PD-1 ("anti-PD-1 antibody"), PD-L1 ("anti-PD-Ll antibody"), CTLA-4, LAG3, TIGIT, TIM3, NKG2a, 0X40, ICOS, CD137, KIR, TGFp, IL- 10, IL-8, IL-2, CD96, VISTA, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, and any combination thereof. In some aspects, the antibody is a chimeric, humanized or human monoclonal antibody or a portion thereof.

[0015] In some aspects, the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1. In some aspects, the anti-PD-1 antibody binds to the same epitope as nivolumab. In some aspects, the anti-PD-1 antibody comprises a heavy chain constant region which is of a human IgGl or IgG4 isotype. In some aspects, the anti-PD-1 antibody is nivolumab. In some aspects, the anti-PD-1 antibody is pembrolizumab.

[0016] In some aspects, the anti-PD-1 antibody is administered at a dose ranging from at least about 0.1 mg/kg to at least about 10.0 mg/kg body weight once about every 1, 2 or 3 weeks. In some aspects, the anti-PD-1 antibody is administered at a dose of at least about 3 mg/kg body weight once about every 2 weeks.

[0017] In some aspects, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose. In some aspects, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, at least about 500 or at least about 550 mg. In some aspects, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of about 240 mg. In some aspects, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of about 480 mg.

[0018] In some aspects, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose about once every 1, 2, 3 or 4 weeks. In some aspects, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose or about 240 mg once about every two weeks. In some aspects, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of about 480 mg once about every four weeks.

[0019] In some aspects, the anti-PD-1 antibody is administered for as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

[0020] In some aspects, the antibody is formulated for intravenous administration. In some aspects, the antibody is administered at a subtherapeutic dose.

[0021] In some aspects, the method further comprises administering an antibody or an antigen binding fragment thereof that binds specifically to CTLA-4 ("an anti-CTLA-4 antibody"). In some aspects, the anti-CTLA-4 antibody cross-competes with ipilimumab or tremelimumab for binding to human CTLA-4. In some aspects, the anti-CTLA-4 antibody binds to the same epitope as ipilimumab or tremelimumab. In some aspects, the anti-CTLA-4 antibody is ipilimumab. In some aspects, the anti-CTLA-4 antibody is tremelimumab.

[0022] In some aspects, the anti-CTLA-4 antibody is administered at a dose ranging from 0.1 mg/kg to 20.0 mg/kg body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks. In some aspects, the anti- CTLA-4 antibody is administered at a dose of 1 mg/kg body weight once every 6 weeks. In some aspects, the anti-CTLA-4 antibody is administered at a dose of 1 mg/kg body weight once every 4 weeks.

[0023] In some aspects, the anti-CTLA-4 antibody is administered at a flat dose. In some aspects, the anti-CTLA-4 antibody is administered at a flat dose of at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, or at least about 200 mg.

[0024] In some aspects, the anti-CTLA-4 antibody is administered as a flat dose about once every 2, 3, 4, 5, 6, 7, or 8 weeks.

[0025] In some aspects, the method further comprises administering an antibody or an antigenbinding fragment thereof that binds specifically to LAG-3 ("an anti-LAG-3 antibody"), n some aspects, the method comprises administering an anti-PD-1 antibody and an anti-LAG-3 antibody. [0026] In some aspects, the method comprises administering an anti-PD-1 antibody, and anti- CTLA-4 antibody, and an anti-LAG-3 antibody. In some aspects, the anti-LAG-3 antibody comprises relatimab.

[0027] In some aspects, the tumor is relapsed, refractory, or both relapsed and refractory. In some aspects, the tumor is refractory to at least one prior therapy comprising administration of at least one anticancer agent. In some aspects, the at least one anticancer agent comprises a standard of care therapy. In some aspects, the at least one anticancer agent comprises an immunotherapy.

[0028] In some aspects, the tumor is locally advanced. In some aspects, the tumor is metastatic. In some aspects, the administering treats the tumor.

[0029] In some aspects, the administering reduces the size of a tumor in the subject. In some aspects, the size of the tumor is reduced by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the tumor size prior to the administration. T In some aspects, the subject exhibits progression-free survival of at least about one month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about one year, at least about eighteen months, at least about two years, at least about three years, at least about four years, or at least about five years after the initial administration. In some aspects, the subject exhibits stable disease after the administration. In some aspects, the subject exhibits a partial response after the administration. In some aspects, the subject exhibits a complete response after the administration. [0030] Some aspects of the present disclosure are directed to a system or device comprising: (i) a loading means for loading a biological sample; and (ii) a means for detecting a level of expression of a panel of genes in the biological sample, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HBO1, and PDHA1. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample comprises a first biological probe, wherein the first biological probe specifically interacts with a transcript of at least one gene in the panel of genes. In some aspects, the system or device further comprises a means for detecting a level of expression of the transcript. In some aspects, the transcript comprises an mRNA or a cDNA.

[0031] In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises a second biological probe. In some aspects, the first biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a first gene of the at least 2 genes; and wherein the second biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a second gene of the at least 2 genes. In some aspects, the transcript of the first gene and/or the transcript of the second gene comprises an mRNA or a cDNA.

[0032] In some aspects, the biological sample comprises a tumor sample from a human subject. In some aspects, the tumor sample is a tumor tissue biopsy. In some aspects, the tumor sample is a formalin-fixed, paraffin-embedded tumor tissue. In some aspects, the tumor sample is a fresh- frozen tumor tissue.

[0033] In some aspects, the panel of genes comprises SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HBO1, and PDHA1. In some aspects, the panel of genes comprises at least one housekeeping gene. In some aspects, the panel of genes comprises at least one control gene.

[0034] Some aspects of the present disclosure are directed to a method of treating a tumor in a human subject in need thereof, comprising administering an effective amount of a checkpoint inhibitor to the subject, wherein the subject is identified as having a tumor suitable for a checkpoint inhibitor therapy, wherein the subject is identified as suitable by obtaining a biological sample from the subject and applying the biological sample to the loading means of a device disclosed herein.

[0035] Some aspects of the present disclosure are directed to a method of identifying a subject suitable for an immunotherapy, comprising obtaining a biological sample from the subject and applying the biological sample to the loading means of a device disclosed herein. [0036] In some aspects, the method further comprises administering an immunotherapy to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGs. 1A-1D are graphical representations of patient progression-free survival (PFS; FIGs. 1A-1B) and overall survival (OS; FIGs. 1C-1D) following treatment with an anti-PD-1 antibody (nivolumab) and an anti-CTLA-4 antibody (ipilimumab) (FIGs. 1A and 1C) or chemotherapy-only (FIGs. IB and ID), wherein the patients are stratified based on the tumor expression level of SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. Patients with a high expression level are those in the upper tertile of expression (marked with "(2)"). Patients with a low expression level are those in the lower tertile of expression (marked with "(1)").

DETAILED DESCRIPTION OF THE DISCLOSURE

[0038] The present disclosure provides a method of identifying a subject suitable for a checkpoint inhibitor therapy, comprising measuring the expression of a panel of genes in a tumor sample obtained from the subject, wherein the panel of genes comprises at least one gene selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, a subject having low tumor expression of one or more of SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1 is likely to be responsive to a therapy comprising an immune checkpoint inhibitor. The inventors of the present application have found that increased expression of certain genes (one or more of SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1) in a tumor sample correlates with a high Warburg effect phenotype, which is associated with poor responsiveness to a therapy with an immune checkpoint inhibitor. As such, some aspects of the present disclosure are directed to a method of identifying a subject suitable for a checkpoint inhibitor therapy, wherein the subject has been diagnosed with NSCLC, the method comprising (i) measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least one gene selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1; and identifying a subject as suitable for an immune checkpoint inhibitor therapy if the subject is identified as having a low expression profile of the panel of genes. In some aspects, the gene panel further comprises GAPDH. I. Terms

[0039] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0040] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and/or "consisting essentially of' are also provided.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0042] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0043] "Administering" refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for an immunotherapy, e.g., a checkpoint inhibitor, e.g., an anti-PD-1 antibody or the anti-PD-Ll antibody, include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Other non-parenteral routes include an oral, topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

[0044] An "adverse event" (AE) as used herein is any unfavorable and generally unintended or undesirable sign (including an abnormal laboratory finding), symptom, or disease associated with the use of a medical treatment. For example, an adverse event can be associated with activation of the immune system or expansion of immune system cells (e.g., T cells) in response to a treatment. A medical treatment can have one or more associated AEs and each AE can have the same or different level of severity. Reference to methods capable of "altering adverse events" means a treatment regime that decreases the incidence and/or severity of one or more AEs associated with the use of a different treatment regime.

[0045] An "antibody" (Ab) shall include, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as Vzz) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, Czzi, Cm and Cm. Each light chain comprises a light chain variable region (abbreviated herein as Vz) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The Vzz and Vz regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each Vzz and Vz comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Therefore, the term "anti-PD-1 antibody" includes a full antibody having two heavy chains and two light chains that specifically binds to PD-1 and antigen-binding portions of the full antibody. Non limiting examples of the antigen-binding portions are shown elsewhere herein.

[0046] An immunoglobulin can derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and non- naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies. A nonhuman antibody can be humanized by recombinant methods to reduce its immunogenicity in man. Where not expressly stated, and unless the context indicates otherwise, the term "antibody" also includes an antigen-binding fragment or an antigen-binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain antibody.

[0047] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds specifically to PD-1 is substantially free of antibodies that bind specifically to antigens other than PD-1). An isolated antibody that binds specifically to PD-1 may, however, have cross-reactivity to other antigens, such as PD-1 molecules from different species. Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.

[0048] The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, /.< ., antibody molecules whose primary sequences are essentially identical, and which exhibits a single binding specificity and affinity for a particular epitope. A monoclonal antibody is an example of an isolated antibody. Monoclonal antibodies can be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art.

[0049] A "human antibody" (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" and are used synonymously.

[0050] A "humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDRs of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one aspect of a humanized form of an antibody, some, most or all of the amino acids outside the CDRs have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDRs are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. A "humanized antibody" retains an antigenic specificity similar to that of the original antibody.

[0051] A "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

[0052] An "anti-antigen antibody" refers to an antibody that binds specifically to the antigen. For example, an anti-PD-1 antibody binds specifically to PD-1, an anti-PD-Ll antibody binds specifically to PD-L1, and an anti-CTLA-4 antibody binds specifically to CTLA-4.

[0053] An "antigen-binding portion" of an antibody (also called an "antigen-binding fragment") refers to one or more fragments of an antibody that retain the ability to bind specifically to the antigen bound by the whole antibody. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody, e.g., an anti- PD-1 antibody or an anti-PD-Ll antibody described herein, include (i) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC and CHI domains; (ii) a F(ab')2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR) and (vii) a combination of two or more isolated CDRs which can optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) roc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigenbinding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0054] A "cancer" refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth divide and grow results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0055] The term "immunotherapy" refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.

[0056] The term "checkpoint inhibitor" refers to any molecule that inhibits or reduces an activity of one or more member of a human immune checkpoint pathway. Examples of proteins within a human immune checkpoint pathway include but are not limited to programmed death- 1 (PD-1), programmed death ligand-1 (PD-L1), PD-L2, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and Lymphocyte Activation Gene-3 (LAG3). A checkpoint inhibitor can comprise a small molecule inhibitor, an antibody or an antigen-binding portion thereof, are a combination thereof. In some aspects, the checkpoint inhibitor comprises an antibody or an antigen-binding portion thereof that specifically binds PD-1 ("an anti-PD-1 antibody"). In some aspects, the checkpoint inhibitor comprises an antibody or an antigen-binding portion thereof that specifically binds PD-L1 ("an anti-PD-Ll antibody"). In some aspects, the checkpoint inhibitor comprises an antibody or an antigen-binding portion thereof that specifically binds LAG3 ("an anti-LAG3 antibody"). In some aspects, the checkpoint inhibitor comprises an antibody or an antigen-binding portion thereof that specifically binds CTLA-4 ("an anti-CTLA-4 antibody"). In some aspects, the checkpoint inhibitor comprises an anti-PD-1 antibody and an anti-LAG3 antibody. In some aspects, the checkpoint inhibitor comprises an anti-PD-Ll antibody and an anti-LAG3 antibody. In some aspects, the checkpoint inhibitor comprises an anti-PD-1 antibody and an anti-CTLA-4 antibody. In some aspects, the checkpoint inhibitor comprises an anti-PD-Ll antibody and an anti- CTLA-4 antibody. In some aspects, the checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-LAG3 antibody, and an anti-CTLA-4 antibody. In some aspects, the checkpoint inhibitor comprises an anti-PD-Ll antibody, an anti-LAG3 antibody, and an anti-CTLA-4 antibody.

[0057] "Programmed Death- 1 " (PD-1) refers to an immunoinhibitory receptor belonging to the

CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo, and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes human PD-1 (hPD- 1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank Accession No. U64863.

[0058] "Programmed Death Ligand- 1 " (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulate T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-Ll), variants, isoforms, and species homologs of hPD-Ll, and analogs having at least one common epitope with hPD-Ll. The complete hPD-Ll sequence can be found under GenBank Accession No. Q9NZQ7. The human PD-L1 protein is encoded by the human CD274 gene (NCBI Gene ID: 29126).

[0059] "Lymphocyte Activation Gene-3," "LAG3," "LAG-3," or "CD223," as used herein, refers to a type I transmembrane protein that is expressed on the cell surface of activated CD4+ and CD8+ T cells and subsets of NK and dendritic cells. LAG-3 protein is closely related to CD4, which is a co-receptor for T helper cell activation. Both molecules have four extracellular Ig-like domains and require binding to their ligand, major histocompatibility complex (MHC) class II, for their functional activity. LAG-3 protein is only expressed on the cell surface of activated T cells and its cleavage from the cell surface terminates LAG-3 signaling. LAG-3 can also be found as a soluble protein, which does not bind to MHC class II. LAG-3 also plays an important role in promoting regulatory T cell (Treg) activity and in negatively regulating T cell activation and proliferation. Both natural and induced Treg express increased LAG-3, which is required for their maximal suppressive function. The complete human LAG-3 amino acid sequence can be found under UniProtKB identification number Pl 8627. The human LAG-3 protein is encoded by the human LAG3 gene (NCBI Gene ID: 3902).

[0060] "Cytotoxic T-Lymphocyte Antigen-4" (CTLA-4) refers to an immunoinhibitory receptor belonging to the CD28 family. CTLA-4 is expressed exclusively on T cells in vivo, and binds to two ligands, CD80 and CD86 (also called B7-1 and B7-2, respectively). The term "CTLA- 4" as used herein includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, and analogs having at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found under GenBank Accession No. AAB59385. [0061] A "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats and guinea pigs. In preferred aspects, the subject is a human. The terms, "subject" and "patient" are used interchangeably herein.

[0062] The use of the term "flat dose" with regard to the methods and dosages of the disclosure means a dose that is administered to a patient without regard for the weight or body surface area (BSA) of the patient. The flat dose is therefore not provided as a mg/kg dose, but rather as an absolute amount of the agent (e.g., the anti-PD-1 antibody). For example, a 60 kg person and a 100 kg person would receive the same dose of an antibody (e.g., 240 mg of an anti-PD-1 antibody).

[0063] The use of the term "fixed dose" with regard to a method of the disclosure means that two or more different antibodies in a single composition (e.g., anti-PD-1 antibody and anti-CTLA- 4 antibody or an anti-PD-Ll antibody and an anti-CTLA-4 antibody) are present in the composition in particular (fixed) ratios with each other. In some aspects, the fixed dose is based on the weight (e.g., mg) of the antibodies. In certain aspects, the fixed dose is based on the concentration (e.g., mg/ml) of the antibodies. In some aspects, the ratio is at least about 1 : 1, about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1 : 10, about 1 : 15, about 1 :20, about 1 :30, about 1 :40, about 1:50, about 1 :60, about 1 :70, about 1 :80, about 1 :90, about 1 :100, about 1 : 120, about 1 : 140, about 1 : 160, about 1 : 180, about 1 :200, about 200: 1, about 180: 1, about 160: 1, about 140: 1, about 120: 1, about 100: 1, about 90: 1, about 80: 1, about 70: 1, about 60: 1, about 50: 1, about 40: 1, about 30: 1, about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8:1, about 7:1, about 6: 1, about 5: 1, about 4:1, about 3:1, or about 2: 1 mg first antibody (e.g., anti-PD-1 antibody or an anti-PD-Ll antibody) to mg second antibody (e.g, anti-CTLA-4 antibody). For example, the 3: 1 ratio of an anti-PD-1 antibody and an anti-CTLA-4 antibody can mean that a vial can contain about 240 mg of the anti-PD-1 antibody and 80 mg of the anti-CTLA-4 antibody or about 3 mg/ml of the anti-PD-1 antibody and 1 mg/ml of the anti-CTLA-4 antibody.

[0064] The term "weight-based dose" as referred to herein means that a dose that is administered to a patient is calculated based on the weight of the patient. For example, when a patient with 60 kg body weight requires 3 mg/kg of an anti-PD-1 antibody, one can calculate and use the appropriate amount of the anti-PD-1 antibody (i.e., 180 mg) for administration.

[0065] A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0066] By way of example, an "anti-cancer agent" promotes cancer regression in a subject. In preferred aspects, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administering an effective amount of the drug, alone or in combination with an anti -neoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a decrease in severity of at least one disease symptom, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. In addition, the terms "effective" and "effectiveness" with regard to a treatment includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the drug to promote cancer regression in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (adverse effects) resulting from administration of the drug.

[0067] By way of example for the treatment of tumors, a therapeutically effective amount of an anti-cancer agent preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. In other preferred aspects of the disclosure, tumor regression can be observed and continue for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days. Notwithstanding these ultimate measurements of therapeutic effectiveness, evaluation of immunotherapeutic drugs must also make allowance for immune-related response patterns.

[0068] An "immune response" is as understood in the art, and generally refers to a biological response within a vertebrate against foreign agents or abnormal, e.g., cancerous cells, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4⁺ cell, a CD8⁺ T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell.

[0069] An "immune-related response pattern" refers to a clinical response pattern often observed in cancer patients treated with immunotherapeutic agents that produce antitumor effects by inducing cancer-specific immune responses or by modifying native immune processes. This response pattern is characterized by a beneficial therapeutic effect that follows an initial increase in tumor burden or the appearance of new lesions, which in the evaluation of traditional chemotherapeutic agents would be classified as disease progression and would be synonymous with drug failure. Accordingly, proper evaluation of immunotherapeutic agents can require longterm monitoring of the effects of these agents on the target disease.

[0070] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process performed on, or administering an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease or enhancing overall survival. Treatment can be of a subject having a disease or a subject who does not have a disease (e.g., for prophylaxis).

[0071] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in overall survival (the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that patients diagnosed with the disease are still alive), or a prevention of impairment or disability due to the disease affliction. A therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or a "prophylactically effective dosage", which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, inhibits the development or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0072] By way of example, an anti-cancer agent is a drug that promotes cancer regression in a subject. In some aspects, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administering an effective amount of the drug, alone or in combination with an antineoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a decrease in severity of at least one disease symptom, an increase in frequency and duration of disease symptom-free periods, an increase in overall survival, a prevention of impairment or disability due to the disease affliction, or otherwise amelioration of disease symptoms in the patient. In addition, the terms "effective" and "effectiveness" with regard to a treatment includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the drug to promote cancer regression in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (adverse effects) resulting from administration of the drug.

[0073] By way of example for the treatment of tumors, a therapeutically effective amount or dosage of the drug inhibits cell growth or tumor growth by at least about 20%, by at least about 40%, by at least about 60%, or by at least about 80% relative to untreated subjects. In some aspects, a therapeutically effective amount or dosage of the drug completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be evaluated using an assay described herein. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth, such inhibition can be measured in vitro by assays known to the skilled practitioner. In some aspects described herein, tumor regression can be observed and continue for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

[0074] The term "biological sample" as used herein refers to biological material isolated from a subject. The biological sample can contain any biological material suitable for analysis, for example, by sequencing nucleic acids in the tumor (or circulating tumor cells) and identifying a genomic alteration in the sequenced nucleic acids. The biological sample can be any suitable biological tissue or fluid such as, for example, tumor tissue, blood, blood plasma, and serum. In one aspect, the sample is a tumor tissue biopsy, e.g., a formalin-fixed, paraffin-embedded (FFPE) tumor tissue or a fresh-frozen tumor tissue or the like. In another aspect, the biological sample is a liquid biopsy that, in some aspects, comprises one or more of blood, serum, plasma, circulating tumor cells, exoRNA, ctDNA, and cfDNA.

[0075] The terms "once about every week," "once about every two weeks," or any other similar dosing interval terms as used herein mean approximate numbers. "Once about every week" can include every seven days ± one day, z.e., every six days to every eight days. "Once about every two weeks" can include every fourteen days ± three days, /.< ., every eleven days to every seventeen days. Similar approximations apply, for example, to once about every three weeks, once about every four weeks, once about every five weeks, once about every six weeks, and once about every twelve weeks. In some aspects, a dosing interval of once about every six weeks or once about every twelve weeks means that the first dose can be administered any day in the first week, and then the next dose can be administered any day in the sixth or twelfth week, respectively. In other aspects, a dosing interval of once about every six weeks or once about every twelve weeks means that the first dose is administered on a particular day of the first week (e.g., Monday) and then the next dose is administered on the same day of the sixth or twelfth weeks (i.e., Monday), respectively.

[0076] As used herein, the term "Warburg phenotype" refers to a subject identified as having an enhanced Warburg effect. The Warburg effect refers to the increased rate of glucose uptake combined with lactate production in the presence of oxygen and fully functioning mitochondria in some tumors. Elevated glucose metabolism decreases the pH in the tumor microenvironment (TME) due to lactate secretion. This in turn activates tumor-associated fibroblasts, which remetabolize lactate and provide energy to tumor cells, in the form of glucose, as well as other energy sources such as fatty acids and proteins. The interaction between tumor cells and tumor-associated fibroblasts depletes sources of energy from the TME. This can "starve" other TME-associated cells and cause direct competition for energy between tumor and tumor infiltrating lymphocytes (TILs). As used herein, the term "low Warburg phenotype" refers to a subject identified as having a tumor that does not display an enhanced Warburg effect.

[0077] As used herein, the term "SIX1" refers to the human gene also known as sine oculis homeobox 1 (Ensemble Gene ID ENSG00000126778). As used herein, the term "SLC2A1/4" refers to the human gene also known as glucose transporter 1/4 or SLC2A1/4 (Ensemble Gene ID ENSG00000117394). As used herein, the term "HK2" refers to the human gene also known as hexokinase 2 (Ensemble Gene ID ENSG00000159399). As used herein, the term "PFKL" refers to the human gene also known as 6-phosphofructokinase, liver type (Ensemble Gene ID ENSG00000141959). As used herein, the term "ALDOA" refers to the human gene also known as aldolase A (Ensemble Gene ID ENSG00000149925). As used herein, the term "GAPDH" refers to the human gene also known as glyceraldehyde 3-phosphate dehydrogenase (Ensemble Gene ID ENSG00000111640). As used herein, the term "LDHA" refers to the human gene also known as lactate dehydrogenase A (Ensemble Gene ID ENSG00000134333). As used herein, the term "HBO1" refers to the human gene also known as histone acetyltransferase KAT7 (Ensemble Gene ID ENSG00000136504). As used herein, the term "PDHA1" refers to the human gene also known as pyruvate dehydrogenase 1 alpha (Ensemble Gene ID ENSG00000131828).

[0078] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

[0079] The terms "about" or "comprising essentially of' refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, ie., the limitations of the measurement system. For example, "about" or "comprising essentially of' can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" or "comprising essentially of' can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of' should be assumed to be within an acceptable error range for that particular value or composition.

[0080] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0081] Abbreviations used herein are defined throughout the present disclosure. A list of additional abbreviations is provided in Table 1.

[0082] Various aspects of the disclosure are described in further detail in the following subsections.

II. Methods of the Disclosure

[0083] Some aspects of the present disclosure are directed to methods of treating a tumor in a human subject, the method comprising administering an effective amount of a checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, and/or an anti- LAG-3 antibody) to the subject, wherein the subject is identified as suitable for the checkpoint inhibitor therapy by measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1; wherein the subject is identified as having a low expression profile of the panel of genes. The present disclosure further provides methods of treating a tumor in a human subject, the method comprising: (a) measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1; and (b) administering an effective amount of a checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, and/or an anti-LAG-3 antibody) to the subject, wherein the subject is identified as having a low expression profile of the panel of genes. Other aspects of the present disclosure are directed to methods of identifying a subject suitable for a checkpoint inhibitor therapy (e.g., comprising an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, and/or an anti-LAG-3 antibody), wherein the subject has been diagnosed with a cancer, the method comprising (i) measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1; and (ii) identifying a subject as suitable for an immune checkpoint inhibitor therapy if the subject is identified as having a low expression profile of the panel of genes. In some aspects, the human subject has been diagnosed with a non-small cell lung cancer (NSCLC)

[0084] In some aspects, decreased expression of one or more genes in the gene panel is indicative of a low Warburg phenotype. As such, some aspects of the present disclosure are directed to a method of treating a tumor in a human subject comprising administering an effective amount of a checkpoint inhibitor to the subject, wherein the subject is identified as having a low Warburg phenotype, wherein the low Warburg phenotype is determined by measuring the expression of a panel of genes in a tumor sample obtained from the subject, the panel of genes comprising at least one gene selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. Some aspects of the present disclosure are directed to a method of treating a tumor in a human subject in need thereof, comprising: (a) determining whether the subject has a low Warburg phenotype by measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least one gene selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1; and (b) administering an effective amount of a checkpoint inhibitor to the subject. Some aspects of the present disclosure are directed to a method of identifying a subject suitable for a checkpoint inhibitor therapy, wherein the subject has been diagnosed with non-small cell lung cancer (NSCLC), and the method comprises determining whether the subject has a low Warburg phenotype by measuring the expression of a panel of genes in a sample from the subject, the panel of genes comprising at least one selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HB01, and PDHA1. In some aspects, the method further comprises administering to the subject a checkpoint inhibitor if the subject is identified as having decreased expression of one or more gene in the gene panel, /.< ., a low Warburg phenotype. In some aspects, the panel of genes further comprises GAPDH.

[0085] In some aspects, the subject is afflicted with a NSCLC tumor. In some aspects, the tumor is a primary NSCLC tumor. In some aspects, the tumor is a tumor that has resulted from the metastasis of a primary NSCLC tumor, z.e., a "tumor derived from a NSCLC." In some aspects, the methods disclosed herein further comprise administering a checkpoint inhibitor (e.g., an anti- PD-1 antibody therapy, an anti-PD-Ll antibody therapy, an anti-CTLA-4 antibody, and/or an anti- LAG-3 antibody) to the subject.

[0086] Any biological sample comprising one or more tumor cell can be used in the methods disclosed herein. In some aspects, the sample is selected from a tumor biopsy, a blood sample, a serum sample, or any combination thereof. In certain aspects, the sample is a tumor biopsy collected from the subject prior to administration of the checkpoint inhibitor therapy. In particular aspects, the sample obtained from the subject is a formalin-fixed tumor biopsy. In some aspects, the sample obtained from the subject is a paraffin-embedded tumor biopsy. In some aspects, the sample obtained from the subject is a fresh-frozen tumor biopsy. In some aspects, the methods disclosed herein can be used to identify a subject suitable for a checkpoint inhibitor therapy using a smaller sample size that other methods. In some aspcts, a suitable tumor sample is a sample (e.g., an FFPE sample) comprising 6 or fewer slides, 5 or fewer slides, 4 or fewer slides, 3 or fewer slides, or 2 or fewer slides. In some embodiments, the tumor sample is a sample comprising 4 or fewer slides.

A. Gene Panels

[0087] In some aspects, the gene panel comprises SIX1 and at least one other gene. In some aspects, the gene panel comprises SLC2A1/4 and at least one other gene. In some aspects, the gene panel comprises HK2 and at least one other gene. In some aspects, the gene panel comprises PFKL and at least one other gene. In some aspects, the gene panel comprises ALDOA and at least one other gene. In some aspects, the gene panel comprises LDHA and at least one other gene. In some aspects, the gene panel comprises HB01 and at least one other gene. In some aspects, the gene panel comprises PDHA1 and at least one other gene.

[0088] In some aspects, the gene panel comprises SIX1 and GAPDH. In some aspects, the gene panel comprises SLC2A1/4 and GAPDH. In some aspects, the gene panel comprises HK2 and GAPDH. In some aspects, the gene panel comprises PFKL and GAPDH. In some aspects, the gene panel comprises ALDO A and GAPDH. In some aspects, the gene panel comprises LDHA and GAPDH. In some aspects, the gene panel comprises HBO1 and at least one other gene. In some aspects, the gene panel comprises PDHA1 and GAPDH.

[0089] In some aspects, the gene panel comprises at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1 and at least one gene selected from SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SLC2A1/4 and at least one gene selected from SIX1, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises HK2 and at least one gene selected from SIX1, SLC2A1/4, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises PFKL and at least one gene selected from SIX1, SLC2A1/4, HK2, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises ALDOA and at least one gene selected from SIX1, SLC2A1/4, HK2, PFKL, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises LDHA and at least one gene selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, HBO1, and PDHA1. In some aspects, the gene panel comprises HBO1 and at least one gene selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and PDHA1. In some aspects, the gene panel comprises PDHA1 and at least one gene selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and HBO1. In some aspects, the gene panel further comprises GAPDH.

[0090] In some aspects, the gene panel comprises at least three genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises GAPDH and at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1 and at least two genes selected from SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SLC2A1/4 and at least two genes selected from SIX1, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises HK2 and at least two genes selected from SIX1, SLC2A1/4, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises PFKL and at least two genes selected from SIX1, SLC2A1/4, HK2, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises ALDOA and at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises LDHA and at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, HBO1, and PDHAL In some aspects, the gene panel comprises HBO1 and at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and PDHAL In some aspects, the gene panel comprises PDHA1 and at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and HBOL

[0091] In some aspects, the gene panel comprises at least four genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises GAPDH and at least three genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises SIX1 and at least three genes selected from SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises SLC2A1/4 and at least three genes selected from SIX1, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises HK2 and at least three genes selected from SIX1, SLC2A1/4, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises PFKL and at least three genes selected from SIX1, SLC2A1/4, HK2, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises ALDOA and at least three genes selected from SIX1, SLC2A1/4, HK2, PFKL, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises LDHA and at least three genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, HBO1, and PDHAL In some aspects, the gene panel comprises HBO1 and at least three genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and PDHAL In some aspects, the gene panel comprises PDHA1 and at least three genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and HBOL

[0092] In some aspects, the gene panel comprises at least five genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises GAPDH and at least four genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises SIX1 and at least four genes selected from SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises SLC2A1/4 and at least four genes selected from SIX1, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises HK2 and at least four genes selected from SIX1, SLC2A1/4, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises PFKL and at least four genes selected from SIX1, SLC2A1/4, HK2, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises ALDOA and at least four genes selected from SIX1, SLC2A1/4, HK2, PFKL, LDHA, HB01, and PDHA1. In some aspects, the gene panel comprises LDHA and at least four genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, HBO1, and PDHA1. In some aspects, the gene panel comprises HBO1 and at least four genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and PDHA1. In some aspects, the gene panel comprises PDHA1 and at least four genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and HBOL

[0093] In some aspects, the gene panel comprises at least six genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises GAPDH and at least five genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1 and at least five genes selected from SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises SLC2A1/4 and at least five genes selected from SIX1, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises HK2 and at least five genes selected from SIX1, SLC2A1/4, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises PFKL and at least five genes selected from SIX1, SLC2A1/4, HK2, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises ALDOA and at least five genes selected from SIX1, SLC2A1/4, HK2, PFKL, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises LDHA and at least five genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, HBO1, and PDHAL In some aspects, the gene panel comprises HBO1 and at least five genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and PDHA1. In some aspects, the gene panel comprises PDHA1 and at least five genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and HBOL

[0094] In some aspects, the gene panel comprises at least seven genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises GAPDH and at least six genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1 and at least six genes selected from SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises SLC2A1/4 and at least six genes selected from SIX1, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises HK2 and at least six genes selected from SIX1, SLC2A1/4, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises PFKL and at least six genes selected from SIX1, SLC2A1/4, HK2, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises ALDOA and at least six genes selected from SIX1, SLC2A1/4, HK2, PFKL, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises LDHA and at least six genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, HBO1, and PDHA1. In some aspects, the gene panel comprises HBO1 and at least six genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and PDHA1. In some aspects, the gene panel comprises PDHA1 and at least six genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and HBOL

[0095] In some aspects, the gene panel comprises SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1, SLC2A1/4, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1, SLC2A1/4, HK2, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1, SLC2A1/4, HK2, PFKL, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1, SLC2A1/4, HK2, PFKL, ALDOA, HBO1, and PDHA1. In some aspects, the gene panel comprises SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and PDHA1. In some aspects, the gene panel comprises SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and HBOL [0096] In some aspects, the gene panel comprises GAPDH, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises GAPDH, SIX1, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHA1. In some aspects, the gene panel comprises GAPDH, SIX1, SLC2A1/4, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises GAPDH, SIX1, SLC2A1/4, HK2, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises GAPDH, SIX1, SLC2A1/4, HK2, PFKL, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises GAPDH, SIX1, SLC2A1/4, HK2, PFKL, ALDOA, HBO1, and PDHAL In some aspects, the gene panel comprises GAPDH, SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and PDHA1. In some aspects, the gene panel comprises GAPDH, SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, and HBOL

[0097] In some aspects, the gene panel comprises SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel comprises GAPDH, SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL

[0098] In some aspects, the gene panel consists of SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL In some aspects, the gene panel consists of GAPDH, SIX1, SLC2A1/4, HK2, PFKL, ALDOA, LDHA, HBO1, and PDHAL

[0099] In some aspects, the panel of genes comprises at least about 5 additional genes, at least about 10 additional genes, at least about 15 additional genes, at least about 20 additional genes, at least about 25 additional genes, at least about 30 additional genes, at least about 35 additional genes, at least about 40 additional genes, at least about 45 additional genes, at least about 50 additional genes, at least about 55 additional genes, at least about 60 additional genes, at least about 65 additional genes, at least about 70 additional genes, at least about 75 additional genes, at least about 80 additional genes, at least about 85 additional genes, at least about 90 additional genes, at least about 95 additional genes, at least about 100 additional genes, at least about 110 additional genes, at least about 120 additional genes, at least about 130 additional genes, at least about 140 additional genes, at least about 150 additional genes, at least about 160 additional genes, at least about 170 additional genes, at least about 180 additional genes, at least about 190 additional genes, or at least about 200 additional genes.

[0100] In some aspects, the panel of genes comprises at least one housekeeping gene. As used herein, a housekeeping gene is a gene that is expressed at a nearly constant level across various cell types. As such, the expression of a housekeeping gene can be relative to the number of cells in a sample, acting as a means of normalizing the expression of other variable genes. In some aspects, the panel of genes comprises at least 2 housekeeping genes, at least 3 housekeeping genes, at least housekeeping genes, at least 4 housekeeping genes, at least 5 housekeeping genes, at least 6 housekeeping genes, at least 7 housekeeping genes, at least 8 housekeeping genes, at least 9 housekeeping genes, at least 10 housekeeping genes, at least 11 housekeeping genes, at least 12 housekeeping genes, at least 13 housekeeping genes, at least 14 housekeeping genes, at least 15 housekeeping genes, at least 16 housekeeping genes, at least 17 housekeeping genes, at least 18 housekeeping genes, at least 19 housekeeping genes, at least 20 housekeeping genes, at least 21 housekeeping genes, at least 22 housekeeping genes, at least 23 housekeeping genes, at least 24 housekeeping genes, at least 25 housekeeping genes, at least 26 housekeeping genes, at least housekeeping genes, at least 27 housekeeping genes, at least 28 housekeeping genes, at least 29 housekeeping genes, at least 30 housekeeping genes, at least 35 housekeeping genes, at least 40 housekeeping genes, at least 45 housekeeping genes, at least 50 housekeeping genes, at least 55 housekeeping genes, at least 60 housekeeping genes, at least 65 housekeeping genes, at least 70 housekeeping genes, at least 75 housekeeping genes, at least 80 housekeeping genes, at least 85 housekeeping genes, at least 85 housekeeping genes, at least 90 housekeeping genes, at least 95 housekeeping genes, or at least 100 housekeeping genes. In some aspects, the panel of genes comprises at least 2 housekeeping genes. In some aspects, the panel of genes comprises at least 3 housekeeping genes. In some aspects, the panel of genes comprises at least 4 housekeeping genes. In some aspects, the panel of genes comprises at least 5 housekeeping genes. In some aspects, the panel of genes comprises at least 6 housekeeping genes. In some aspects, the panel of genes comprises at least 7 housekeeping genes. In some aspects, the panel of genes comprises at least 8 housekeeping genes. In some aspects, the panel of genes comprises at least 9 housekeeping genes. In some aspects, the panel of genes comprises at least 10 housekeeping genes. In some aspects, the panel of genes comprises at least 11 housekeeping genes. In some aspects, the panel of genes comprises at least 12 housekeeping genes. In some aspects, the panel of genes comprises at least 13 housekeeping genes. In some aspects, the panel of genes comprises at least 14 housekeeping genes. In some aspects, the panel of genes comprises at least 15 housekeeping genes.

[0101] Any housekeeping genes known in the art can be used in the panel of genes disclosed herein. In some aspects, the housekeeping genes are selected from the group consisting of ACTB, ATP5F1, DDX5, EEF 1G, NCL, OAZ1, PPIA, RPI.38, RPL6, RPS7, SLC25A3, SOD1, YWHAZ, and any combination thereof

[0102] In some aspects, the panel of genes comprises at least one control gene. In some aspects, the panel of genes comprises at least 2 control genes, at least 3 control genes, at least control genes, at least 4 control genes, at least 5 control genes, at least 6 control genes, at least 7 control genes, at least 8 control genes, at least 9 control genes, at least 10 control genes, at least 11 control genes, at least 12 control genes, at least 13 control genes, at least 14 control genes, at least 15 control genes, at least 16 control genes, at least 17 control genes, at least 18 control genes, at least 19 control genes, at least 20 control genes, at least 21 control genes, at least 22 control genes, at least 23 control genes, at least 24 control genes, at least 25 control genes, at least 26 control genes, at least control genes, at least 27 control genes, at least 28 control genes, at least 29 control genes, at least 30 control genes, at least 35 control genes, at least 40 control genes, at least 45 control genes, at least 50 control genes, at least 55 control genes, at least 60 control genes, at least 65 control genes, at least 70 control genes, at least 75 control genes, at least 80 control genes, at least 85 control genes, at least 85 control genes, at least 90 control genes, at least 95 control genes, or at least 100 control genes. In some aspects, the panel of genes comprises at least 2 control genes. In some aspects, the panel of genes comprises at least 3 control genes. In some aspects, the panel of genes comprises at least 4 control genes. In some aspects, the panel of genes comprises at least 5 control genes. In some aspects, the panel of genes comprises at least 6 control genes. In some aspects, the panel of genes comprises at least 7 control genes. In some aspects, the panel of genes comprises at least 8 control genes. In some aspects, the panel of genes comprises at least 9 control genes. In some aspects, the panel of genes comprises at least 10 control genes. In some aspects, the panel of genes comprises at least 11 control genes. In some aspects, the panel of genes comprises at least 12 control genes. In some aspects, the panel of genes comprises at least 13 control genes. In some aspects, the panel of genes comprises at least 14 control genes. In some aspects, the panel of genes comprises at least 15 control genes. In some aspects, the panel of genes comprises at least 16 control genes. In some aspects, the panel of genes comprises at least 17 control genes. In some aspects, the panel of genes comprises at least 18 control genes. In some aspects, the panel of genes comprises at least 19 control genes. In some aspects, the panel of genes comprises at least 20 control genes. In some aspects, the control genes are selected from the group consisting of ANTI, ANT2, ANT3, ANT4, PCL-1, PCL-10, PCL-2, PCL-3, PCL-4, PCL-5, PCL-6, PCL-7, PCL-8, PCL- 9, POS1, POS2, POSS, POS4, and any combination thereof.

[0103] The methods disclosed herein generally provide a quicker means of identifying patients suitable for a therapy comprising a checkpoint inhibitor, as compared to conventional means, e.g., analysis of tumor mutational burden (TMB).

B. Expression Profiles

[0104] Some aspects of the present disclosure are directed to methods of treating a subject in need thereof, comprising determining the expression profile of a panel of genes in a tumor sample obtained from the subject. The present disclosure provides that a subject having an expression profile characterized by decreased tumor expression, i.e., a low signature score, of one or more genes in the gene panel is more likely to be responsive to a therapy comprising a checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, and/or an anti-LAG3 antibody).

[0105] In some aspects, the expression profile is expressed as a gene signature score. A signature score, as used herein, is a measurement of the composite expression of the genes in the gene panel in a tumor sample from the subject, wherein the profile is compared to the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, the average composite signature score is determined by measuring the composite expression of the panel of genes in tumor samples obtained from the population of subjects.

[0106] Any method known in the art for measuring the expression of a particular gene or a panel of genes can be used in the methods of the present disclosure. In some aspects, the expression of one or more of the genes in the panel of genes is determined by detecting the presence of mRNA transcribed from the gene, the presence of a protein encoded by the gene, or both.

[0107] In some aspects, the expression of one or more of the genes is determined by measuring the level of gene mRNA in a tumor sample obtained from the subject. Any method can be used to measure the level of the gene mRNA. In some aspects, the gene mRNA is measured using reverse transcriptase PCR. In some aspects, the gene mRNA is measured using RNA in situ hybridization. [0108] In some aspects, the expression of one or more of the genes is determined by measuring the level of protein expressed from the gene in a tumor sample obtained from the subject. Any method can be used to measure the level of the protein. In some aspects, the protein is measured using an immunohistochemistry (IHC) assay. In certain aspects, the IHC is an automated IHC.

[0109] In some aspects, the expression of the genes in the panel of genes is determined by exome RNA-seq analysis.

[0110] In some aspects, the expression of one or more of the genes of the gene panel is normalized relative to the expression of one or more housekeeping genes. In some aspects, the one or more housekeeping genes comprises any housekeeping gene disclosed herein or any combination thereof.

[0111] In some aspects, raw gene expression values are normalized following standard gene expression profiling (GEP) protocols. In these aspects, gene signature scores can be calculated as the median or average of the log2 -transformed normalized and scaled expression values across all of the target genes in the profile, and presented on a linear scale. In certain aspects, scores have positive or negative values, depending on whether gene expression is up- or down-regulated under a particular condition.

[0112] In certain aspects, a low signature score is characterized by a score that is lower than a reference score. In some aspects, the signature score is an average signature. In some aspects, the average signature score is determined by measuring the expression of the genes present in the panel of genes in tumor samples obtained from a population of subjects, and calculating the average for the population of subjects. In some aspects, each member of the population of subjects is afflicted with the same tumor as the subject being administered the checkpoint inhibitor, e.g., the anti-PD- 1 antibody, the anti-PD-Ll antibody, the anti-CTLA-4 antibody, the anti -LAG-3 antibody, or any combination thereof.

[0113] In some aspects, a low expression profile, e.g., a low signature score, is characterized by composite gene expression of the panel of genes that is lower than the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% lower than the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low gene signature is characterized by composite gene expression that is in the lowest tertile. In some aspects, a low gene signature is characterized by composite gene expression that is below a low tertile cutoff. In some aspects, a high gene signature is characterized by composite gene expression that is in the top tertile. In some aspects, a low gene signature is characterized by composite gene expression that is below a high tertile cutoff.

[0114] In some aspects, a low expression profile is characterized by composite gene expression that is less than about 95% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 90% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression is that less than about 85% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 80% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 75% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 70% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 65% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 60% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 55% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 50% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 45% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 40% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 35% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 30% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 25% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 20% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 15% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 10% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC. In some aspects, a low expression profile is characterized by composite gene expression that is less than about 5% that of the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with the tumor, e.g., NSCLC.

C. Checkpoint Inhibitors

[0115] The present disclosure is directed to methods for treating a human subject afflicted with a NSCLC comprising administering to the subject a checkpoint inhibitor. Any checkpoint inhibitor art can be used in the methods disclosed herein. In some aspects, the checkpoint inhibitor is any reagent that blocks, inhibits, or reduces the activity of one or more immune checkpoint protein. In some aspects, the checkpoint protein is selected from the group selected from the group consisting of PD-1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, 0X40, ICOS, CD137, KIR, IL-2, CD96, VISTA, B7-H4, CXCR4, CD27, GITR, and any combination thereof. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of PD-1. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of CTLA-4. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of LAG3. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of TIGIT. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of TIM3. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of NKG2a. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of 0X40. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of ICOS. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of CD137. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of KIR. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of TGFp. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of IL- 10. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of IL-8. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of IL-2. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of CD96. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of VISTA. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of B7-H4. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of Fas ligand. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of CXCR4. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of mesothelin. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of CD27. In some aspects, the checkpoint inhibitor blocks, inhibits, or reduces the activity of GITR. [0116] Any inhibitor can be used in the methods disclosed herein. In some aspects, the inhibitor is a small molecule. In some aspects, the inhibitor is a protein. In some aspects, the inhibitor is an antibody or an antigen-binding portion thereof. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds PD-1. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds CTLA-4. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds LAG3. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds TIGIT. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds TIM3. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds NKG2a. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds 0X40. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds ICOS. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds CD137. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds KIR. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds TGFp. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds IL- 10. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds IL-8. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds IL-2. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds CD96. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds VISTA. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds B7-H4. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds Fas ligand. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds CXCR4. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds mesothelin. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds CD27. In some aspects, the checkpoint inhibitor is an antibody or antigen-binding portion thereof that specifically binds GITR.

[0117] In some aspects, the subject is administered a monotherapy, e.g., an anti-PD-1 monotherapy, e.g., wherein the subject is not administered one or more additional anti-cancer agent. In some aspects, the subject is administered a combination therapy, e.g., wherein the subject is administered a first checkpoint inhibitor, e.g., an anti-PD-1 antibody, and one or more additional anti-cancer agents. In certain aspects, the subject is administered a combination therapy comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody. In certain aspects, the subject is administered a combination therapy comprising an anti-PD-1 antibody and an anti -LAG-3 antibody.

[0118] In other aspects of the present disclosure, an anti-PD-Ll antibody is substituted for the anti-PD-1 antibody. In certain aspects, the methods comprise administering an anti-PD-Ll antibody to a subject. In some aspects, the subject is administered an anti-PD-Ll monotherapy. In some aspects, the subject is administered a combination therapy comprising an anti-PD-Ll antibody and a second anti-cancer agent, e.g., an anti-CTLA-4 antibody. In some aspects, the subject is administered a combination therapy comprising an anti-PD-Ll antibody and a second anti-cancer agent, e.g., an anti -LAG-3 antibody.

[0119] In some aspects, the antibody is a chimeric, humanized or human monoclonal antibody or a portion thereof.

/. Anti-PD-1 Antibodies Useful for the Disclosure

[0120] Any Anti-PD-1 antibody or an antigen-binding portion thereof can be used in the presently described compositions and methods. Various human monoclonal antibodies that bind specifically to PD-1 with high affinity have been disclosed in U.S. Patent No. 8,008,449. Anti-PD- 1 human antibodies disclosed in U.S. Patent No. 8,008,449 have been demonstrated to exhibit one or more of the following characteristics: (a) bind to human PD-1 with a KD of 1 x 10'⁷ M or less, as determined by surface plasmon resonance using a Biacore biosensor system; (b) do not substantially bind to human CD28, CTLA-4 or ICOS; (c) increase T-cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay; (d) increase interferon-y production in an MLR assay; (e) increase IL-2 secretion in an MLR assay; (f) bind to human PD-1 and cynomolgus monkey PD- 1; (g) inhibit the binding of PD-L1 and/or PD-L2 to PD-1; (h) stimulate antigen-specific memory responses; (i) stimulate antibody responses; and (j) inhibit tumor cell growth in vivo. Anti-PD-1 antibodies usable in the present disclosure include monoclonal antibodies that bind specifically to human PD-1 and exhibit at least one, in some aspects, at least five, of the preceding characteristics. [0121] Other anti-PD-1 monoclonal antibodies have been described in, for example, U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757 and 8,354,509, US Publication No. 2016/0272708, and PCT Publication Nos. WO 2012/145493, WO 2008/156712, WO 2015/112900, WO 2012/145493, WO 2015/112800, WO 2014/206107, WO 2015/35606, WO 2015/085847, WO 2014/179664, WO 2017/020291, WO 2017/020858, WO 2016/197367, WO 2017/024515, WO 2017/025051, WO 2017/123557, WO 2016/106159, WO 2014/194302, WO 2017/040790, WO 2017/133540, WO 2017/132827, WO 2017/024465, WO 2017/025016, WO 2017/106061, WO 2017/19846, WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540 each of which is incorporated by reference in its entirety.

[0122] In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008/156712), PDR001 (Novartis; see WO 2015/112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012/145493), cemiplimab (Regeneron; also known as REGN-2810; see WO 2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; also known as toripalimab; see Si- Yang Liu et al., J. Hematol. Oncol. 70: 136 (2017)), BGB-A317 (Beigene; also known as Tislelizumab; see WO 2015/35606 and US 2015/0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015/085847; Si-Yang Liu et al., J. Hematol. Oncol. 70: 136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014/179664), GLS-010 (Wuxi/Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 70: 136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014/194302), AGEN2034 (Agenus; see WO 2017/040790), MGA012 (Macrogenics, see WO 2017/19846), BCD-100 (Biocad; Kaplon et al., mAbs 70(2 183-203 (2018), and IBI308 (Innovent; see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540).

[0123] In one aspect, the anti-PD-1 antibody comprises nivolumab. Nivolumab is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the down-regulation of antitumor T-cell functions (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9/846-56).

[0124] In another aspect, the anti-PD-1 antibody comprises pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody directed against human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0125] Anti-PD-1 antibodies usable in the disclosed compositions and methods also include isolated antibodies that bind specifically to human PD-1 and cross-compete for binding to human PD-1 with any anti-PD-1 antibody disclosed herein, e.g., nivolumab (see, e.g., U.S. Patent No. 8,008,449 and 8,779,105; WO 2013/173223). In some aspects, the anti-PD-1 antibody binds the same epitope as any of the anti-PD-1 antibodies described herein, e.g., nivolumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other crosscompeting antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g., nivolumab, by virtue of their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

[0126] In certain aspects, the antibodies that cross-compete for binding to human PD-1 with, or bind to the same epitope region of human PD-1 antibody, nivolumab, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art. [0127] Anti-PD-1 antibodies usable in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0128] Anti-PD-1 antibodies suitable for use in the disclosed compositions and methods are antibodies that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and or PD-L2, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-1 "antibody" includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits the functional properties similar to those of whole antibodies in inhibiting ligand binding and up-regulating the immune system. In certain aspects, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1.

[0129] In some aspects, the anti-PD-1 antibody is administered at a dose ranging from 0.1 mg/kg to 20.0 mg/kg body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks, e.g., 0.1 mg/kg to 10.0 mg/kg body weight once every 2, 3, or 4 weeks. In other aspects, the anti-PD-1 antibody is administered at a dose of about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or 10 mg/kg body weight once every 2 weeks. In other aspects, the anti-PD-1 antibody is administered at a dose of about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or 10 mg/kg body weight once every 3 weeks. In one aspect, the anti-PD-1 antibody is administered at a dose of about 5 mg/kg body weight about once every 3 weeks. In another aspect, the anti-PD-1 antibody, e.g., nivolumab, is administered at a dose of about 3 mg/kg body weight about once every 2 weeks. In other aspects, the anti-PD-1 antibody, e.g., pembrolizumab, is administered at a dose of about 2 mg/kg body weight about once every 3 weeks.

[0130] The anti-PD-1 antibody useful for the present disclosure can be administered as a flat dose. In some aspects, the anti-PD-1 antibody is administered at a flat dose of from about 100 to about 1000 mg, from about 100 mg to about 900 mg, from about 100 mg to about 800 mg, from about 100 mg to about 700 mg, from about 100 mg to about 600 mg, from about 100 mg to about 500 mg, from about 200 mg to about 1000 mg, from about 200 mg to about 900 mg, from about 200 mg to about 800 mg, from about 200 mg to about 700 mg, from about 200 mg to about 600 mg, from about 200 mg to about 500 mg, from about 200 mg to about 480 mg, or from about 240 mg to about 480 mg, In one aspect, the anti-PD-1 antibody is administered as a flat dose of at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 320 mg, at least about 340 mg, at least about 360 mg, at least about 380 mg, at least about 400 mg, at least about 420 mg, at least about 440 mg, at least about 460 mg, at least about 480 mg, at least about 500 mg, at least about 520 mg, at least about 540 mg, at least about 550 mg, at least about 560 mg, at least about 580 mg, at least about 600 mg, at least about 620 mg, at least about 640 mg, at least about 660 mg, at least about 680 mg, at least about 700 mg, or at least about 720 mg at a dosing interval of about I, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In another aspects, the anti-PD-I antibody is administered as a flat dose of about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 200 mg to about 500 mg, at a dosing interval of about 1, 2, 3, or 4 weeks.

[0131] In some aspects, the anti-PD-1 antibody is administered as a flat dose of about 200 mg at about once every 3 weeks. In other aspects, the anti-PD-1 antibody is administered as a flat dose of about 200 mg at about once every 2 weeks. In other aspects, the anti-PD-1 antibody is administered as a flat dose of about 240 mg at about once every 2 weeks. In certain aspects, the anti-PD-1 antibody is administered as a flat dose of about 480 mg at about once every 4 weeks.

[0132] In some aspects, nivolumab is administered at a flat dose of about 240 mg once about every 2 weeks. In some aspects, nivolumab is administered at a flat dose of about 240 mg once about every 3 weeks. In some aspects, nivolumab is administered at a flat dose of about 360 mg once about every 3 weeks. In some aspects, nivolumab is administered at a flat dose of about 480 mg once about every 4 weeks. [0133] In some aspects, pembrolizumab is administered at a flat dose of about 200 mg once about every 2 weeks. In some aspects, pembrolizumab is administered at a flat dose of about 200 mg once about every 3 weeks. In some aspects, pembrolizumab is administered at a flat dose of about 400 mg once about every 4 weeks.

2. Anti-PD-Ll Antibodies Useful for the Disclosure

[0134] In certain aspects, an anti-PD-Ll antibody is substituted for the anti-PD-1 antibody in any of the methods disclosed herein. Any anti-PD-Ll antibody can be used in the compositions and methods of the present disclosure. Examples of anti-PD-Ll antibodies useful in the compositions and methods of the present disclosure include the antibodies disclosed in US Patent No. 9,580,507. Anti-PD-Ll human monoclonal antibodies disclosed in U.S. Patent No. 9,580,507 have been demonstrated to exhibit one or more of the following characteristics: (a) bind to human PD-L1 with a KD of 1 x 10'⁷ M or less, as determined by surface plasmon resonance using a Biacore biosensor system; (b) increase T-cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay; (c) increase interferon-y production in an MLR assay; (d) increase IL-2 secretion in an MLR assay; (e) stimulate antibody responses; and (f) reverse the effect of T regulatory cells on T cell effector cells and/or dendritic cells. Anti-PD-Ll antibodies usable in the present disclosure include monoclonal antibodies that bind specifically to human PD-L1 and exhibit at least one, in some aspects, at least five, of the preceding characteristics.

[0135] In certain aspects, the anti-PD-Ll antibody is selected from the group consisting of BMS-936559 (also known as 12A4, MDX-1105; see, e.g, U.S. Patent No. 7,943,743 and WO 2013/173223), atezolizumab (Roche; also known as TECENTRIQ®; MPDL3280A, RG7446; see US 8,217,149; see, also, Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI™, MEDL4736; see WO 2011/066389), avelumab (Pfizer; also known as BAVENCIO®, MSB-0010718C; see WO 2013/079174), STI-1014 (Sorrento; see WO2013/181634), CX-072 (Cytomx; see WO2016/149201 ), KN035 (3D Med/Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017/034916), BGB-A333 (BeiGene; see Desai et al., JCO 36 (15suppl)A S3 3 (2018)), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR:Abstract 4606 (Apr 2016)).

[0136] In certain aspects, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgGl monoclonal anti-PD-Ll antibody.

[0137] In certain aspects, the PD-L1 antibody is durvalumab (IMFINZI™). Durvalumab is a human IgGl kappa monoclonal anti-PD-Ll antibody. [0138] In certain aspects, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgGl lambda monoclonal anti-PD-Ll antibody.

[0139] Anti-PD-Ll antibodies usable in the disclosed compositions and methods also include isolated antibodies that bind specifically to human PD-L1 and cross-compete for binding to human PD-L1 with any anti-PD-Ll antibody disclosed herein, e.g., atezolizumab, durvalumab, and/or avelumab. In some aspects, the anti-PD-Ll antibody binds the same epitope as any of the anti-PD- Ll antibodies described herein, e.g., atezolizumab, durvalumab, and/or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g., atezolizumab and/or avelumab, by virtue of their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and/or avelumab in standard PD-L1 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

[0140] In certain aspects, the antibodies that cross-compete for binding to human PD-L1 with, or bind to the same epitope region of human PD-L1 antibody as, atezolizumab, durvalumab, and/or avelumab, are monoclonal antibodies. For administration to human subjects, these crosscompeting antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0141] Anti-PD-Ll antibodies usable in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0142] Anti-PD-Ll antibodies suitable for use in the disclosed compositions and methods are antibodies that bind to PD-L1 with high specificity and affinity, block the binding of PD-1, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-Ll "antibody" includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits the functional properties similar to those of whole antibodies in inhibiting receptor binding and up-regulating the immune system. In certain aspects, the anti-PD-Ll antibody or antigen-binding portion thereof cross-competes with atezolizumab, durvalumab, and/or avelumab for binding to human PD-L1. [0143] The anti-PD-Ll antibody useful for the present disclosure can be any PD-L1 antibody that specifically binds to PD-L1, e.g., antibodies that cross-compete with durvalumab, avelumab, or atezolizumab for binding to human PD-1, e.g., an antibody that binds to the same epitope as durvalumab, avelumab, or atezolizumab. In a particular aspect, the anti-PD-Ll antibody is durvalumab. In other aspects, the anti-PD-Ll antibody is avelumab. In some aspects, the anti-PD- Ll antibody is atezolizumab.

[0144] In some aspects, the anti-PD-Ll antibody is administered at a dose ranging from about 0.1 mg/kg to about 20.0 mg/kg body weight, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, or about 20 mg/kg, about once every 2, 3, 4, 5, 6, 7, or 8 weeks.

[0145] In some aspects, the anti-PD-Ll antibody is administered at a dose of about 15 mg/kg body weight at about once every 3 weeks. In other aspects, the anti-PD-Ll antibody is administered at a dose of about 10 mg/kg body weight at about once every 2 weeks.

[0146] In other aspects, the anti-PD-Ll antibody useful for the present disclosure is a flat dose. In some aspects, the anti-PD-Ll antibody is administered as a flat dose of from about 200 mg to about 1600 mg, about 200 mg to about 1500 mg, about 200 mg to about 1400 mg, about 200 mg to about 1300 mg, about 200 mg to about 1200 mg, about 200 mg to about 1100 mg, about 200 mg to about 1000 mg, about 200 mg to about 900 mg, about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 700 mg to about 1300 mg, about 800 mg to about 1200 mg, about 700 mg to about 900 mg, or about 1100 mg to about 1300 mg. In some aspects, the anti-PD-Ll antibody is administered as a flat dose of at least about 240 mg, at least about 300 mg, at least about 320 mg, at least about 400 mg, at least about 480 mg, at least about 500 mg, at least about 560 mg, at least about 600 mg, at least about 640 mg, at least about 700 mg, at least 720 mg, at least about 800 mg, at least about 840 mg, at least about 880 mg, at least about 900 mg, at least 960 mg, at least about 1000 mg, at least about 1040 mg, at least about 1100 mg, at least about 1120 mg, at least about 1200 mg, at least about 1280 mg, at least about 1300 mg, at least about 1360 mg, or at least about 1400 mg, at a dosing interval of about 1, 2, 3, or 4 weeks. In some aspects, the anti-PD-Ll antibody is administered as a flat dose of about 1200 mg at about once every 3 weeks. In other aspects, the anti-PD-Ll antibody is administered as a flat dose of about 800 mg at about once every 2 weeks. In other aspects, the anti-PD-Ll antibody is administered as a flat dose of about 840 mg at about once every 2 weeks. [0147] In some aspects, atezolizumab is administered as a flat dose of about 1200 mg once about every 3 weeks. In some aspects, atezolizumab is administered as a flat dose of about 800 mg once about every 2 weeks. In some aspects, atezolizumab is administered as a flat dose of about 840 mg once about every 2 weeks.

[0148] In some aspects, avelumab is administered as a flat dose of about 800 mg once about every 2 weeks.

[0149] In some aspects, durvalumab is administered at a dose of about 10 mg/kg once about every 2 weeks. In some aspects, durvalumab is administered as a flat dose of about 800 mg/kg once about every 2 weeks. In some aspects, durvalumab is administered as a flat dose of about 1200 mg/kg once about every 3 weeks.

3. Anti-CTLA-4 Antibodies

[0150] Any anti-CTLA-4 antibody can be used in the compositions and methods of the present disclosure. Anti-CTLA-4 antibodies of the instant disclosure bind to human CTLA-4 so as to disrupt the interaction of CTLA-4 with a human B7 receptor. Because the interaction of CTLA-4 with B7 transduces a signal leading to inactivation of T-cells bearing the CTLA-4 receptor, disruption of the interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhancing or prolonging an immune response.

[0151] Human monoclonal antibodies that bind specifically to CTLA-4 with high affinity have been disclosed in U.S. Patent Nos. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described in, for example, U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121 and International Publication Nos. WO 2012/122444, WO 2007/113648, WO 2016/196237, and WO 2000/037504, each of which is incorporated by reference herein in its entirety. The anti- CTLA-4 human monoclonal antibodies disclosed in U.S. Patent No. Nos. 6,984,720 have been demonstrated to exhibit one or more of the following characteristics: (a) binds specifically to human CTLA-4 with a binding affinity reflected by an equilibrium association constant (K_a) of at least about 10⁷ M'¹, or about 10⁹ M'¹, or about 10¹⁰ M'¹ to 10¹¹ M'¹ or higher, as determined by Biacore analysis; (b) a kinetic association constant (k_a) of at least about 10³, about 10⁴, or about 10⁵ m'¹ s'¹; (c) a kinetic disassociation constant (k /) of at least about 10³, about 10⁴, or about 10⁵ m'¹ s'¹; and (d) inhibits the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies useful for the present disclosure include monoclonal antibodies that bind specifically to human CTLA-4 and exhibit at least one, at least two, or at least three of the preceding characteristics. [0152] In certain aspects, the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab (also known as YERVOY®, MDX-010, 10D1; see U.S. Patent No. 6,984,720), MK- 1308 (Merck), AGEN-1884 (Agenus Inc.; see WO 2016/196237), and tremelimumab (AstraZeneca; also known as ticilimumab, CP-675,206; see WO 2000/037504 and Ribas, Update Cancer Ther. 2(3): 133-39 (2007)). In particular aspects, the anti-CTLA-4 antibody is ipilimumab. [0153] In particular aspects, the CTLA-4 antibody is ipilimumab for use in the compositions and methods disclosed herein. Ipilimumab is a fully human, IgGl monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligands, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.

[0154] In particular aspects, the CTLA-4 antibody is tremelimumab.

[0155] In particular aspects, the CTLA-4 antibody is MK-1308.

[0156] In particular aspects, the CTLA-4 antibody is AGEN-1884.

[0157] In some aspects, the antibody that binds CTLA-4 is non-fucosylated. In some aspects, the antibody that binds CTLA-4 is a non-fucosylated antibody that comprises the 6 CDRs of ipilimumab. In some aspects, the antibody that binds CTLA-4 comprises the heavy chain variable region of ipilimumab and the light chain variable region of ipilimumab. In some aspects, the antibody that binds CTLA-4 is a non-fucosylated variant of ipilimumab. In some aspects, the antibody that binds CTLA-4 is a non-fucosylated anti-CTLA-4 antibody disclosed in International Publication No. WO 14/089113, which is incorporated by reference herein in its entirety.

[0158] In some aspects, the antibody is an activatable antibody disclosed in International Publication No. WO 18/085555 that when activated binds human CTLA-4. In some aspects, the activatable antibody comprises (a) a variable heavy domain and (b) a light chain, wherein the light chain comprises (i) a masking moiety (MM), (ii) a cleavable moiety (CM), and (iii) a variable light domain (VL). In some aspects, the light chain has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-VL. In some aspects, the masking moiety inhibits binding of the antibody to CTLA-4, and the cleavable moiety is cleaved at the site of a tumor, removing the masking moiety, and activating the anti-CTLA-4 antibody. In some aspects, the antibody that binds CTLA-4 comprises (a) a variable heavy domain, comprising the variable heavy chain CDRs of ipilimumab; and (b) a light chain, wherein the light chain comprises (i) a masking moiety, (ii) a cleavable moiety, and (iii) a variable light domain, comprising the variable light chain CDRs of ipilimumab. In some aspects, the antibody that binds CTLA-4 comprises (a) the variable heavy domain of ipilimumab; and (b) a light chain, wherein the light chain comprises (i) a masking moiety, (ii) a cleavable moiety, and (iii) the variable light domain of ipilimumab. In some embodiments, the light chain has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-VL. In some aspects, the antibody that binds CTLA-4 comprises any antibody disclosed in International Publication No. WO 18/085555, which is incorporated by reference herein in its entirety.

[0159] In some aspects, the antibody that binds CTLA-4 is modified to enhance its activity in an acidic environment and/or to enhance its affinity for CTLA-4 in an acidic environment, as disclosed in International Publication No. WO 2020/214748. In some aspects, the antibody that binds CTLA-4 is modified to enhance its activity in an acidic environment. In some aspects, the antibody that binds CTLA-4 is modified to enhance its affinity for CTLA-4 in an acidic environment. In some aspects, the modified anti-CTLA-4 antibody comprises one or more mutations in the variable domain that enhance target binding at low/acidic pH (e.g., pH 6.0) compared with neutral pH (e.g., pH 7.4). In some aspects, the modified anti-CTLA-4 antibody comprises (a) a heavy chain variable region comprising (i) a histidine (H) substitution at one, two, three or more non-histidine positions, or (ii) at least one histidine residue in two or more of HC- CDR1, HC-CDR2, and HC-CDR3 of the anti-CTLA-4 antibody; (b) a light chain variable region comprising (i) a LC-CDR1 sequence having an acidic residue (e.g., aspartic acid or glutamic acid) substituted at one, two, three or more positions that were not previously aspartic acid or glutamic acid, or (ii) having one, two, three or more aspartic acid or glutamic acid residues in LC-CDRI; or (c) both (a) and (b). In some embodiments of these aspects, the anti-CLTA-4 antibody is ipiliumumab or tremelimumab. In some aspects the anti-CTLA-4 antibody comprises an anti- CTLA-4 antibody disclosed in International Publication No. WO 2020/214748, which is incorporated by reference herein in its entirety.

[0160] Additional anti-CTLA-4 antibodies usable in the disclosed compositions and methods also include isolated antibodies that bind specifically to human CTLA-4 and cross-compete for binding to human CTLA-4 with any anti-CTLA-4 antibody disclosed herein, e.g., ipilimumab and/or tremelimumab. In some aspects, the anti-CTLA-4 antibody binds the same epitope as any of the anti-CTLA-4 antibodies described herein, e.g., ipilimumab and/or tremelimumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g., ipilimumab and/or tremelimumab, by virtue of their binding to the same epitope region of CTLA-4. Cross-competing antibodies can be readily identified based on their ability to cross-compete with ipilimumab and/or tremelimumab in standard CTLA-4 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

[0161] In certain aspects, the antibodies that cross-compete for binding to human CTLA-4 with, or bind to the same epitope region of human CTLA-4 antibody as, ipilimumab and/or tremelimumab, are monoclonal antibodies. For administration to human subjects, these crosscompeting antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0162] Anti-CTLA-4 antibodies usable in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0163] Anti-CTLA-4 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to CTLA-4 with high specificity and affinity, block the activity of CTLA-4, and disrupt the interaction of CTLA-4 with a human B7 receptor. In any of the compositions or methods disclosed herein, an anti-CTLA-4 "antibody" includes an antigen-binding portion or fragment that binds to CTLA-4 and exhibits the functional properties similar to those of whole antibodies in inhibiting the interaction of CTLA-4 with a human B7 receptor and up-regulating the immune system. In certain aspects, the anti-CTLA-4 antibody or antigen-binding portion thereof cross-competes with ipilimumab and/or tremelimumab for binding to human CTLA-4.

[0164] In some aspects, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose ranging from 0.1 mg/kg to 10.0 mg/kg body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks. In some aspects, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 1 mg/kg or 3 mg/kg body weight once every 3, 4, 5, or 6 weeks. In one aspect, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 3 mg/kg body weight once every 2 weeks. In another aspect, the anti-PD-1 antibody or antigenbinding portion thereof is administered at a dose of 1 mg/kg body weight once every 6 weeks.

[0165] In some aspects, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered as a flat dose. In some aspects, the anti-CTLA-4 antibody is administered at a flat dose of from about 10 to about 1000 mg, from about 10 mg to about 900 mg, from about 10 mg to about 800 mg, from about 10 mg to about 700 mg, from about 10 mg to about 600 mg, from about 10 mg to about 500 mg, from about 100 mg to about 1000 mg, from about 100 mg to about 900 mg, from about 100 mg to about 800 mg, from about 100 mg to about 700 mg, from about 100 mg to about 100 mg, from about 100 mg to about 500 mg, from about 100 mg to about 480 mg, or from about 240 mg to about 480 mg. In one aspect, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered as a flat dose of at least about 60 mg, at least about 80 mg, at least about 100 mg, at least about 120 mg, at least about 140 mg, at least about 160 mg, at least about 180 mg, at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 320 mg, at least about 340 mg, at least about 360 mg, at least about 380 mg, at least about 400 mg, at least about 420 mg, at least about 440 mg, at least about 460 mg, at least about 480 mg, at least about 500 mg, at least about 520 mg at least about 540 mg, at least about 550 mg, at least about 560 mg, at least about 580 mg, at least about 600 mg, at least about 620 mg, at least about 640 mg, at least about 660 mg, at least about 680 mg, at least about 700 mg, or at least about 720 mg. In another aspect, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered as a flat dose about once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0166] In some aspects, ipilimumab is administered at a dose of about 3 mg/kg once about every 3 weeks. In some aspects, ipilimumab is administered at a dose of about 10 mg/kg once about every 3 weeks. In some aspects, ipilimumab is administered at a dose of about 10 mg/kg once about every 12 weeks. In some aspects, the ipilimumab is administered for four doses.

4. Anti-LAG-3 Antibodies

[0167] As used herein a LAG-3 antagonist includes, but is not limited to, LAG-3 binding agents, e.g., a LAG-3 antibody, and soluble LAG-3 polypeptides, e.g., a fusion protein comprising the extracellular portion of LAG-3.

[0168] In some aspects, the LAG-3 inhibitor is a soluble LAG-3 polypeptide, for example, a LAG-3 -Fc fusion polypeptide capable of binding to MHC Class II.

[0169] In some aspects, the LAG-3 antagonist comprises IMP321 (eftilagimod alpha).

[0170] In some aspects, the LAG-3 antagonist is an anti-LAG-3 antibody or an antigen-binding fragment thereof that specifically binds to LAG-3 ("anti-LAG-3 antibody").

[0171] Anti-LAG-3 antibodies (or VH/VL domains derived therefrom) suitable for use herein can be generated using any methods. Alternatively, art recognized anti-LAG-3 antibodies can be used.

[0172] In some aspects, the anti-LAG-3 antibody is a chimeric, humanized, or human monoclonal antibody, or a portion thereof. In other aspects, the anti-LAG-3 antibody is a bispecific antibody or a multispecific antibody. [0173] In some aspects, the anti-LAG-3 antibody is relatlimab, e.g., BMS-986016 as described in PCT/US13/48999, the teachings of which are hereby incorporated by reference.

[0174] In other aspects, the antibody has the heavy and light chain CDRs or variable regions of relatlimab. Accordingly, in one aspect, the antibody comprises CDR1, CDR2, and CDR3 domains of the VH region of relatlimab, and CDR1, CDR2 and CDR3 domains of the VL region of relatlimab. In another aspect, the antibody comprises VH and/or VL regions of relatlimab.

[0175] In some aspects, the anti-LAG-3 antibody cross-competes with relatlimab for binding to human LAG-3. The ability of antibodies to cross-compete for binding to an antigen indicates that the antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g., relatlimab, by virtue of their binding to the same epitope region. Cross-competing antibodies can be readily identified based on their ability to cross-compete in standard binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

[0176] In some aspects, the anti-LAG-3 antibody binds to the same epitope as relatlimab.

[0177] In some aspects, the anti-LAG-3 antibody is a biosimilar of relatlimab.

[0178] In some aspects, the anti- LAG-3 antibody is LAG-525, MK-4280, REGN3767, TSR- 033, TSR-075, Sym022, FS-118, or any combination thereof.

[0179] In some aspects, art recognized anti-LAG-3 antibodies can be used in the therapeutic methods of the disclosure. For example, the anti-human LAG-3 antibody described in US2011/0150892 Al, which is herein incorporated by reference, and referred to as monoclonal antibody 25F7 (also known as "25F7" and "LAG-3.1) can be used. Other art recognized anti-LAG- 3 antibodies that can be used include IMP731 (H5L7BW) described in US 2011/007023, MK-4280 (28G-10) described in WO2016028672, REGN3767 described in Journal for ImmunoTherapy of Cancer, (2016) Vol. 4, Supp. Supplement 1 Abstract Number: P195, BAP050 described in WO20 17/019894, IMP-701 (LAG-525), aLAG3(0414), aLAG3(0416), Sym022, TSR-033, TSR- 075, XmAb22841, MGD013, BI754111, FS118, P 13B02-30, AVA-017 and GSK2831781. These and other anti-LAG-3 antibodies useful in the claimed invention can be found in, for example: US 10,188,730, WO2016/028672, W02017/106129, WO2017/062888, W02009/044273,

WO20 18/069500, WO2016/126858, WO2014/179664, WO2016/200782, WO2015/200119, WO20 17/019846, WO2017/198741, WO2017/220555, WO2017/220569, WO2018/071500, W02017/015560, WO2017/025498, WO2017/087589, WO2017/087901, W02018/083087, WO2017/149143, WO2017/219995, US2017/0260271, WO2017/086367, WO2017/086419, WO20 18/034227, WO18/185046, WO18/185043, WO2018/217940, W019/011306, WO2018/208868, and W02014/140180. The contents of each of these references are incorporated by reference herein in their entirety. Antibodies that compete with any of the art-recognized antibodies for binding to LAG-3 also can be used.

[0180] In some aspects, the anti-LAG-3 antibody cross-competes with, binds to the same epitope as, or is a biosimilar of an anti-LAG-3 antibody that is described herein or that is known in the art.

[0181] In certain aspects, an anti-LAG-3 antibody is used to determine LAG-3 expression. In some aspects, an anti-LAG-3 antibody is selected for its ability to bind to LAG-3 in formalin-fixed, paraffin-embedded (FFPE) tissue specimens. In other aspects, an anti-LAG-3 antibody is capable of binding to LAG-3 in frozen tissues. In further aspects, an anti-LAG-3 antibody is capable of distinguishing membrane bound, cytoplasmic, and/or soluble forms of LAG-3.

[0182] In some aspects, an anti-LAG-3 antibody useful for assaying, detecting, and/or quantifying LAG-3 expression in accordance with the methods described herein is the 17B4 mouse IgGl anti -human LAG-3 monoclonal antibody, or an antigen-binding fragment thereof. See, e.g., J. Matsuzaki, etal., PNAS 107, 7875 (2010).

[0183] In some aspects, the anti-LAG-3 antibody or antigen-binding portion thereof is administered at a dose ranging from about 0.1 mg/kg to about 10.0 mg/kg body weight once about every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some aspects, the anti-LAG-3 antibody or antigen-binding portion thereof is administered at a dose of at least about 1 mg/kg, at least about 2 mg/kg, at least about 3 mg/kg, at least about 4 mg/kg, at least about 5 mg/kg, at least about 6 mg/kg, at least about 7 mg/kg, at least about 8 mg/kg, at least about 9 mg/kg, or at least about 10 mg/kg body weight about once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0184] In some aspects, the anti-LAG-3 antibody or antigen-binding portion thereof is administered at a flat dose. In some aspects, the anti-LAG-3 antibody is administered at a flat dose of from about 20 mg to about 2000 mg. In one aspect, the anti-LAG-3 antibody or antigen-binding portion thereof is administered as a flat dose of at least about 80 mg or at least about 160 mg. In another aspect, the anti-LAG-3 antibody or antigen-binding portion thereof is administered at a flat dose once about every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0185] In some aspects, the anti-LAG-3 antibody or antigen-binding portion thereof is administered at a flat dose.

[0186] In some aspects, the anti-LAG-3 antibody or antigen-binding portion thereof is administered at a flat dose of about 80 mg. [0187] In some aspects, the anti-LAG-3 antibody or antigen-binding portion thereof is administered at a flat dose of about 160 mg.

5. Combination Therapies

[0188] In certain aspects, the anti-PD-1 antibody, the anti-PD-Ll antibody, the anti-LAG-3 antibody, and/or the anti-CTLA-4 antibody are administered at a therapeutically effective amount. In some aspects, the method comprises administering a therapeutically effective amount of an anti- PD-1 antibody and an anti-CTLA-4 antibody. In some aspects, the method comprises administering a therapeutically effective amount of anti-PD-1 antibody and an anti-LAG-3 antibody. In some aspects, the method comprises administering a therapeutically effective amount of anti-PD-Ll antibody and an anti-CTLA-4 antibody. In some aspects, the method comprises administering a therapeutically effective amount of anti-PD-Ll antibody and an anti-LAG-3 antibody. Any anti-PD-1, anti-PD-Ll, anti-LAG-3 antibody, or anti-CTLA-4 antibody disclosed herein can be used in the method. In certain aspects, the anti-PD-1 antibody comprises nivolumab. In some aspects, the anti-PD-1 antibody comprises pembrolizumab. In some aspects, the anti-PD- Ll antibody comprises atezolizumab. In some aspects, the anti-PD-Ll antibody comprises durvalumab. In some aspects, the anti-PD-Ll antibody comprises avelumab. In some aspects, the anti-CTLA-4 antibody comprises ipilimumab. In some aspects, the anti-CTLA-4 antibody comprises tremelimumab.

[0189] In some aspects, the (a) anti-PD-1 antibody or the anti-PD-Ll antibody and the (b) anti- CTLA-4 antibody are each administered once about every 2 weeks, once about every 3 weeks, once about every 4 weeks, once about every 5 weeks, or once about every 6 weeks. In some aspects, the anti-PD-1 antibody or the anti-PD-Ll antibody is administered once about every 2 weeks, once about every 3 weeks, or once about every 4 weeks; and the anti-CTLA-4 antibody is administered once about every 6 weeks. In some aspects, the anti-PD-1 antibody or anti-PD-Ll antibody is administered on the same day as the anti-CTLA-4 antibody. In some aspects, the anti-PD-1 antibody or the anti-PD-Ll antibody is administered on a different day than the anti-CTLA-4 antibody.

[0190] In some aspects, the anti-CTLA-4 antibody is administered at a dose ranging from about 0.1 mg/kg to about 20.0 mg/kg body weight once about every 2, 3, 4, 5, 6, 7, or 8 weeks. In some aspects, the anti-CTLA-4 antibody is administered at a dose of about 0.1 mg/kg, about 0.3 mg/kg, about 0.6 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 3 mg/kg, about 6 mg/kg, about 9 mg/kg, about 10 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, or about 20 mg/kg. In certain aspects, the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 4 weeks. In some aspects, the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks.

[0191] In some aspects, the anti-CTLA-4 antibody is administered at a flat dose. In some aspects, the anti-CTLA-4 antibody is administered at a flat dose ranging from at least about 40 mg to at least about 1600 mg. In some aspects, the anti-CTLA-4 antibody is administered at a flat dose of at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, or at least about 200 mg. In some aspects, the CTLA-4 antibody is administered at a flat dose of at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 320 mg, at least about 360 mg, at least about 400 mg, at least about 440 mg, at least about 480 mg, at least about 520 mg, at least about 560 mg, or at least about 600 mg. In some aspects, the CTLA-4 antibody is administered at a flat dose of at least about 640 mg, at least about 720 mg, at least about 800 mg, at least about 880 mg, at least about 960 mg, at least about 1040 mg, at least about 1120 mg, at least about 1200 mg, at least about 1280 mg, at least about 1360 mg, at least about 1440 mg, or at least about 1600 mg. In some aspects, the anti-CTLA-4 antibody is administered in a flat dose at least once about every 2, 3, 4, 5, 6, 7, or 8 weeks.

[0192] In certain aspects, the anti-PD-1 antibody is administered at a dose of about 2 mg/kg once about every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks. In some aspects, the anti-PD-1 antibody is administered at a dose of about 3 mg/kg once about every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks. In some aspects, the anti-PD-1 antibody is administered at a dose of about 6 mg/kg once about every 4 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks.

[0193] In certain aspects, the anti-PD-1 antibody is administered at a flat dose of about 200 mg once about every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks. In some aspects, the anti-PD-1 antibody is administered at a flat dose of about 200 mg once about every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks. In some aspects, the anti-PD-1 antibody is administered at a flat dose of about 240 mg once about every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks. In some aspects, the anti-PD-1 antibody is administered at a flat dose of about 480 mg once about every 4 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks.

[0194] In certain aspects, the anti-PD-1 antibody is administered at a flat dose of about 200 mg once about every 3 weeks and the anti-CTLA-4 antibody is administered at a flat dose of about 80 mg once about every 6 weeks. In some aspects, the anti-PD-1 antibody is administered at a flat dose of about 200 mg once about every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 80 mg once about every 6 weeks. In some aspects, the anti-PD-1 antibody is administered at a flat dose of about 240 mg once about every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 80 mg once about every 6 weeks. In some aspects, the anti-PD-1 antibody is administered at a flat dose of about 480 mg once about every 4 weeks and the anti-CTLA-4 antibody is administered at a dose of about 80 mg once about every 6 weeks.

[0195] In certain aspects, the anti-PD-Ll antibody is administered at a dose of about 10 mg/kg once about every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks. In some aspects, the anti-PD-Ll antibody is administered at a dose of about 15 mg/kg once about every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks.

[0196] In certain aspects, the anti-PD-Ll antibody is administered at a flat dose of about 800 mg once about every 2 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks. In some aspects, the anti-PD-Ll antibody is administered at a flat dose of about 1200 mg once about every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg once about every 6 weeks.

[0197] In certain aspects, the anti-PD-Ll antibody is administered at a flat dose of about 800 mg once about every 2 weeks and the anti-CTLA-4 antibody is administered at a flat dose of about 80 mg once about every 6 weeks. In some aspects, the anti-PD-Ll antibody is administered at a flat dose of about 1200 mg once about every 3 weeks and the anti-CTLA-4 antibody is administered at a dose of about 80 mg once about every 6 weeks.

[0198] In some aspects, the anti-PD-1 antibody, e.g., nivolumab, is administered at a dose of about 3 mg/kg and the anti-CTLA-4 antibody is administered at a dose of about 1 mg/kg on the same day, once about every 3 weeks for 4 doses, then the anti-PD-1 antibody, e.g., nivolumab, is administered at a flat dose of 240 mg once about every 2 weeks or 480 mg once about every 4 weeks. In some aspects, the anti-PD-1 antibody, e.g., nivolumab, is administered at a dose of about 1 mg/kg and the anti-CTLA-4 antibody is administered at a dose of about 3 mg/kg on the same day, once about every 3 weeks for 4 doses, then the anti-PD-1 antibody, e.g., nivolumab, is administered at a flat dose of 240 mg once about every 2 weeks or 480 mg once about every 4 weeks.

6. Additional Anticancer Therapies

[0199] In some aspects of the present disclosure, the methods disclosed herein further comprise administering a checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti- CTLA-4 antibody, and/or an anti-LAG-3 antibody) and an additional anticancer therapy. In certain aspects, the method comprises administering an anti-PD-1 antibody (or an anti-PD-Ll antibody), an anti-CTLA-4 antibody, and an additional anticancer therapy The additional anticancer therapy can comprise any therapy for the treatment of a tumor in a subject and/or any standard-of-care therapy, as disclosed herein. In some aspects, the additional anticancer therapy comprises a surgery, a radiation therapy, a chemotherapy, an immunotherapy, or any combination thereof. In some aspects, the additional anticancer therapy comprises a chemotherapy, including any chemotherapy disclosed herein. In some aspects, the chemotherapy comprises a platinum-based therapy. In some aspects, the platinum-based therapy comprises a platinum-based antineoplastic selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof. In certain aspects, the platinum-based therapy comprises cisplatin. In one particular aspect, the platinumbased therapy comprises carboplatin.

[0200] In some aspects, the additional anticancer therapy comprises a platinum agent (e.g., cisplatin, carboplatin), a taxane agent (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, gemcitabine, bevacizumab (AVASTIN®), erlotinib (TARCEVA®), crizotinib (XALKORI®), cetuximab (ERBITUX®), and any combination thereof. In certain aspects, the at least one prior therapy comprises a platinum-based doublet chemotherapy.

[0201] In some aspect, the additional anticancer therapy comprises an immunotherapy. In some aspects, the additional anticancer therapy comprises administration of an antibody or antigenbinding portion thereof that specifically binds TIGIT, TIM3, NKG2a, 0X40, ICOS, MICA, CD137, KIR, TGFp, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, or any combination thereof.

7. Tumors

[0202] In some aspects, the tumor is derived from a cancer selected from the group consisting of hepatocellular cancer, gastroesophageal cancer, melanoma, bladder cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, and any combination thereof. In certain aspects, the tumor is a NSCLC tumor. In certain aspects, the subject has received one, two, three, four, five or more prior cancer treatments. In other aspects, the subject is treatment-naive. In some aspects, the subject has progressed on other cancer treatments. In certain aspects, the prior cancer treatment comprised an immunotherapy. In other aspects, the prior cancer treatment comprised a chemotherapy. In some aspects, the tumor has reoccurred. In some aspects, the tumor is metastatic. In other aspects, the tumor is not metastatic. In some aspects, the tumor is locally advanced.

[0203] In some aspects, the subj ect has received a prior therapy to treat the tumor and the tumor is relapsed or refractory. In certain aspects, the at least one prior therapy comprises a standard-of- care therapy. In some aspects, the at least one prior therapy comprises a surgery, a radiation therapy, a chemotherapy, an immunotherapy, or any combination thereof. In some aspects, the at least one prior therapy comprises a chemotherapy. In some aspects, the subject has received a prior immuno-oncology (I-O) therapy to treat the tumor and the tumor is relapsed or refractory. In some aspects, the subject has received more than one prior therapy to treat the tumor and the subject is relapsed or refractory. In other aspects, the subject has received either an anti-PD-1 or anti-PD-Ll antibody therapy.

[0204] In some aspects, the previous line of therapy comprises a chemotherapy. In some aspects, the chemotherapy comprises a platinum-based therapy. In some aspects, the platinumbased therapy comprises a platinum-based antineoplastic selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof. In certain aspects, the platinum-based therapy comprises cisplatin. In one particular aspect, the platinum-based therapy comprises carboplatin.

[0205] In some aspects, the at least one prior therapy is selected from a therapy comprising administration of an anticancer agent selected from the group consisting of a platinum agent (e.g., cisplatin, carboplatin), a taxanes agent (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, gemcitabine, bevacizumab (AVASTIN®), erlotinib (TARCEVA®), crizotinib (XALKORI®), cetuximab (ERBITUX®), and any combination thereof. In certain aspects, the at least one prior therapy comprises a platinum-based doublet chemotherapy.

[0206] In some aspects, the subject has experienced disease progression after the at least one prior therapy. In certain aspects, the subject has received at least two prior therapies, at least three prior therapies, at least four prior therapies, or at least five prior therapies. In certain aspects, the subject has received at least two prior therapies. In one aspect, the subject has experienced disease progression after the at least two prior therapies. In certain aspects, the at least two prior therapies comprises a first prior therapy and a second prior therapy, wherein the subject has experienced disease progression after the first prior therapy and/or the second prior therapy, and wherein the first prior therapy comprises a surgery, a radiation therapy, a chemotherapy, an immunotherapy, or any combination thereof; and wherein the second prior therapy comprises a surgery, a radiation therapy, a chemotherapy, an immunotherapy, or any combination thereof. In some aspects, the first prior therapy comprises a platinum-based doublet chemotherapy, and the second prior therapy comprises a single-agent chemotherapy. In certain aspects, the single-agent chemotherapy comprises docetaxel.

8. Pharmaceutical Compositions and Dosages

[0207] Therapeutic agents of the present disclosure can be constituted in a composition, e.g., a pharmaceutical composition containing an antibody and/or a cytokine and a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier for a composition containing an antibody is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion), whereas the carrier for a composition containing an antibody and/or a cytokine is suitable for non-parenteral, e.g., oral, administration. In some aspects, the subcutaneous injection is based on Halozyme Therapeutics’ ENHANZE® drug-delivery technology (see U.S. Patent No. 7,767,429, which is incorporated by reference herein in its entirety). ENHANZE® uses a co-formulation of an antibody with recombinant human hyaluronidase enzyme (rHuPH20), which removes traditional limitations on the volume of biologies and drugs that can be delivered subcutaneously due to the extracellular matrix (see U.S. Patent No. 7,767,429). A pharmaceutical composition of the disclosure can include one or more pharmaceutically acceptable salts, anti-oxidant, aqueous and non-aqueous carriers, and/or adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Therefore, in some aspects, the pharmaceutical composition for the present disclosure can further comprise recombinant human hyaluronidase enzyme, e.g., rHuPH20.

[0208] In some aspects, the method comprises administering an anti-PD-1 antibody (or an anti- PD-L1 antibody) and an anti-CTLA-4 antibody, wherein the anti-PD-1 antibody (or the anti-PD- L1 antibody) is administered in a fixed dose with the anti-CTLA-4 antibody in a single composition. In some aspects, the anti-PD-1 antibody is administered in a fixed dose with the anti- CTLA-4 antibody. In some aspects, the anti-PD-Ll antibody is administered in a fixed dose with the anti-CTLA-4 antibody in a single composition. In some aspects, the ratio of the anti-PD-1 antibody (or the anti-PD-Ll antibody) to the anti-CTLA-4 antibody is at least about 1 : 1, about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1 : 10, about 1 : 15, about 1 :20, about 1 :30, about 1 :40, about 1 :50, about 1 :60, about 1 :70, about 1 :80, about 1 :90, about 1 : 100, about 1 : 120, about 1 : 140, about 1 : 160, about 1 : 180, about 1:200, about 200: 1, about 180: 1, about 160: 1, about 140: 1, about 120: 1, about 100: 1, about 90: 1, about 80: 1, about 70: 1, about 60: 1, about 50: 1, about 40: 1, about 30: l, about 20:l, about 15: 1, about 10: 1, about 9: l, about 8: 1, about 7: 1, about 6: 1, about 5:1, about 4: 1, about 3: 1, or about 2: 1.

[0209] Although higher nivolumab monotherapy dosing up to 10 mg/kg every two weeks has been achieved without reaching the maximum tolerated does (MTD), the significant toxicities reported in other trials of checkpoint inhibitors plus anti -angiogenic therapy (see, e.g., Johnson el al., 2013; Rini et al., 2011) support the selection of a nivolumab dose lower than 10 mg/kg.

[0210] Generally, treatment is continued as long as clinical benefit is observed or until unacceptable toxicity or disease progression occurs. Nevertheless, in certain aspects, the dosages of the anti-PD-1 antibody, the anti-PD-Ll antibody, and/or the anti-CTLA-4 antibody administered are significantly lower than the approved dosage, i.e., a subtherapeutic dosage, of the agent. The anti-PD-1 antibody, the anti-PD-Ll antibody, and/or the anti-CTLA-4 antibody can be administered at the dosage that has been shown to produce the highest efficacy as monotherapy in clinical trials, e.g., about 3 mg/kg of nivolumab administered once every three weeks (Topalian et al., 2012a; Topalian et al., 2012), or at a significantly lower dose, i.e., at a subtherapeutic dose.

[0211] Dosage and frequency vary depending on the half-life of the antibody in the subject. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is typically administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0212] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being unduly toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A composition of the present disclosure can be administered via one or more routes of administration using one or more of a variety of methods well known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

III. Kits

[0213] Also within the scope of the present disclosure are kits comprising (a) one or more checkpoint inhibitors described elsewhere herein (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, and/or an anti-LAG-3 antibody), for therapeutic uses wherein the kits are suitable for the subjects identified as described herein. Kits typically include a label indicating the intended use of the contents of the kit and instructions for use. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit. Accordingly, this disclosure provides a kit for treating a subject afflicted with a tumor, the kit comprising: (a) a therapeutically effective dosage of a checkpoint inhibitor, e.g., a dosage ranging from 0.1 to 10 mg/kg body weight of an anti-PD-1 antibody or a dosage ranging from 0.1 to 20 mg/kg body weight of an anti-PD-Ll antibody; and (b) instructions for using the checkpoint inhibitor, e.g., the anti-PD-1 antibody or the anti-PD-Ll antibody, in the methods disclosed herein. This disclosure further provides a kit for treating a subject identified as a suitable candidate for a checkpoint inhibitor therapy as described herein, the kit comprising: (a) a therapeutically effective dosage of a checkpoint inhibitor, e.g., a dosage ranging from about 4 mg to about 500 mg of an anti-PD-1 antibody or a dosage ranging from about 4 mg to about 2000 mg of an anti-PD-Ll antibody; and (b) instructions for using the checkpoint inhibitor, e.g., the anti- PD-1 antibody or the anti-PD-Ll antibody in the methods disclosed herein. In some aspects, this disclosure provides a kit for treating a subject identified as a suitable candidate for a checkpoint inhibitor therapy as described herein, the kit comprising: (a) a therapeutically effective dosage of a checkpoint inhibitor, e.g., a dosage ranging from 200 mg to 800 mg of an anti-PD-1 antibody or a dosage ranging from 200 mg to 1800 mg of an anti-PD-Ll antibody; and (b) instructions for using the checkpoint inhibitor, e.g., the anti-PD-1 antibody or the anti-PD-Ll antibody, in the methods disclosed herein.

[0214] In certain aspects for treating human patients, the kit comprises an anti-human PD-1 antibody disclosed herein, e.g. , nivolumab or pembrolizumab. In certain aspects for treating human patients, the kit comprises an anti-human PD-L1 antibody disclosed herein, e.g., atezolizumab, durvalumab, or avelumab.

[0215] In some aspects, the kit further comprises an anti-CTLA-4 antibody. In certain aspects for treating human patients, the kit comprises an anti-human CTLA-4 antibody disclosed herein, e.g., ipilimumab, tremelimumab, MK-1308, or AGEN-1884.

[0216] In some aspects, the kit further includes a gene panel assay disclosed herein. In some aspects, the kit further includes instructions to administer the anti-PD-1 antibody or the anti-PD- Ll antibody to a subject identified as having a low expression profile of the one or more genes of the gene panel, according to the methods disclosed herein. In other aspects, the kit further includes an anti-CTLA-4 antibody and instructions to administer (a) the anti-PD-1 antibody or the anti-PD- Ll antibody and (b) the anti-CTLA-4 antibody to a subject identified as having a low expression profile of the one or more genes of the gene panel, according to the methods disclosed herein.

IV. Devices

[0217] Certain aspects of the present disclosure are directed to a system or a device comprising: (i) a loading means for loading a biological sample; and (ii) a detection means for detecting a level of expression of a panel of genes in the biological sample. Certain aspects of the present disclosure are directed to a system or a device comprising: (i) a loading means for loading a biological sample; and (ii) a first biological probe, wherein the biological probe specifically interacts with a transcript of at least one gene in a panel of genes.

[0218] In some aspects, the panel of genes comprises any panel of genes disclosed herein. In some aspects, the panel of genes comprises at least 2 genes, wherein the at least 2 genes comprise are selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HBO1, and PDHA1. [0219] In some aspects, the device further comprises a means for detecting a level of expression of the transcript. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample comprises a first biological probe, wherein the first biological probe specifically interacts with a transcript of at least one gene in the panel of genes. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises a second biological probe, a third biological probe, a fourth biological probe, a fifth biological probe, a sixth biological probe, a seventh biological probe, an eight biological probe, a ninth biological probe, or a tenth biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises a second biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises a third biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises a fourth biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises a fifth biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises a sixth biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises a seventh biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises an eight biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises a ninth biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample further comprises or a tenth biological probe. In some aspects, the means for detecting a level of expression of a panel of genes in the biological sample comprises more than ten biological probes.

[0220] In some aspects, the first biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a first gene of the panel of genes. In some aspects, the second biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a second gene of the panel of genes. In some aspects, the third biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a third gene of the panel of genes. In some aspects, the fourth biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a fourth gene of the panel of genes. In some aspects, the fourth biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a fourth gene of the panel of genes. In some aspects, the fifth biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a fifth gene of the panel of genes.

[0221] In some aspects, the transcript comprises an mRNA. In some aspects, the transcript comprises a cDNA.

[0222] In some aspects, the probe comprises an RNA-hybridization probe. In some aspects, the probe comprises an oligonucleotide (e.g., a DNA primer). [0223] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0224] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1

[0225] Tumors are known to have various means of evading attack by the immune system. Some of these evasion tactics can make it less likely for a patient to respond to certain therapies, including administration of checkpoint inhibitors. Currently, both tumor mutational burden (TMB) and PD-L1 expression are used to independently to identify patients who will respond to immunotherapies in non-small cell lung cancer (NSCLC). For example, in various lung cancer trials, where an anti-PD-1 antibody was used as treatment, patients with both high TMB and PD- L1 expression had a greater response rate (75%) compared to those with only high TMB (32%) or high PD-L1 (34%). See New England Journal of Medicine, 2018; 378:2093-104. Furthermore, patients with a low TMB score (<14 mutations/MB), exhibited very poor response to IO treatment comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody. In the same trial, tumors with low TMB (<14 mutations/MB) score and low PD-L1 (<50%) expression were further associated with increased rates of rapid progression (within 3 months of starting treatment) in the IO treatment arm but not in the chemotherapy arm.

[0226] For stratification approaches relying on TMB mutational score and PD-L1 expression, separate diagnostic tests (NGS and IHC) are performed in clinical practice to identify patients likely to respond to IO therapy or patients for whom IO may not be beneficial. One key disadvantage of this approach is that it requires relatively large amounts of tissue to generate the clinical biomarker results (generally more than 10 tissue slides). Also, it can require long turnaround times (e.g., more than two weeks). The NGS uses on average 7-14 tissue slides and IHC uses on average 3-5 slides, which means a total of approximately 10-19 slides from each tumor biopsy, which can be very difficult to obtain, especially from advanced lung cancer patients, because the biopsies are often obtained from metastatic lesions and are an invasive procedure associated with significant risk. The present example describes the identification of biomarkers indicative of favorable/unfavorable response to IO using an alternative gene expression profiling approach. [0227] RNA from formalin-fixed, paraffin-embedded (FFPE) tumor samples from patients enrolled in an anti-PD-1 antibody NSCLC trial were isolated and subjected to exome RNAseq analysis. For this assay, a tissue input of only 2-4 slides per patient was needed.

[0228] Patients were randomly assigned in a 1 : 1 : 1 ratio to receive nivolumab (at a dose of 3 mg per kilogram of body weight every 2 weeks) plus ipilimumab (at a dose of 1 mg per kilogram every 6 weeks), nivolumab monotherapy (240 mg every 2 weeks), or platinum-doublet chemotherapy every 3 weeks for up to four cycles.

[0229] Gene signature analysis was performed via NGS sequencing of RNA extracted from baseline formalin-fixed, paraffin-embedded tumor samples (samples obtained prior to the treatment), and z-scores were calculated using log-transformed counts per million. High, middle, and low scores were stratified by tertiles of z-score across the entire dataset.

[0230] Nine genes were identified as correlated with favorable response to treatment with an anti-PD-1 antibody (nivolumab) and an anti-CTLA-4 antibody (ipilimumab): SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HBO1, and PDHA1. Low gene expression levels (i.e., below the low tertile cut off) measured by exome RNA-seq of the nine genes correlated with favorable Progression-free Survival (PFS; FIG. 1A) and Overall Survival (OS; FIG. 1C). Conversely, high gene expression levels (i.e., above the high tertile cut off) of the 9 signature genes correlated with poor response to IO treatment, as indicated by unfavorable clinical endpoints including lower PFS (FIG. 1A) and OS (FIG. 1C). No such correlation was observed in the chemotherapy arm (FIGs. IB and ID), suggesting that the signature is specifically predictive of IO response, as opposed to behaving as a general prognostic marker.

[0231] These results indicate that a novel gene panel can be used for identifying patients for treatment with checkpoint inhibitor therapy. Furthermore, these results indicate that a single diagnostic assay that can be completed more quickly, more economically and with much smaller amounts of tissue can be used for patient stratification.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a tumor in a human subject diagnosed with a non-small cell lung cancer (NSCLC), the method comprising administering an effective amount of a checkpoint inhibitor to the subject, wherein the subject is identified as suitable for the checkpoint inhibitor therapy by measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1; wherein the subject is identified as having a low expression profile of the panel of genes.

2. A method of treating a tumor in a human subject diagnosed with a non-small cell lung cancer (NSCLC), the method comprising:

(a) measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1; and

(b) administering an effective amount of a checkpoint inhibitor to the subject, wherein the subject is identified as having a low expression profile of the panel of genes.

3. A method of identifying a subject suitable for a checkpoint inhibitor therapy, wherein the subject has been diagnosed with non-small cell lung cancer (NSCLC), and the method comprises (i) measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1; and (ii) identifying a subject as suitable for an immune checkpoint inhibitor therapy if the subject is identified as having a low expression profile of the panel of genes.

4. The method of claim 3, further comprising administering to the subject a checkpoint inhibitor.

5. A checkpoint inhibitor for treating a tumor in a human subject diagnosed with a non-small cell lung cancer (NSCLC), wherein the subject is identified as being suitable for a checkpoint inhibitor therapy by measuring the expression of a panel of genes in a tumor sample from the subject, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1; and wherein the subject is identified as having a low expression profile of the panel of genes. The method or checkpoint inhibitor of any preceding claim, wherein the panel of genes comprises at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, or at least 9 genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1. The method or checkpoint inhibitor of any preceding claim, wherein the panel of genes comprises SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHAL The method or checkpoint inhibitor of any preceding claim, wherein the panel of genes comprises at least one housekeeping gene. The method or checkpoint inhibitor of any preceding claim, wherein the panel of genes comprises at least one control gene. The method or checkpoint inhibitor of any preceding claim, wherein a high expression profile is characterized by measuring the composite expression of the genes in the gene panel in a tumor sample from the subject, and comparing the composite expression of the genes in the gene panel to the average composite expression of the panel of genes in tumor samples obtained from a population of subjects afflicted with NSCLC. The method or checkpoint inhibitor of claim 10, wherein the average composite expression is determined by measuring the composite expression of the panel of genes in tumor samples obtained from the population of subjects. The method or checkpoint inhibitor of any preceding claim, wherein the expression of the genes in the panel of genes is determined by measuring the level of mRNA, the level of a protein encoded by the gene, or both. The method or checkpoint inhibitor of claim 12, wherein the level of mRNA is determined using reverse transcriptase PCR. The method or checkpoint inhibitor of claim 12 or 13, wherein the level of the protein encoded by the gene is determined using an IHC assay. The method or checkpoint inhibitor of claim 14, wherein the IHC assay is an automated IHC assay. The method of any one of claims 1 to 10, wherein the expression of the genes in the panel of genes is determined by whole transcriptome RNA-seq. The method or checkpoint inhibitor of any preceding claim, wherein the checkpoint inhibitor comprises a polypeptide, a small molecule, a polynucleotide, or any combination thereof. The method or checkpoint inhibitor of any preceding claim, wherein the checkpoint inhibitor comprises an antibody or an antigen-binding fragment thereof. The method or checkpoint inhibitor of claim 18, wherein the antibody or antigen-binding fragment thereof specifically binds a target selected from PD-1 ("anti-PD-1 antibody"), PD- L1 ("anti-PD-Ll antibody"), CTLA-4, LAG3, TIGIT, TIM3, NKG2a, 0X40, ICOS, CD 137, KIR, TGFp, IL- 10, IL-8, IL-2, CD96, VISTA, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, and any combination thereof. The method or checkpoint inhibitor of claim 18 or 19, wherein the antibody is a chimeric, humanized or human monoclonal antibody or a portion thereof. The method or checkpoint inhibitor of claim 19 or 20, wherein the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1. The method or checkpoint inhibitor of any one of claims 19 to 21, wherein the anti-PD-1 antibody binds to the same epitope as nivolumab. The method or checkpoint inhibitor of any one of claims 19 to 22, wherein the anti-PD-1 antibody comprises a heavy chain constant region which is of a human IgGl or IgG4 isotype. The method or checkpoint inhibitor of any one of claims 19 to 23, wherein the anti-PD-1 antibody is nivolumab. The method or checkpoint inhibitor of any one of claims 19 to 24, wherein the anti-PD-1 antibody is pembrolizumab. The method or checkpoint inhibitor of any one of claims 19 to 25, wherein the anti-PD-1 antibody is administered at a dose ranging from at least about 0.1 mg/kg to at least about 10.0 mg/kg body weight once about every 1, 2 or 3 weeks. The method or checkpoint inhibitor of claim 26, wherein the anti-PD-1 antibody is administered at a dose of at least about 3 mg/kg body weight once about every 2 weeks. The method or checkpoint inhibitor of any one of claims 19 to 27, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose. The method or checkpoint inhibitor of any one of claims 19 to 25 and 28, wherein the anti- PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, at least about 500 or at least about 550 mg. The method or checkpoint inhibitor of any one of claims 19 to 25, 28, and 29, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of about 240 mg. The method or checkpoint inhibitor of any one of claims 19 to 25, 28, and 29, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of about 480 mg. The method or checkpoint inhibitor of any one of claims 19 to 25, and 28 to 31, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose about once every 1, 2, 3 or 4 weeks. The method or checkpoint inhibitor of claims 19 to 25, 28, 29, and 32 wherein the anti-PD- 1 antibody or antigen-binding portion thereof is administered at a flat dose or about 240 mg once about every two weeks. The method or checkpoint inhibitor of any one of claims 19 to 25, 28, and 29, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered at a flat dose of about 480 mg once about every four weeks. The method or checkpoint inhibitor of any one of claims 19 to 34, wherein the anti-PD-1 antibody is administered for as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs. The method or checkpoint inhibitor of any one of claims 18 to 35, wherein the antibody is formulated for intravenous administration. The method or checkpoint inhibitor of any one of claims 18 to 36, wherein the antibody is administered at a subtherapeutic dose. The method or checkpoint inhibitor of any one of claims 19 to 37, further comprising administering an antibody or an antigen binding fragment thereof that binds specifically to CTLA-4 ("an anti-CTLA-4 antibody"). The method or checkpoint inhibitor of claim 38, wherein the anti-CTLA-4 antibody crosscompetes with ipilimumab or tremelimumab for binding to human CTLA-4. The method or checkpoint inhibitor of claim 38 or 39, wherein the anti-CTLA-4 antibody binds to the same epitope as ipilimumab or tremelimumab. The method or checkpoint inhibitor of any one of claims 38 to 40, wherein the anti-CTLA- 4 antibody is ipilimumab. The method or checkpoint inhibitor of any one of claims 38 to 40, wherein the anti-CTLA- 4 antibody is tremelimumab. The method or checkpoint inhibitor of any one of claims 38 to 40, wherein the anti-CTLA- 4 antibody is administered at a dose ranging from 0.1 mg/kg to 20.0 mg/kg body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks. The method or checkpoint inhibitor of any one of claims 38 to 43, wherein the anti-CTLA- 4 antibody is administered at a dose of 1 mg/kg body weight once every 6 weeks. The method or checkpoint inhibitor of any one of claims 38 to 43, wherein the anti-CTLA- 4 antibody is administered at a dose of 1 mg/kg body weight once every 4 weeks. The method or checkpoint inhibitor of any one of claims 38 to 64, wherein the anti-CTLA- 4 antibody is administered at a flat dose. The method or checkpoint inhibitor of claim 46, wherein the anti-CTLA-4 antibody is administered at a flat dose of at least about 40 mg, at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, or at least about 200 mg. The method or checkpoint inhibitor of claim 46 or 47, wherein the anti-CTLA-4 antibody is administered as a flat dose about once every 2, 3, 4, 5, 6, 7, or 8 weeks. The method or checkpoint inhibitor of any one of claims 19 to 48, further comprising administering an antibody or an antigen-binding fragment thereof that binds specifically to LAG-3 ("an anti-LAG-3 antibody"). The method or checkpoint inhibitor of any one of claims 19 to 49, comprising administering an anti-PD-1 antibody and an anti-LAG-3 antibody. The method or checkpoint inhibitor of any one of claims 19 to 50, comprising administering an anti-PD-1 antibody, and anti-CTLA-4 antibody, and an anti-LAG-3 antibody. The method or checkpoint inhibitor of any one of claims 49 to 51, wherein the anti-LAG- 3 antibody comprises relatimab. The method or checkpoint inhibitor of any preceding claim, wherein the tumor is relapsed, refractory, or both relapsed and refractory. The method or checkpoint inhibitor of any preceding claim, wherein the tumor is refractory to at least one prior therapy comprising administration of at least one anticancer agent. The method or checkpoint inhibitor of claim 54, wherein the at least one anticancer agent comprises a standard of care therapy. The method or checkpoint inhibitor of claim 54 or 55, wherein the at least one anticancer agent comprises an immunotherapy. The method or checkpoint inhibitor of any preceding claim, wherein the tumor is locally advanced. The method or checkpoint inhibitor of any preceding claim, wherein the tumor is metastatic. The method or checkpoint inhibitor of any preceding claim, wherein the administering treats the tumor. The method or checkpoint inhibitor of any preceding claim, wherein the administering reduces the size of a tumor in the subject. The method or checkpoint inhibitor of claim 60, wherein the size of the tumor is reduced by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the tumor size prior to the administration. The method or checkpoint inhibitor of any preceding claim, wherein the subject exhibits progression-free survival of at least about one month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about one year, at least about eighteen months, at least about two years, at least about three years, at least about four years, or at least about five years after the initial administration. The method or checkpoint inhibitor of any preceding claim, wherein the subject exhibits stable disease after the administration. The method or checkpoint inhibitor of any preceding claim, wherein the subject exhibits a partial response after the administration. The method or checkpoint inhibitor of any preceding claim, wherein the subject exhibits a complete response after the administration. A system or device comprising:

(i) a loading means for loading a biological sample; and (ii) a means for detecting a level of expression of a panel of genes in the biological sample, the panel of genes comprising at least two genes selected from SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1. The system or device of claim 66, wherein the means for detecting a level of expression of a panel of genes in the biological sample comprises a first biological probe, wherein the first biological probe specifically interacts with a transcript of at least one gene in the panel of genes. The system or device of claim 67, further comprising a means for detecting a level of expression of the transcript. The system or device of claim 67 or 68, wherein the transcript comprises an mRNA or a cDNA. The system or device of any one of claims 67 to 69, wherein the means for detecting a level of expression of a panel of genes in the biological sample further comprises a second biological probe. The system or device of claim 70, wherein the first biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a first gene of the at least 2 genes; and wherein the second biological probe is capable of specifically binding to a nucleotide sequence within a transcript of a second gene of the at least 2 genes. The system or device of claim 71, wherein the transcript of the first gene and/or the transcript of the second gene comprises an mRNA or a cDNA. The system or device of any one of claims 66 to 72, wherein the biological sample comprises a tumor sample from a human subject. The system or device of claim 73, wherein the tumor sample is a tumor tissue biopsy. The system or device of claim 73 or 74, wherein the tumor sample is a formalin-fixed, paraffin-embedded tumor tissue. The system or device of claim 73 or 74, wherein the tumor sample is a fresh-frozen tumor tissue. The system or device of any one of claims 66 to 76, wherein the panel of genes comprises SIX1, SLC2A1/4, HK2, PFKL, ALDOA, GAPDH, LDHA, HB01, and PDHA1. The system or device of any one of claims 66 to 77, wherein the panel of genes comprises at least one housekeeping gene. The system or device of any one of claims 66 to 78, wherein the panel of genes comprises at least one control gene. A method of treating a tumor in a human subject in need thereof, comprising administering an effective amount of a checkpoint inhibitor to the subj ect, wherein the subj ect is identified as having a tumor suitable for a checkpoint inhibitor therapy, wherein the subject is identified as suitable by obtaining a biological sample from the subject and applying the biological sample to the loading means of the device of any one of claims 66 to 79. A method of identifying a subject suitable for an immunotherapy, comprising obtaining a biological sample from the subject and applying the biological sample to the loading means of the device of any one of claims 66 to 79. The method of claim 81, further comprising administering an immunotherapy to the subject.