Methods for treating colorectal cancer using anti-CTLA4 antibodies

Abstract

Methods of treating colon cancer with antibodies that specifically bind to human cytotoxic T-lymphocyte antigen 4 (CTLA-4) are provided.

Description

[Background technology]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 381,621, filed October 31, 2022, and U.S. Provisional Patent Application No. 63 / 497,392, filed April 20, 2023, the entire disclosures of each of which are incorporated herein by reference.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in ST.26 format, which is hereby incorporated by reference in its entirety (the copy of ST.26 created on October 24, 2023 is named "205370_seqlist.xml" and is 19,846 bytes in size).

[0003] Colorectal cancer (CRC) is one of the most common cancers and a leading cause of cancer-related death worldwide. In metastatic CRC (mCRC), the 5-year survival rate for patients with distant metastases is less than 20%.

[0004] Currently, the standard initial treatment for patients with mCRC is systemic combination chemotherapy using drugs that have been used for decades (e.g., 5-fluorouracil, oxaliplatin, and irinotecan). Recent initial treatment options used in combination with chemotherapy include vascular endothelial growth factor (VEGF)-targeted therapy (e.g., bevacizumab) and epidermal growth factor receptor (EGFR)-targeted therapy (e.g., cetuximab or panitumumab). However, the efficacy of these treatments is modest. Furthermore, while immunotherapeutic approaches have shown success in various cancer types, including microsatellite instability-high / mismatch repair-deficient (MSI-H / dMMR) mCRC, immunotherapy has not yet demonstrated success in the treatment of non-MSI-H / dMMR mCRC. Among mCRC patients, over 95% are non-MSI-H / dMMR, and approximately 30–35% of these patients, including those enrolled in clinical trials, do not have liver metastases.

[0005] The majority of CRC patients (including those without liver metastases) are ineligible for immune checkpoint inhibitors as standard of care due to their non-MSI-H / dMMR status, and this patient population is dependent on chemotherapy and targeted therapy regimens with modest efficacy, representing an area of ​​significant unmet medical need.

[0006] Thus, there remains a need for new and effective methods for treating metastatic colorectal cancer. Summary of the Invention

[0007] The present disclosure relates to methods for treating colorectal cancer using antibodies that specifically bind to human cytotoxic T-lymphocyte antigen 4 (CTLA-4). Also provided herein are specific methods for administering antibodies that specifically bind to human CTLA-4 and reduce tumor burden in a subject. In one embodiment, the method comprises a therapeutically effective amount that safely and effectively treats metastatic colorectal cancer. In one embodiment, the method comprises a therapeutically effective amount that safely and effectively reduces tumor burden in a subject with metastatic colorectal adenocarcinoma.

[0008] In one aspect, the present specification provides a method for treating colorectal cancer in a subject in need of such treatment, the method comprising administering to the subject a dose of 25 mg to 200 mg of an antibody that specifically binds to human cytotoxic T-lymphocyte antigen 4 (CTLA-4), wherein the antibody comprises a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 8.

[0009] In one aspect, the present specification provides a method for enhancing T cell activation in a subject with colorectal cancer, the method comprising administering to the subject 25 mg to 200 mg of an antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 8.

[0010] In one embodiment, the antibody is administered at a dose of 50 mg to 175 mg. In one embodiment, the antibody is administered at a dose of 75 mg to 150 mg. In one embodiment, the antibody is administered at a dose of about 25 mg, 50 mg, 75 mg, 100 mg, or 150 mg. In one embodiment, the antibody is administered at a dose of 25 mg, 50 mg, 75 mg, 100 mg, or 150 mg.

[0011] In one embodiment, the antibody is administered intravenously. In one embodiment, the antibody is administered by intravenous infusion over about 30 minutes.

[0012] In one embodiment, the antibody is administered once per week. In one embodiment, the antibody is administered once every two weeks. In one embodiment, the antibody is administered once every three weeks. In one embodiment, the antibody is administered once every four weeks. In one embodiment, the antibody is administered once every five weeks. In one embodiment, the antibody is administered once every six weeks.

[0013] In one embodiment, the antibody is administered intravenously at a dose of 25 mg once every six weeks. In one embodiment, the antibody is administered intravenously at a dose of 50 mg once every six weeks. In one embodiment, the antibody is administered intravenously at a dose of 75 mg once every six weeks. In one embodiment, the antibody is administered intravenously at a dose of 100 mg once every six weeks. In one embodiment, the antibody is administered intravenously at a dose of 150 mg once every six weeks.

[0014] In one embodiment, the dose is a therapeutically effective amount.

[0015] In one embodiment, the colon cancer is colorectal adenocarcinoma. In one embodiment, the colon cancer is unresectable. In one embodiment, the colon cancer is metastatic. In one embodiment, the colon cancer is metastatic unresectable colorectal adenocarcinoma. In one embodiment, the subject does not have liver metastases.

[0016] In one embodiment, the antibody is administered to the subject prior to surgical resection of the primary tumor. In one embodiment, the subject has not previously received any chemotherapy. In one embodiment, the subject has not previously received any radiation therapy.

[0017] In one embodiment, the colorectal cancer is recurrent and / or refractory.

[0018] In one embodiment, the subject has received at least one prior chemotherapy. In one embodiment, the at least one prior chemotherapy is a fluoropyrimidine, irinotecan, oxaliplatin, or an anti-EGFR antibody. In one embodiment, the anti-EGFR antibody is cetuximab or panitumumab.

[0019] In one embodiment, the subject has previously been treated with folinic acid, 5-fluorouracil, oxaliplatin, and / or irinotecan, hi one embodiment, the subject is unable to tolerate standard of care treatment.

[0020] In one embodiment, the subject has a RAS mutation. In one embodiment, the RAS mutation is a KRAS or NRAS mutation.

[0021] In one embodiment, the colon cancer is not microsatellite instability-high (MSI-H). In one embodiment, the colon cancer is microsatellite stable (MSS). In one embodiment, the colon cancer is not mismatch repair deficient (dMMR).

[0022] In one embodiment, the subject has not previously received treatment with an anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody. In one embodiment, the subject has not previously received regorafenib, trifluridine, and / or tipiracil therapy.

[0023] In one embodiment, the cancer is refractory to standard care treatment. In one embodiment, the standard care treatment is chemotherapy or radiation therapy. In one embodiment, the standard care treatment is folinic acid, 5-fluorouracil, oxaliplatin, irinotecan, a fluoropyrimidine, cetuximab, and / or panitumumab.

[0024] In one embodiment, administration of the antibody reduces tumor size in the subject. In one embodiment, administration of the antibody increases activation of T cells, memory T cells, myeloid cells, and / or antigen-presenting cells in the subject. In one embodiment, administration of the antibody reduces the number of Treg cells in the subject.

[0025] In one embodiment, prior to administration of the antibody, the subject has measurable disease on baseline imaging according to RECIST 1.1. In one embodiment, prior to administration of the antibody, the subject has an Eastern Cooperative Oncology Group performance status (PS) of 0 to 1. In one embodiment, prior to administration of the antibody, the subject has a predicted life expectancy of 12 weeks or greater.

[0026] In one embodiment, prior to administration of the antibody, the subject has adequate organ function as defined by one or more of: a) neutrophils > 1500 / μL; b) platelets > 100 x 103 / μL; c) hemoglobin > 8.0 g / dL; d) creatinine clearance > 30 mL / min measured or calculated according to local institutional standards; e) AST / ALT < 2.5 x upper limit of normal (ULN); f) total bilirubin < 1.5 x ULN (except for patients with Gilbert's syndrome, in which total bilirubin values ​​should be < 3.0 x ULN); and / or g) albumin > 3.0 g / dL.

[0027] In one embodiment, the subject has no partial or complete bowel obstruction, signs / symptoms of bowel obstruction, or known radiological evidence of impending obstruction within the past three months.

[0028] In one embodiment, the subject does not have refractory ascites, defined as requiring two or more therapeutic paracentesis within the past four weeks, or four or more therapeutic paracentesis within the past 90 days, or one or more therapeutic paracentesis within the past two weeks prior to administration of the antibody.

[0029] In one embodiment, the subject does not have clinically significant cardiovascular disease.

[0030] In one embodiment, the subject does not have active brain or leptomeningeal metastases. In one embodiment, the subject does not have a concurrent malignancy requiring treatment or a history of an active malignancy within two years prior to administration of the antibody.

[0031] In one embodiment, the subject has not received a cytotoxic therapy, targeted therapy, or other investigational therapy within three weeks prior to administration of the antibody. In one embodiment, the subject has not received another monoclonal antibody therapy, antibody-drug conjugate therapy, or radioimmunoconjugate therapy within four weeks prior to administration of the antibody. In one embodiment, the subject has not received a small molecule tyrosine kinase inhibitor therapy within two weeks prior to administration of the antibody.

[0032] In one embodiment, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0033] In one embodiment, the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8.

[0034] In one embodiment, the antibody comprises a human IgG1 heavy chain constant region comprising the S239D / A330L / I332E mutations numbered according to the EU numbering system.

[0035] In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0036] In one embodiment, the antibody is botencilimab.

[0037] In one aspect, provided herein is a method of treating metastatic colorectal adenocarcinoma in a subject in need thereof, the method comprising administering to the subject an antibody that specifically binds human CTLA-4 at a dose of 75 mg or 150 mg once every six weeks, wherein the antibody is botencilimab.

[0038] In one embodiment, the metastatic colorectal adenocarcinoma is unresectable. In one embodiment, the metastatic colorectal adenocarcinoma is non-MSI-H / dMMR.

[0039] In one embodiment, the method further comprises administering to the subject an antibody that specifically binds human PD-1.

[0040] In one embodiment, an antibody that specifically binds to human PD-1 comprises a heavy chain variable region (VH) comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region (VL) comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 18.

[0041] In one embodiment, an antibody that specifically binds to human PD-1 comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 11, 12, 13, 14, 15, and 16, respectively.

[0042] In one embodiment, an antibody that specifically binds to human PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18. In one embodiment, an antibody that specifically binds to human PD-1 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0043] In one embodiment, the antibody that specifically binds to human PD-1 is balstilimab.

[0044] In one embodiment, the antibody that specifically binds to human PD-1 is administered at a dose of 200 mg to 300 mg. In one embodiment, the antibody that specifically binds to human PD-1 is administered at a dose of 240 mg.

[0045] In one embodiment, the antibody that specifically binds human PD-1 is administered once a week or once every two weeks. In one embodiment, the antibody that specifically binds human PD-1 is administered at a dose of 240 mg once every two weeks.

[0046] In one aspect, there is provided herein an antibody that specifically binds to human CTLA-4 for use in the treatment of colorectal cancer, wherein the treatment is carried out according to a method according to any one of the preceding claims.

[0047] In one aspect, there is provided herein an antibody that specifically binds to human CTLA-4 for use in the manufacture of a medicament for the treatment of colorectal cancer, wherein the treatment is carried out according to a method according to any one of the preceding claims.

[0048] In one aspect, there is provided herein the use of an antibody that specifically binds to human CTLA-4 for the treatment of colorectal cancer, the treatment being carried out according to a method according to any one of the preceding claims.

[0049] In one aspect, there is provided herein an antibody that specifically binds human CTLA-4 and an antibody that specifically binds human PD-1 for use in the treatment of colorectal cancer, wherein the treatment is carried out according to a method according to any one of the preceding claims.

[0050] In one aspect, there is provided herein an antibody that specifically binds human CTLA-4 and an antibody that specifically binds human PD-1 for use in the manufacture of a medicament for the treatment of colorectal cancer, wherein the treatment is carried out according to a method according to any one of the preceding claims.

[0051] In one aspect, there is provided herein the use of an antibody that specifically binds human CTLA-4 and an antibody that specifically binds human PD-1 for the treatment of colorectal cancer, wherein the treatment is carried out according to a method according to any one of the preceding claims. [Brief explanation of the drawings]

[0052] [Figure 1] FIG. 1 shows the percent change in total target lesions over time from the start of treatment in an efficacy-evaluable population (n=70) without active liver metastases treated with botencilimab (either 1 or 2 mg / kg every 6 weeks) and balstilimab (3 mg / kg every 2 weeks) over time from the start of combination / salvage therapy, according to embodiments of the present disclosure. [Figure 2]

[0023] Figure 1 shows the best percent change in total target lesions in the efficacy-evaluable population (n=70) without active liver metastases treated with botencilimab (either 1 or 2 mg / kg every 6 weeks) and balstilimab (3 mg / kg every 2 weeks) according to an embodiment of the present disclosure. Asterisks indicate patients who experienced a complete response (CR) or partial response (PR). [Figure 3] FIG. 1 shows overall survival probability over time from treatment initiation in the efficacy-evaluable (EE) population without active liver metastases (n=70) and the intent-to-treat (ITT) population without active liver metastases (n=77) treated with botencilimab (either 1 or 2 mg / kg every 6 weeks) and balstilimab (3 mg / kg every 2 weeks), according to an embodiment of the disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0053] The present disclosure relates to methods for treating colorectal cancer using antibodies that specifically bind to human cytotoxic T-lymphocyte antigen 4 (CTLA-4). Also provided herein are specific methods for administering antibodies that specifically bind to human CTLA-4 and reduce tumor burden in a subject. In one embodiment, the method comprises a therapeutically effective amount that safely and effectively treats metastatic colorectal cancer. In one embodiment, the method comprises a therapeutically effective amount that safely and effectively reduces tumor burden in a subject with metastatic colorectal adenocarcinoma.

[0054] definition As used herein, the terms "antibody" and "antibodies" include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, the antibody described herein refers to a polyclonal antibody population. An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. In one embodiment, the antibody is a humanized monoclonal antibody. In one embodiment, the antibody is a human monoclonal antibody.

[0055] As used herein, the term "CDR" or "complementarity determining region" refers to the noncontiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. These particular regions are described, for example, in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of Proteins of Immunological Interest. (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entireties, and the definition includes overlapping or subsets of amino acid residues relative to each other (see Table 1 below). In certain embodiments, the term "CDR" refers to the CDRs defined in MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A., "Protein Sequence and Structural Analysis of Antibody Variable Domains," Antibody Engineering, Kontermann and Duebel (eds.), Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" refers to the CDRs defined in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of Proteins of Immunological Interest. (1991). In certain embodiments, the heavy chain CDRs and light chain CDRs of an antibody are defined using different rules. In certain embodiments, the heavy and / or light chain CDRs are defined by performing a structural analysis of the antibody and identifying residues in the variable region(s) that are predicted to contact the epitope region of the target molecule (e.g., human CTLA-4). CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs. [Table 1]

[0056] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. The variable region refers to a portion of an antibody, generally a portion of either the light or heavy chain, that differs significantly in sequence among antibodies and is used in the binding and specificity of a particular antibody for its particular antigen, typically the approximately amino-terminal 110-120 or 110-125 amino acids in mature heavy chains and approximately 90-115 amino acids in mature light chains. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while the more highly conserved regions within the variable region are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In one embodiment, the variable region is a primate (e.g., non-human primate) variable region. In one embodiment, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0057] As used herein, the terms "VH" and "VL" refer to antibody heavy and light chain variable regions, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety.

[0058] As used herein, the term "constant region" is as is common in the art. The constant region is a portion of an antibody, e.g., the carboxyl-terminal portion of the light chain and / or heavy chain, that is not directly involved in binding the antibody to an antigen but can exert various effector functions, such as interacting with Fc receptors (e.g., Fc gamma receptors).

[0059] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region, which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0060] As used herein, the term "light chain," when used in reference to an antibody, can refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant region. Light chain amino acid sequences are well known in the art. In one embodiment, the light chain is a human light chain.

[0061] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," and "immunospecifically recognize" are analogous terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex), and such binding is understood by those of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), or other assays known in the art. In one embodiment, a molecule that specifically binds to an antigen binds to the antigen with a K that is at least 2 logs (e.g., 10-fold), 2.5 logs, 3 logs, 4 logs, or more, greater than the K when the molecule nonspecifically binds to another antigen.

[0062] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as described in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 5th ed., 1991, each of which is incorporated herein by reference in its entirety.

[0063] As used herein, the term "subject" includes any human or non-human animal. In one embodiment, the subject is a human.

[0064] As used herein, in the context of administering therapy, the term "effective amount" refers to the amount of therapy that achieves the desired prophylactic or therapeutic effect.

[0065] As used herein, the term "standard of care" refers to the most common treatment prescribed for a particular type of cancer. In one embodiment, the standard of care for metastatic colon cancer includes fluorouracil, capecitabine, oxaliplatin, irinotecan, or trifluridine-tipiracil.

[0066] As used herein, the term "targeted therapy" refers to a therapy that inhibits a specific protein. In one embodiment, the targeted therapy inhibits a protein (e.g., EGFR) known to be important for the growth and / or survival of colon cancer cells.

[0067] As used herein, the term "cytotoxic therapy" refers to a therapy that blocks or slows cell division. In one embodiment, the cytotoxic therapy kills cancer cells. In one embodiment, the cytotoxic therapy is fluorouracil, capecitabine, oxaliplatin, irinotecan, or trifluridine-tipiracil.

[0068] As used herein, the term "tumor burden" refers to the number of cancer cells, the size of the tumor, or the amount of cancer in a subject's body.

[0069] As used herein, the term "about" when referring to a measurable value, such as a dosage, encompasses variations of ±20%, ±15%, ±10%, ±5%, ±1%, or ±0.1% of the given value or range, as appropriate for practicing the methods disclosed herein.

[0070] Anti-CTLA-4 antibody Antibodies that specifically bind to human CTLA-4 (ie, anti-CTLA-4 antibodies) useful in the methods and uses described herein include, but are not limited to, those listed below.

[0071] In one embodiment, the antibody comprises a heavy chain variable region (VH) comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7. In one embodiment, the antibody comprises a heavy chain variable region (VH) comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region (VL) comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO:8.

[0072] In one embodiment, the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively. In one embodiment, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0073] In one embodiment, the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 7. In one embodiment, the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and a VL comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8.

[0074] In one embodiment, the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8.

[0075] In one embodiment, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In one embodiment, the heavy chain constant region is IgG1. In one embodiment, the heavy chain constant region is IgG2. In one embodiment, the antibody comprises a light chain constant region selected from the group consisting of a human kappa light chain constant region and a human lambda light chain constant region.

[0076] In one embodiment, the antibody comprises an IgG1 heavy chain constant region. In one embodiment, the amino acid sequence of the IgG1 heavy chain constant region comprises S239D / I332E mutations numbered according to the EU numbering system. In one embodiment, the amino acid sequence of the IgG1 heavy chain constant region comprises S239D / A330L / I332E mutations numbered according to the EU numbering system. In one embodiment, the amino acid sequence of the IgG1 heavy chain constant region comprises L235V / F243L / R292P / Y300L / P396L mutations numbered according to the EU numbering system. In one embodiment, the IgG1 heavy chain constant region is an afucosylated IgG1.

[0077] In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0078] In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10. In one embodiment, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 9 and the amino acid sequence of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 10.

[0079] In one embodiment, the antibody is botencilimab (also known as AGEN1181), the amino acid sequence of which is shown in Table 2 below. [Table 2]

[0080] Anti-PD-1 antibody In one embodiment, any one of the methods disclosed herein further comprises administering to the subject an antibody that specifically binds human PD-1. Antibodies that specifically bind human PD-1 (i.e., anti-PD-1 antibodies) useful in the methods and uses described herein include, but are not limited to, those listed below.

[0081] In one embodiment, an antibody that specifically binds to human PD-1 comprises a heavy chain variable region (VH) comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 17. In one embodiment, an antibody that specifically binds to human PD-1 comprises a light chain variable region (VL) comprising the CDRL1, CDRH2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 18. In one embodiment, an antibody that specifically binds to human PD-1 comprises a heavy chain variable region (VH) comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region (VL) comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 18.

[0082] In one embodiment, an antibody that specifically binds to human PD-1 comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 11, 12, and 13, respectively. In one embodiment, an antibody that specifically binds to human PD-1 comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 11, 12, 13, 14, 15, and 16, respectively. In one embodiment, an antibody that specifically binds to human PD-1 comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 11, 12, 13, 14, 15, and 16, respectively.

[0083] In one embodiment, an antibody that specifically binds to human PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 17. In one embodiment, an antibody that specifically binds to human PD-1 comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 18. In one embodiment, an antibody that specifically binds to human PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18.

[0084] In one embodiment, an antibody that specifically binds to human PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0085] In one embodiment, an antibody that specifically binds to human PD-1 comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In one embodiment, the heavy chain constant region is IgG1. In one embodiment, the heavy chain constant region is IgG2. In one embodiment, the antibody comprises a light chain constant region selected from the group consisting of a human kappa light chain constant region and a human lambda light chain constant region.

[0086] In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19. In one embodiment, the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:20.

[0087] In one embodiment, an antibody that specifically binds to human PD-1 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20. In one embodiment, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 19 and the amino acid sequence of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 20.

[0088] In one embodiment, the antibody that specifically binds to human PD-1 is balstilimab (also known as AGEN2034), the amino acid sequence of which is shown in Table 3 below. [Table 3]

[0089] Treatment method The present disclosure demonstrates that antibodies that specifically bind to human CTLA-4 (e.g., botencilimab) are highly effective in treating colorectal cancer. The present disclosure also demonstrates that antibodies that specifically bind to human CTLA-4 (e.g., botencilimab) are highly effective in treating metastatic, unresectable colorectal adenocarcinoma (e.g., non-MSI-H / dMMR metastatic colorectal adenocarcinoma). Thus, the present disclosure broadly relates to methods of treating colorectal cancer using antibodies that specifically bind to human CTLA-4. Also provided herein are specific dosing regimens for administering antibodies that specifically bind to human CTLA-4 and reduce tumor burden in a subject.

[0090] In one aspect, a method for treating colorectal cancer in a subject in need of such treatment is provided, the method comprising administering to the subject an effective amount of an antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 8.

[0091] In one aspect, provided herein is a method for enhancing T cell activation in a subject with colorectal cancer, the method comprising administering to the subject an effective amount of an antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 8.

[0092] In one aspect, the present specification provides a method for treating colorectal cancer in a subject in need of such treatment, the method comprising administering to the subject a dose of about 25 mg to about 200 mg of an antibody that specifically binds to human cytotoxic T-lymphocyte antigen 4 (CTLA-4), wherein the antibody comprises a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 8.

[0093] In one aspect, the present specification provides a method for enhancing T cell activation in a subject with colorectal cancer, the method comprising administering to the subject a dose of about 25 mg to about 200 mg of an antibody that specifically binds to human CTLA-4, the antibody comprising a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 8.

[0094] In one embodiment, the antibody is administered at a dose of about 50 mg to about 175 mg. In one embodiment, the antibody is administered at a dose of about 25 mg to about 150 mg. In one embodiment, the antibody is administered at a dose of about 50 mg to about 150 mg. In one embodiment, the antibody is administered at a dose of about 75 mg to about 150 mg.

[0095] In one embodiment, the antibody is administered at a dose of 50 mg to 175 mg. In one embodiment, the antibody is administered at a dose of 25 mg to 150 mg. In one embodiment, the antibody is administered at a dose of 50 mg to 150 mg. In one embodiment, the antibody is administered at a dose of 75 mg to 150 mg.

[0096] In one embodiment, the antibody is administered at a dose of about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, or about 150 mg.

[0097] In one embodiment, the antibody is administered at a dose of 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, or 150 mg.

[0098] In one embodiment, the antibody is administered intravenously. In one embodiment, the antibody is administered intratumorally. In one embodiment, the antibody is administered peritumorally.

[0099] In one embodiment, the antibody is administered by intravenous infusion over about 30 minutes. In one embodiment, the antibody is administered by intravenous infusion over about 45 minutes. In one embodiment, the antibody is administered by intravenous infusion over about 60 minutes. In one embodiment, the antibody is administered by intravenous infusion over about 90 minutes.

[0100] In one embodiment, the antibody is administered about once per week. In one embodiment, the antibody is administered about once per two weeks. In one embodiment, the antibody is administered about once per three weeks. In one embodiment, the antibody is administered about once per four weeks. In one embodiment, the antibody is administered about once per five weeks. In one embodiment, the antibody is administered about once per six weeks. In one embodiment, the antibody is administered about once per seven weeks. In one embodiment, the antibody is administered about once per eight weeks. In one embodiment, the antibody is administered about once per nine weeks. In one embodiment, the antibody is administered about once per ten weeks. In one embodiment, the antibody is administered about once per eleven weeks. In one embodiment, the antibody is administered about once per twelve weeks. In one embodiment, the antibody is administered about once per thirteen weeks.

[0101] In one embodiment, the antibody is administered once per week. In one embodiment, the antibody is administered once per two weeks. In one embodiment, the antibody is administered once per three weeks. In one embodiment, the antibody is administered once per four weeks. In one embodiment, the antibody is administered once per five weeks. In one embodiment, the antibody is administered once per six weeks. In one embodiment, the antibody is administered once per seven weeks. In one embodiment, the antibody is administered once per eight weeks. In one embodiment, the antibody is administered once per nine weeks. In one embodiment, the antibody is administered once per ten weeks. In one embodiment, the antibody is administered once per eleven weeks. In one embodiment, the antibody is administered once per twelve weeks. In one embodiment, the antibody is administered once per thirteen weeks.

[0102] In one embodiment, the antibody is administered intravenously at a dose of about 25 mg once every six weeks. In one embodiment, the antibody is administered intravenously at a dose of about 50 mg once every six weeks. In one embodiment, the antibody is administered intravenously at a dose of 75 mg once every six weeks. In one embodiment, the antibody is administered intravenously at a dose of 100 mg once every six weeks. In one embodiment, the antibody is administered intravenously at a dose of 150 mg once every six weeks.

[0103] In one embodiment, the dose is a therapeutically effective amount.

[0104] In one embodiment, the colon cancer is colorectal adenocarcinoma. In one embodiment, the colon cancer is metastatic. In one embodiment, the colon cancer is unresectable. In one embodiment, the colon cancer is metastatic unresectable colorectal adenocarcinoma. In one embodiment, the subject does not have liver metastases. In one embodiment, the subject has liver metastases.

[0105] In one embodiment, the antibody is administered to the subject prior to surgical resection of the primary tumor. In one embodiment, the subject has not previously received any chemotherapy. In one embodiment, the subject has not previously received any radiation therapy.

[0106] In one embodiment, the colorectal cancer is recurrent and / or refractory. In one embodiment, the subject has received at least one previous chemotherapy. In one embodiment, the at least one previous chemotherapy is a fluoropyrimidine, irinotecan, oxaliplatin, or an anti-EGFR antibody. In one embodiment, the anti-EGFR antibody is cetuximab or panitumumab.

[0107] In one embodiment, the subject has previously been treated with folinic acid, 5-fluorouracil, oxaliplatin, and irinotecan (ie, FOLFOXIRI therapy).

[0108] In one embodiment, the subject is unable to tolerate standard care treatment.

[0109] In one embodiment, the subject has a RAS mutation. In one embodiment, the RAS mutation is a KRAS or NRAS mutation.

[0110] In one embodiment, the colon cancer is not microsatellite instability-high (MSI-H). In one embodiment, the colon cancer is microsatellite stable (MSS). In one embodiment, the colon cancer is not mismatch repair deficient (dMMR).

[0111] In one embodiment, the subject has not received prior immune checkpoint inhibitor therapy, hi one embodiment, the subject has not received prior treatment with an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody.

[0112] In one embodiment, the subject has previously received immune checkpoint inhibitor therapy, hi one embodiment, the subject has previously received treatment with an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody.

[0113] In one embodiment, the cancer may be positive or negative for PD-L1 expression (e.g., if tumor-infiltrating immune cells stained for PD-L1 cover less than 5% of the tumor area as determined by immunohistochemistry or by a commercially available companion diagnostic, including diagnostics with FDA premarket approval numbers P160002, P150013, or P150025).

[0114] In one embodiment, the subject has not previously received regorafenib, trifluridine, and / or tipiracil therapy.

[0115] In one embodiment, the cancer is refractory to standard care treatment. In one embodiment, the standard care treatment is chemotherapy or radiation therapy. In one embodiment, the standard care treatment is folinic acid, 5-fluorouracil, oxaliplatin, irinotecan, a fluoropyrimidine, cetuximab, and / or panitumumab.

[0116] In one embodiment, administration of the antibody reduces tumor size in the subject. In one embodiment, administration of the antibody increases activation of T cells, memory T cells, myeloid cells, and / or antigen-presenting cells in the subject. In one embodiment, administration of the antibody reduces the number of Treg cells in the subject. In one embodiment, administration of the antibody increases the expansion of new T cell clones.

[0117] In one embodiment, administration of the antibody increases the level of one or more cytokines. In one embodiment, the one or more cytokines are CXCL9 or CXCL10. In one embodiment, administration of the antibody increases the level of IFN-γ, HLA-DR, and / or ICOS expression.

[0118] In one embodiment, prior to administration of the antibody, the subject has measurable disease on baseline imaging according to RECIST 1.1.

[0119] In one embodiment, prior to administration of the antibody, the subject has an Eastern Cooperative Oncology Group performance status (PS) of 0 to 1. In one embodiment, prior to administration of the antibody, the subject has a predicted life expectancy of 12 weeks or greater. In one embodiment, prior to administration of the antibody, the subject has adequate organ function as defined by one or more of: a) neutrophils > 1500 / μL, b) platelets > 100 x 10 / μL, c) hemoglobin > 8.0 g / dL, d) creatinine clearance > 30 mL / min as measured or calculated according to local institutional standards, e) AST / ALT < 2.5 x upper limit of normal (ULN), f) total bilirubin < 1.5 x ULN (except for patients with Gilbert's syndrome, in which total bilirubin values ​​should be < 3.0 x ULN), and / or g) albumin > 3.0 g / dL.

[0120] In one embodiment, the subject has no partial or complete bowel obstruction, signs / symptoms of bowel obstruction, or known radiological evidence of impending obstruction within the past three months.

[0121] In one embodiment, the subject does not have refractory ascites, defined as requiring two or more therapeutic paracentesis within the past four weeks, or four or more therapeutic paracentesis within the past 90 days, or one or more therapeutic paracentesis within the past two weeks prior to administration of the antibody.

[0122] In one embodiment, the subject does not have clinically significant cardiovascular disease. In one embodiment, the subject does not have active brain or leptomeningeal metastases.

[0123] In one embodiment, the subject does not have a history of a concurrent malignancy requiring treatment or a malignancy that was active within two years prior to administration of the antibody.

[0124] In one embodiment, the subject has not received a cytotoxic, targeted, or other investigational therapy within three weeks prior to administration of the antibody.

[0125] In one embodiment, the subject has not received any other monoclonal antibody therapy, antibody drug conjugate therapy, or radioimmunoconjugate therapy within four weeks prior to administration of the antibody.

[0126] In one embodiment, the subject has not received small molecule tyrosine kinase inhibitor therapy within two weeks prior to administration of the antibody.

[0127] In one embodiment, subjects' objective response rate (ORR), duration of response (DOR), disease control rate (DCR), and progression-free survival (PFS) are assessed according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1).

[0128] In one embodiment, the method results in a complete response as defined by RECIST 1.1. In one embodiment, the method results in a partial response as defined by RECIST 1.1. In one embodiment, the method results in stable disease as defined by RECIST 1.1.

[0129] In one embodiment, the method results in about a 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% reduction in tumor burden in the subject. In one embodiment, the method results in no change in tumor burden in the subject. In one embodiment, the method results in about a 1% reduction in tumor burden in the subject. In one embodiment, the method results in about a 5% reduction in tumor burden in the subject. In one embodiment, the method results in about a 10% reduction in tumor burden in the subject. In one embodiment, the method results in about a 20% reduction in tumor burden in the subject. In one embodiment, the method results in about a 30% reduction in tumor burden in the subject. In one embodiment, the method results in about a 40% reduction in tumor burden in the subject. In one embodiment, the method results in about a 50% reduction in tumor burden in the subject. In one embodiment, the method results in about a 60% reduction in tumor burden in the subject. In one embodiment, the method results in about a 70% reduction in tumor burden in the subject. In one embodiment, the method results in about a 80% reduction in tumor burden in the subject. In one embodiment, the method results in about a 90% reduction in tumor burden in the subject. In one embodiment, the method results in about a 100% reduction in tumor burden in the subject.

[0130] In one embodiment, the method reduces tumor burden. In one embodiment, the method increases survival. In one embodiment, the method increases overall survival. In one embodiment, the method increases progression-free survival.

[0131] In one aspect, provided herein is a method of treating metastatic colorectal adenocarcinoma in a subject in need thereof, the method comprising administering to the subject an antibody that specifically binds human CTLA-4, wherein the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8, at a dose of 75 mg or 150 mg once every six weeks.

[0132] In one aspect, provided herein is a method of treating metastatic colorectal adenocarcinoma in a subject in need thereof, the method comprising: a) administering to the subject an antibody that specifically binds human CTLA-4, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8, at a dose of 75 mg once every six weeks; and b) administering to the subject an antibody that specifically binds human PD-1, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:17 and a VL comprising the amino acid sequence set forth in SEQ ID NO:18, at a dose of 240 mg once every two weeks.

[0133] In one aspect, provided herein is a method of treating metastatic colorectal adenocarcinoma in a subject in need thereof, the method comprising: a) administering to the subject an antibody that specifically binds human CTLA-4, wherein the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8, at a dose of 150 mg once every six weeks; and b) administering to the subject an antibody that specifically binds human PD-1, wherein the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:17 and a VL comprising the amino acid sequence set forth in SEQ ID NO:18, at a dose of 240 mg once every two weeks.

[0134] In one aspect, provided herein is a method of treating metastatic colorectal adenocarcinoma in a subject in need thereof, the method comprising: a) administering to the subject an antibody that specifically binds human CTLA-4, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8, at a dose of 75 mg once every six weeks; and b) administering to the subject an antibody that specifically binds human PD-1, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:17 and a VL comprising the amino acid sequence set forth in SEQ ID NO:18, at a dose of 450 mg once every three weeks.

[0135] In one aspect, provided herein is a method of treating metastatic colorectal adenocarcinoma in a subject in need thereof, the method comprising: a) administering to the subject an antibody that specifically binds human CTLA-4, wherein the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8, at a dose of 150 mg once every six weeks; and b) administering to the subject an antibody that specifically binds human PD-1, wherein the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:17 and a VL comprising the amino acid sequence set forth in SEQ ID NO:18, at a dose of 450 mg once every three weeks.

[0136] In one embodiment, the metastatic colorectal adenocarcinoma is unresectable. In one embodiment, the metastatic colorectal adenocarcinoma is non-MSI-H / dMMR.

[0137] In one embodiment, any one of the methods disclosed herein further comprises administering to a subject an antibody that specifically binds to human PD-1. In one embodiment, the antibody that specifically binds to human PD-1 is administered at a dose of 200 mg to 500 mg. In one embodiment, the antibody that specifically binds to human PD-1 is administered at a dose of 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, or 500 mg. In one embodiment, the antibody that specifically binds to human PD-1 is administered at a dose of 240 mg. In one embodiment, the antibody that specifically binds to human PD-1 is administered at a dose of 450 mg.

[0138] In one embodiment, the antibody that specifically binds human PD-1 is administered once a week, once every two weeks, or once every three weeks. In one embodiment, the antibody that specifically binds human PD-1 is administered at a dose of 240 mg once every two weeks. In one embodiment, the antibody that specifically binds human PD-1 is administered at a dose of 450 mg once every three weeks.

[0139] In one embodiment, any one of the methods disclosed herein further comprises administering an antibody that specifically binds to human PD-1 at a dose of about 240 mg once every two weeks. In one embodiment, any one of the methods disclosed herein further comprises administering an antibody that specifically binds to human PD-1 at a dose of 240 mg once every two weeks. In one embodiment, any one of the methods disclosed herein further comprises administering an antibody that specifically binds to human PD-1 at a dose of about 450 mg once every three weeks. In one embodiment, any one of the methods disclosed herein further comprises administering an antibody that specifically binds to human PD-1 at a dose of 450 mg once every three weeks.

[0140] In one embodiment, any one of the methods disclosed herein further comprises administering balstilimab to the subject. In one embodiment, any one of the methods disclosed herein further comprises administering balstilimab to the subject at a dose of about 240 mg once every two weeks. In one embodiment, any one of the methods disclosed herein further comprises administering balstilimab to the subject at a dose of 240 mg once every two weeks. In one embodiment, any one of the methods disclosed herein further comprises administering balstilimab to the subject at a dose of about 450 mg once every three weeks. In one embodiment, any one of the methods disclosed herein further comprises administering balstilimab to the subject at a dose of 450 mg once every three weeks.

[0141] In one embodiment, the antibody that specifically binds human PD-1 is administered intravenously. In one embodiment, the antibody that specifically binds human PD-1 is administered by intravenous infusion over about 30 minutes.

[0142] In one embodiment, the antibody that specifically binds human PD-1 is administered before the antibody that specifically binds human CTLA-4.

[0143] In one aspect, provided herein is an antibody that specifically binds human CTLA-4 for use in treating colorectal cancer, wherein the treatment is carried out according to the methods disclosed herein.

[0144] In one aspect, provided herein is an antibody that specifically binds human CTLA-4 for use in the manufacture of a medicament for the treatment of colorectal cancer, wherein the treatment is carried out according to the methods disclosed herein.

[0145] 10. Use of an antibody that specifically binds to human CTLA-4 for the treatment of colorectal cancer, the treatment being carried out according to a method according to any one of the preceding claims.

[0146] In one aspect, there is provided herein an antibody that specifically binds human CTLA-4 and an antibody that specifically binds human PD-1 for use in the treatment of colorectal cancer, wherein the treatment is carried out according to a method according to any one of the preceding claims.

[0147] In one aspect, there is provided herein an antibody that specifically binds human CTLA-4 and an antibody that specifically binds human PD-1 for use in the manufacture of a medicament for the treatment of colorectal cancer, wherein the treatment is carried out according to a method according to any one of the preceding claims.

[0148] In one aspect, there is provided herein the use of an antibody that specifically binds human CTLA-4 and an antibody that specifically binds human PD-1 for use in the treatment of colorectal cancer, wherein the treatment is carried out according to a method according to any one of the preceding claims. [Example]

[0149] Example 1 - Phase 2 Study of Botencilimab (AGEN1181) Monotherapy and Combination with Balstilimab (AGEN2034) in the Treatment of Refractory Metastatic Colorectal Cancer This phase 2 study further evaluated the safety and efficacy of botencilimab as monotherapy and in combination with balstilimab (AGEN2034, an anti-PD-1 antibody) in patients with adenocarcinoma of the colon or rectum who had received at least one prior chemotherapy regimen for metastatic or recurrent disease. The study also aimed to optimize the dosing of botencilimab in patients with metastatic CRC.

[0150] A. Study Design Overall Design This was a multicenter, randomized, open-label, phase 2 study investigating botencilimab as monotherapy or in combination with balstilimab, or the investigator's choice of standard of care (SoC), regorafenib or trifluridine in combination with tipiracil. The study enrolled patients with adenocarcinoma of the colon or rectum who had received at least one prior chemotherapy regimen for metastatic or recurrent disease.

[0151] Patients included in this study had progressed on or were intolerant to fluoropyrimidines, irinotecan, oxaliplatin, and, where applicable, anti-EGFR-directed therapy. Most patients had received multiple lines of prior therapy. Only RAS-mutated patients who received FOLFOXIRI in the first line were eligible for this study as early as the second line. Patients with active liver metastases were excluded from this study.

[0152] Eligible patients were randomly assigned in a 1:1:1:1:1 ratio to Group A, B, C, D, or E. Baseline study procedures included radiological imaging (computed tomography [CT] or magnetic resonance imaging [MRI] of the chest, abdomen, and pelvis [C / A / P], and central nervous system [CNS] imaging for patients with a history of brain metastases) and routine clinical examinations.

[0153] Test treatment Treatment groups included: Arm A: Botencilimab 75 mg administered intravenously every 6 weeks for up to 4 doses and balstilimab 240 mg administered intravenously every 2 weeks. Balstilimab may be continued for up to 2 years (17 6-week cycles). Arm B: Botencilimab 150 mg administered intravenously every 6 weeks for up to 4 doses and balstilimab 240 mg administered intravenously every 2 weeks. Balstilimab may be continued for up to 2 years (17 6-week cycles). -Arm C: Botencilimab 75 mg administered intravenously every 6 weeks for up to 4 doses. -Arm D: Botencilimab 150 mg IV every 6 weeks for up to 4 doses. -Arm E: Investigator selected SoC: Oral administration of regorafenib: 160 mg (4 x 40 mg tablets) once daily for the first 21 days of each 28-day cycle, or ■ 80 mg (2 × 40 mg tablets) was administered once daily on days 1–7 of cycle 1, followed by 120 mg (3 × 40 mg tablets) once daily on days 8–14 of cycle 1, followed by 160 mg (4 × 40 mg tablets) once daily on days 15–21 of cycle 1 (28-day cycle). Subsequent 28-day cycles consisted of 160 mg (4 × 40 mg tablets) administered once daily on days 1–21. 35 mg / m 2 / dose of trifluridine and tipiracil [rounded to the nearest 5 mg] was administered orally twice daily on days 1 to 5 and 8 to 12 of a 28-day cycle. [Table 4]

[0154] During the treatment period, patients received study treatment and underwent regular clinical visits to monitor safety, health status, and changes in their medical condition.

[0155] Antitumor efficacy was determined by imaging assessments performed at weeks 8, 16, and 24, and then every 12 weeks thereafter. Disease response assessments were performed by the investigator and, at the sponsor's discretion, may be assessed by an independent central review.

[0156] Safety was assessed through monitoring of AEs, SAEs, treatment discontinuation due to AEs, physical examination, vital signs, hematology, and chemistry. Patients who discontinued study treatment for reasons other than progressive disease (PD) were allowed to continue with imaging evaluation.

[0157] Patients who discontinued study visits and imaging studies were followed up every 12 weeks by telephone to assess their survival status.Futility analyses were performed for both botencilimab arms, with the condition that if one arm was discontinued at a prespecified time point, the other arm could be continued.

[0158] In the monotherapy group, patients were treated for approximately 24 weeks or until one of the criteria for discontinuation of study treatment was met. In the combination therapy group, patients were treated with balstilimab for up to 2 years (17 6-week cycles) or until one of the criteria for discontinuation of study treatment was met. In the SoC group, patients were treated with regorafenib or trifluridine and tipiracil until disease progression or unacceptable toxicity.

[0159] Administration of test drug Botencilimab was administered as monotherapy via IV infusion over 30 (±5) minutes. Patients were observed for 30 minutes after the end of the infusion. Vital signs were measured before and at the end of each infusion in each cycle. Saline flushing of the IV line was performed immediately after the infusion, according to institutional guidelines.

[0160] In the combination group, balstilimab was administered before botencilimab. Balstilimab was administered by IV infusion over 30 (± 5) minutes. Patients were observed for IRR for 30 minutes after balstilimab infusion (no botencilimab was administered during this observation period). Botencilimab was administered by IV infusion over 30 (± 5) minutes. Patients were observed for 30 minutes after the end of the infusion. Vital signs were measured before the start of each infusion and at the end of each infusion in each cycle. Saline flushing of the IV line was performed immediately after the infusion, according to institutional guidelines.

[0161] If the dose level of botencilimab (monotherapy or combination therapy) was changed by the SMC and a different dose level was subsequently opened, patients had the option to change their botencilimab dose to the new dose level, as determined by the investigator and approved by the sponsor. Dose reductions and / or increases of botencilimab or balstilimab were not permitted for any other reason in this study. Patients received the full dose of botencilimab and balstilimab according to the protocol or until treatment was appropriately withheld or discontinued. If treatment was withheld and the patient was ready to resume treatment, the subsequent treatment was administered on the next protocol-specified treatment date, i.e., treatments were not postponed, skipped, or administered unscheduled.

[0162] Number of patients Approximately 230 patients will be enrolled, with the potential for expansion to approximately 50 patients per botencilimab arm, with approximately 30 patients per arm. Patients who discontinued the study were not replaced. Existing arms may be stopped early due to futility, and additional patients may be enrolled at different dose levels of botencilimab, per SMC recommendations.

[0163] Treatment after disease progression Patients who tolerated the drug and were deemed by the investigator to be experiencing clinical benefit were permitted to continue treatment beyond initial PD as defined by RECIST 1.1, with sponsor approval, if they met the following clinical stability criteria: - Absence of clinical symptoms and signs of PD (including deterioration of laboratory values), i.e., patient is clinically stable as judged by the investigator. - No decline in Eastern Cooperative Oncology Group (ECOG) performance status due to underlying malignancy. - Absence of rapidly progressing disease or aggressive tumors in critical anatomical locations (e.g., spinal cord compression) requiring urgent alternative medical intervention.

[0164] Patients treated after progression had to first provide written informed consent using their designated ICF before receiving additional study drug infusions. New lesions were considered measurable at the time of initial progression if their longest diameter was 10 mm or greater (excluding pathological lymph nodes, which must have a short axis of 15 mm or greater). New lesions that were considered nonmeasurable at the time of initial progression became measurable and could be included in tumor burden measurements if their longest diameter increased to 10 mm or greater (excluding pathological lymph nodes, which must have a short axis of 15 mm or greater).

[0165] Treatment duration In groups A–D, patients received study drug for up to four doses over 24 weeks in the monotherapy group and for up to two years (17 6-week cycles) in the combination group, continuing until disease progression (with the exceptions noted above), unacceptable toxicity, or the patient withdrew consent for any reason. In group E (i.e., SoC), patients received study drug until disease progression or unacceptable toxicity.

[0166] After treatment completion or discontinuation (e.g., due to toxicity), patients were followed for safety at 30 and 90 days, with long-term follow-up every 3 months until study completion or patient withdrawal.

[0167] Prohibited Drugs and Treatments Any medication or vaccination specifically prohibited by the exclusion criteria was not permitted during the ongoing study. A clinical indication for a specifically prohibited medication or vaccination during the study could necessitate discontinuation of study treatment or vaccination. The final decision regarding supportive care or vaccination was made by the investigator and / or the patient's physician. However, the decision to continue a patient on study treatment required mutual agreement between the investigator, sponsor, and patient.

[0168] Specific restrictions regarding concomitant therapy during the study period are as follows: -Anti-tumor systemic chemotherapy or biologic therapy other than SoC. - Immuno-oncology therapy not specified in this protocol. - Investigational drug other than the assigned study treatment. -Live vaccines. -Systemic glucocorticoids (more than 10 mg prednisone equivalent for more than 1 week) for any purpose other than treatment of immune-related adverse events. Note: Prophylactic corticosteroid use to avoid allergic reactions (e.g., IV contrast or blood transfusions) is permitted, as is the use of inhaled steroids or intranasal or local injection of corticosteroids.

[0169] In addition to the above, patients taking regorafenib were required to avoid strong CYP3A4 inducers or inhibitors.

[0170] Patients treated with prohibited medications for clinical management (with the exceptions noted above) were excluded from the study. Rare exceptions, such as the use of glucocorticoids for reasons other than irAEs, were considered with the approval of the medical monitor. All treatments deemed necessary for the patient's welfare by the investigator were available at the investigator's discretion in accordance with community standards of care. All concomitant medications, including prescription and over-the-counter medications, herbal supplements, and IV medications and fluids, were recorded on the electronic case report form (eCRF). Medication doses, frequency, route of administration, and dates were recorded on the eCRF if they changed during the study period.

[0171] Surgery and radiation therapy If patients required surgery for management of progressive malignancy, study treatment was discontinued. In the case of surgery for bowel obstruction, patients were allowed to continue study treatment if PD was not confirmed. Palliative radiation therapy for non-target lesions was permitted after consultation with the sponsor based on specific clinical scenarios.

[0172] Permanent discontinuation of study drug treatment Botencilimab or balstilimab treatment was permanently discontinued for any of the following reasons: - Occurrence of immune-related adverse events meeting discontinuation criteria. - Confirmed PD unless the investigator deems the patient to be deriving clinical benefit from treatment, the patient is clinically stable, and approved by the sponsor. - In the absence of radiological progression per RECIST 1.1, clinical progression as indicated by one or more of the following: Signs and / or symptoms consistent with clinically significant disease progression (including worsening laboratory values, appearance of new lesion(s) / worsening of the most common clinical lesion, etc.). - Decline in ECOG performance status due to worsening malignancy. Tumor progression in critical anatomical locations requiring urgent medical intervention (e.g., CNS metastases with possible spinal progression). - Missed two or more consecutive study treatment sessions due to non-compliance, unless otherwise approved by the sponsor. -pregnancy

[0173] A tumor flare event was defined as localized pain, irritation, or rash confined to the site of a known or suspected tumor and did not require treatment discontinuation.

[0174] Regorafenib or trifluridine and tipiracil was permanently discontinued if patients experienced disease progression or unacceptable toxicity. Please refer to the approved prescribing information for additional information regarding discontinuation of regorafenib or trifluridine and tipiracil.

[0175] Definition of Exam Completion If the study was not terminated for the reasons mentioned above, the overall study ended 24 months after the last patient completed the 90-day safety follow-up visit. If the last patient withdrew from the study or was lost to follow-up before the 90-day safety follow-up visit (i.e., the patient could not be contacted by the investigator), the study ended approximately 24 months after the date of that event.

[0176] B. Study Population Inclusion criteria To participate in the study, patients must meet all of the following inclusion criteria: 1. Histologically confirmed diagnosis of unresectable metastatic colorectal adenocarcinoma. 2. Tumors must have been evaluated for MSI-H or dMMR status according to standard local testing methods. 3. The patient, or if the patient is unable to do so, their legally authorized representative, must have voluntarily agreed to participate by providing signed and dated written informed consent prior to any study-specific procedures. 4. 18 years of age or older. 5. Patients must have received at least one of the following chemotherapy regimens for metastatic or recurrent CRC: Standard chemotherapy including all of the following agents (if eligible and without contraindications): fluoropyrimidine, irinotecan, oxaliplatin, and, if applicable, an anti-EGFR antibody (cetuximab or panitumumab). These agents may be given in combination; for example, if FOLFOXIRI is given as the first line of treatment, RAS-mutated patients may be eligible for this trial as the second line of treatment; more commonly, the agents are given sequentially, with most patients eligible as the third line or later. b. Patients must have progressed while receiving or within 3 months of their last line of standard therapy, or be intolerant of any of these standard therapies due to toxicity that justifies discontinuation of treatment and precludes retreatment with the same agent. c. Patients who received adjuvant chemotherapy and relapse during or within 6 months of completing adjuvant chemotherapy may count this as a line of treatment. 6. Measurable disease on baseline imaging per RECIST 1.1. 7. Life expectancy ≥ 12 weeks. 8.ECOG performance status 0 or 1. 9. Adequate organ function as defined by the following laboratory values ​​within 7 days of C1D1: a. Neutrophil counts ≥ 1500 / μL (must be stable and growth factor-free within 4 weeks of first study treatment administration). b. Platelets ≥ 100 x 103 / μL (transfusions to achieve this level were not permitted within 2 weeks of the first study treatment administration). c. Hemoglobin ≥ 8.0 g / dL (transfusions to achieve this level were not permitted within 2 weeks of the first study drug administration). d. Creatinine clearance ≥ 30 mL / min measured or calculated according to local institutional standards. e.AST / ALT≦2.5×upper limit of normal (ULN). f. Total bilirubin ≤ 1.5 × ULN (except for patients with Gilbert's syndrome, for whom total bilirubin levels should be ≤ 3.0 × ULN). g. Albumin ≥ 3.0 g / dL. 10. A recent biopsy of the tumor lesion available as a formalin-fixed, paraffin-embedded (FFPE) tumor tissue block was required. If recent tumor tissue is unavailable or insufficient, the patient must be willing to provide a fresh biopsy if deemed safe and feasible. Once a sufficient number of biopsies have been collected, the sponsor may waive the screening biopsy requirement. 11. Women of childbearing potential (WOCBP) must have a negative serum pregnancy test at screening (within 72 hours of the first dose of study drug) and before study drug administration. 12. Male patients with female partners of childbearing potential must agree to use highly effective contraception for the duration of the study, from the screening visit through 3 months after the final dose of study medication. Men with pregnant partners must agree to use condoms. No additional contraception is required for pregnant partners. 13. Willing and able to comply with the requirements of the Protocol.

[0177] Exclusion criteria Participants were excluded from the study if they met any of the following criteria: 1. The tumor was MSI-H / dMMR by standard local testing. 2. Received any ICI or experimental immunological agent including PD-1, PD-L1, or CTLA-4 antibodies. 3. Previous treatment with regorafenib or trifluridine / tipiracil. 4. Partial or complete bowel obstruction, signs / symptoms of bowel obstruction, or known radiological evidence of impending obstruction within the past 3 months. 5. Refractory ascites is defined as two or more therapeutic paracentesis within the past four weeks, four or more therapeutic paracentesis within the past 90 days, one or more therapeutic paracentesis within two weeks prior to study entry, or the need for diuretics within two weeks prior to study entry. 6. Liver metastases by CT or MRI. Note: Patients with definitively treated liver metastases (including surgical resection or stereotactic body radiotherapy [SBRT], but not including Y-90 or chemotherapy alone) are eligible if they received treatment at least 6 months prior to enrollment and have no evidence of metastatic disease in the liver on subsequent imaging. However, they must be excluded if: a.Received more than one SBRT field to the liver. b. Underwent major hepatectomy (right, extended right, or extended left) and SBRT was performed on the remnant liver. c. History of variceal bleeding, history of ascites related to cirrhosis, or signs of hepatic insufficiency, including severe portal hypertension. 7. Clinically significant (i.e., active) cardiovascular disease: cerebrovascular accident / stroke or myocardial infarction within 6 months of enrollment, unstable angina, congestive heart failure (New York Heart Association class ≥ III), or severe uncontrolled arrhythmia requiring medical therapy. a.QTcF (QTc interval corrected using Fridericia's formula) is greater than 480 ms. 8. Active brain or leptomeningeal metastases with the following exceptions: a. Treated brain metastases required a) surgical resection or b) stereotactic radiosurgery. These patients were required to have discontinued steroid therapy for the purpose of managing brain metastases at least 10 days before randomization. If the patient's last brain MRI was within the screening window, repeat brain imaging after surgical resection or stereotactic radiosurgery was not required. Whole-brain radiation therapy was not permitted. b. Untreated solitary brain metastases too small (e.g., 1-2 mm) and / or of unknown origin to be treated by surgical resection or stereotactic radiosurgery were potentially eligible but had to be approved in consultation with the trial medical monitor. 9. A concurrent malignancy requiring treatment (present at screening) or a history of a previous malignancy that was active within 2 years prior to the first dose of study treatment. Patients with a history of a previous malignancy were eligible if treatment was completed at least 2 years prior to the first dose of study treatment and the patient had no evidence of disease. Patients with a history of previous early-stage basal / squamous cell skin cancer, low-risk prostate cancer eligible for active surveillance, or noninvasive or in situ cancer that had received curative treatment at any time were also eligible. 10. Treatment with any of the following classes of medications within the specified time frame prior to C1D1: a. Have received cytotoxic therapy, targeted therapy, or other investigational therapy within the past 3 weeks. b. Have received monoclonal antibody, antibody-drug conjugate, radioimmunoconjugate, or similar therapy within 4 weeks or 5 half-lives, whichever is shorter. c. Have received a small molecule / tyrosine kinase inhibitor within 2 weeks or less than 5 circulating half-lives of the study drug. 11. Have received a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine or booster vaccination within 7 days prior to C1D1. 12. Known allergy or hypersensitivity to the study drug or any of the study drug excipients. 13. Current evidence of interstitial lung disease (ILD) or pneumonia, or a history of ILD or non-infectious pneumonia requiring glucocorticoids. 14. History of allogeneic organ transplantation. 15. Mental illness or substance abuse disorder that prevents cooperation with the requirements of the examination. 16. Patients who require systemic treatment with corticosteroids (more than 10 mg prednisone equivalent per day) within 14 days of the first dose of study treatment, or treatment with another immunosuppressant within 30 days. In the absence of active autoimmune disease, inhaled or topical steroids and adrenal replacement steroids (less than 10 mg prednisone equivalent per day) were permitted. 17. Active autoimmune disease or a history of autoimmune disease that required systemic treatment (i.e., use of disease-modifying or immunosuppressive drugs) within 2 years of starting study treatment. 18. Past or current evidence of any medical history, comorbidity, treatment, active infection, or laboratory abnormality that may confound the results of the study or interfere with the patient's participation throughout the study, or that, in the opinion of the treating investigator, would not be in the patient's best interest to participate in the study. 19. Prior to C1D1, you have been infected with SARS-CoV-2 within 10 days if you had mild or asymptomatic symptoms, or within 20 days if you had severe / critical symptoms. 20. Uncontrolled human immunodeficiency virus (HIV) infection. Patients were eligible if they had been on stable highly active antiretroviral therapy for at least 6 months prior to study entry, had an undetectable viral load, and had a normal CD4 count. HIV serology testing at screening was not required. 21. Known hepatitis B virus (HBV) surface antigen positivity or other positive test for HBV indicating acute or chronic infection. Patients who have received or have received anti-HBV therapy and have had undetectable HBV DNA for at least 6 months prior to study entry were eligible. HBV serology testing at screening was not required. 22. Known active hepatitis C virus (HCV) as determined by positive serology and confirmed by PCR. Patients who have received or have received antiretroviral therapy were eligible if they had no detectable virus by PCR for at least 6 months prior to study entry. HCV serology was not required at screening. 23. Urinary protein was 1 gram / 24 hours or more. 24. Uncontrolled hypertension: Repeated measurements of systolic blood pressure ≥ 150 mmHg or diastolic blood pressure ≥ 90 mmHg that cannot be controlled with standard antihypertensive medication within 28 days of the first dose of study drug(s). 25. Patients who required treatment with strong CYP3A4 inducers or inhibitors. 26. Had gastrointestinal disease such as malabsorption that may affect the absorption of the study drug(s). 27. Non-healing wound(s). 28. Symptomatic active bleeding.

[0178] C. Test Evaluation and Procedures All screening assessments were completed and reviewed to ensure potential patients met all eligibility criteria. Investigators maintained a screening log to record details of all patients screened and to confirm eligibility and, where applicable, record reasons for failure to screen.

[0179] Procedures performed as part of the patient's routine clinical management (e.g., blood count) and obtained before signing the informed consent form (ICF) may be used for screening or baseline purposes if the procedure meets the criteria specified in the protocol and is performed within the time frame of other baseline assessments.

[0180] Repeat or unscheduled samples and images may be taken for safety reasons or due to technical problems with the sample.

[0181] screening The investigator or a qualified representative obtained written informed consent from each potential patient before participation in the clinical trial. After the patient signed the ICF, they were assigned a unique, consecutive patient number. Once assigned, a number cannot be reassigned, even if the original patient proves ineligible or withdraws consent.

[0182] Patients who met all inclusion criteria and none of the exclusion criteria were enrolled in the study. Patients who did not meet the inclusion and exclusion criteria were considered screen failures, and their demographic information and reasons for screen failure were documented.

[0183] During the screening period, attention was paid to previous treatments and washout periods of prohibited drugs.

[0184] Treatment and Evaluation Period Patients continued to undergo all evaluations while actively receiving treatment.

[0185] Treatment discontinuation visit Discontinuation visits (within 7 days of meeting the criteria for permanent discontinuation) were conducted only if study medication was discontinued for reasons other than completion of the entire study treatment period. If the discontinuation visit occurred approximately 30 days after the last dose of study medication, the procedure did not need to be repeated at the time of the mandatory 30-day safety follow-up visit. If a patient discontinued study treatment at the clinic visit, a treatment discontinuation visit assessment was conducted. Patients were not required to return for a separate treatment discontinuation visit.

[0186] 30-day safety follow-up visit A mandatory 30-day safety follow-up visit was conducted for all patients 30 days (± 7 days) after the last dose of study drug or before the initiation of new anti-tumor treatment, whichever occurred first. Patients with AEs > Grade 1 were further followed until the AE resolved from Grade 0 to 1 or new anti-tumor treatment was initiated (whichever occurred first). Patients with ongoing TRAEs at the safety visit were followed until the TRAE resolved, stabilized, or was deemed clinically insignificant by the investigator.

[0187] All patients who discontinued treatment for any reason also underwent a 90-day safety follow-up visit (±7 days).

[0188] Efficacy follow-up (treatment discontinued, ongoing) Patients who discontinued study treatment for reasons other than PD continued to undergo imaging evaluation as scheduled.

[0189] Survival Tracking For patients who discontinued study visits and imaging (e.g., after initiating subsequent antitumor therapy), patients were contacted by telephone every 12 weeks (± 14 days) until the end of the entire study to assess their survival status. Survival data for patients lost to follow-up were obtained from official records.

[0190] Anti-cancer therapy after clinical trials The investigator or a qualified representative reviewed all new anticancer therapies initiated after the last dose of study drug. If a patient initiated a new anticancer therapy within 4 weeks of the last dose of study drug, a 30-day safety follow-up visit must be conducted before the first dose of the new therapy. Once the new anticancer therapy was initiated, the patient was transitioned to survival follow-up.

[0191] Efficacy evaluation Response assessments were performed according to RECIST 1.1 (Eisenhauer 2009). For all patients, tumor response assessments were obtained by radiological imaging, with assessment of C / A / P (and other areas as needed depending on the specific tumor type or medical history) by CT (preferred) or MRI (if CT was contraindicated). Generally, lesions detected at baseline were followed up at subsequent tumor assessment visits using the same imaging technique, preferably the same imaging device. During disease assessment, if a lesion was deemed to represent fibrosis or scarring, the site could be examined for the presence of residual disease using fine-needle aspiration biopsy or fluorodeoxyglucose PET. If no active disease was present, the patient could be considered in CR according to RECIST 1.1. Tumor response to treatment was assigned based on the assessment of target, non-target, and new lesion response according to RECIST 1.1 (all measurements were recorded in metric notation) (Eisenhauer 2009). To assess objective response, baseline tumor burden was estimated and used for comparison with subsequent measurements. At baseline, tumor lesions were classified as target and non-target lesions (Eisenhauer 2009). The results of these assessments were recorded in as much detail as possible to ensure that pre- and post-treatment results provided the best opportunity to accurately assess tumor response.

[0192] CR or PR was confirmed at the next scheduled scan (Eisenhauer 2009). If patients discontinued treatment but continued on the study, scans were performed according to the treatment schedule. Additional imaging was performed if clinically indicated at the investigator's discretion.

[0193] Tumor Imaging The first tumor imaging scan was performed during the screening period to establish a baseline disease burden. Scans performed as part of routine clinical management were eligible for use as screening scans as long as they were of sufficient quality and were less than 21 days before the first dose.

[0194] Investigators, at their discretion, could perform scans in addition to scheduled study scans if deemed clinically necessary. Timing of imaging during the study was according to calendar days and was not adjusted for delayed treatment administration or clinic visits. For consistency, the same imaging modality was used for patients throughout the study.

[0195] Brain imaging MRI (with or without contrast) was the preferred modality for brain imaging; however, CT was acceptable if MRI was clinically contraindicated. Patients with a history of CNS metastases underwent brain imaging on the same schedule as chest / abdominal / pelvic imaging.

[0196] During the study, brain CT / MRI scans were performed if clinically indicated due to the development of new symptoms.

[0197] Safety evaluation The safety profile of the study treatment was assessed through recording, reporting, and analysis of baseline medical conditions, AEs, physical examination findings including vital signs, and clinical laboratory tests. A comprehensive assessment of overt toxicities experienced by patients was conducted from the time patients signed informed consent and throughout the course of the study. Study site personnel reported any AEs, whether observed by the investigator or reported by the patient.

[0198] Primary efficacy analysis Objective response rate (ORR) ORR was defined as the proportion of patients who achieved a best overall response rate (BOR) among objective responses (CR or PR). BOR was defined as the best recorded response rate from randomization to data cutoff, disease progression, or the initiation of new anticancer treatment. Patients without post-baseline response assessments were considered non-responders to the BOR. Confirmed ORRs assessed by the investigator according to RECIST 1.1 were reported for each group with corresponding Clopper-Pearson 95% CIs. Furthermore, differences in ORR between groups were calculated with 95% CIs constructed using the Miettinen-Nurminen method (Miettinen 1985). There were two types of comparisons: 1) a comparison between the low-dose group and the high-dose group (combination: groups A and B; monotherapy: groups C and D), and 2) a comparison between the combination therapy group and the monotherapy group (low-dose: groups A and C; high-dose: groups B and D). The analysis also included a comparison with Arm E to better demonstrate the contribution of the components and characterize the treatment effect of botencilimab with or without balstilimab. This study was not powered for hypothesis testing; therefore, lack of statistical significance should not be interpreted as evidence of no difference. Subgroup analyses were performed for the primary efficacy endpoint in the intention-to-treat analysis set.

[0199] The primary analysis was performed approximately 6 months after the last patient was randomized and is considered the final analysis of the study. A supplemental analysis including updated efficacy and safety data was performed at the completion of the study.

[0200] Secondary efficacy analysis Duration of response (DOR) DOR was defined as the time from the first objective response to the first documented investigator-assessed progression according to RECIST 1.1 or death, whichever occurred first. DOR was summarized using the Kaplan-Meier method for responding patients only. All censoring rules for PFS analysis also applied to DOR. When estimable, median DOR and its 95% CI were constructed using the generalized Brookmeyer and Crowley method (Brookmeyer 1982). Cumulative probabilities of DOR at 3-month intervals were calculated and presented with two-sided 95% CIs using the Greenwood formula.

[0201] Progression-free survival (PFS) PFS was defined as the time from randomization to investigator-assessed progression or death, whichever occurred first, according to RECIST 1.1. Patients without an event (death or PD) at the analysis cutoff date were censored at the date of their last tumor assessment or the start date of new anticancer therapy. For each treatment group, the median PFS and cumulative probability of PFS at 3-month intervals were calculated using the Kaplan-Meier method and presented with two-sided 95% CIs. PFS censoring rules followed the FDA's "Guidance for Industry Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologics" (Food and Drug Administration, 2007). Data for patients without disease progression or death at the time of analysis were censored at the date of their last adequate tumor assessment. Data for patients lost to follow-up before documented disease progression were censored at the date of their last adequate tumor assessment when the patient was found to be progression-free. Data for patients who initiated new anticancer therapy were censored at the date of their last adequate tumor assessment before the introduction of the new therapy.

[0202] In the final analysis, PFS distributions between treatment groups were compared descriptively using the log-rank test. Hazard ratios between groups were estimated from Cox regression models.

[0203] Overall survival (OS) OS was defined as the time to death from any cause and was analyzed in the intention-to-treat analysis set. Patients were censored at the date the patient was last known to be alive or the data cutoff date, whichever came first. Median OS and cumulative OS probabilities estimated at 6-month intervals were calculated using Kaplan-Meier estimates for each treatment group and presented with two-sided 95% CIs. Descriptive comparisons of OS between groups were performed similarly to those for PFS in the final analysis.

[0204] Safety analysis All safety endpoints were analyzed in the safety analysis set using actual treatment assignment.

[0205] Extent of exposure The extent of exposure to each study drug was summarized descriptively as number of doses (number and percentage of patients), duration of exposure (days), cumulative total dose per patient (mg), dose intensity, and relative dose intensity. The number (percentage) of patients requiring dose interruptions, dose delays, and discontinuations due to AEs was summarized for each study drug. The frequency of the above dose adjustments and discontinuations was summarized by category.

[0206] Patient data listings were provided for all medication records and summary statistics were calculated.

[0207] Adverse events (AEs) All AEs were coded using MedDRA v25.0 or later and classified according to NCI CTCAE v5.0. AEs that occurred on or after the first dose of study drug up to 90 days after discontinuation of study treatment (i.e., after the last dose of randomized treatment) or up to the start of the first anticancer therapy (including radiation therapy, excluding palliative radiation therapy) after discontinuation of study treatment (whichever occurred first) or that worsened in severity from baseline (pretreatment) were considered treatment-related adverse events (TEAEs) and were included in summary tables. All AEs, whether or not they were caused by treatment, were included in the listings.

[0208] The incidence of TEAEs was reported as the number (proportion) of patients experiencing a TEAE by system, organ, class, and preferred term. The number (proportion) of patients experiencing a TEAE was also summarized by relationship to study drug. TRAEs included AEs that the investigator considered to be related to study drug or AEs for which causality assessment was missing.

[0209] SAEs, deaths, TEAEs of grade 3 or higher severity, irAEs, TRAEs, and TEAEs leading to treatment discontinuation, dose interruption, or dose delay were summarized.

[0210] D. Pharmacokinetics PK parameters of serum botencilimab and balstilimab included C max-ss , C min-ss , the area under the drug concentration-time curve (AUC (t1-t2)-ss ), the area under the drug concentration-time curve from time 0 to time t (AUC (0-t) ), the area under the drug concentration-time curve from time 0 to infinity (AUC (0-∞) ), t max , terminal disappearance rate constant (λz), t 1 / 2 , systemic drug clearance (CL), and Vd.

[0211] Both non-compartmental and compartmental modeling (e.g., PopPK) can be used for PK analysis. Additional PK exposure metrics for serum botencilimab and balstilimab were assessed via PopPK.

[0212] E. Immunogenicity Immunogenicity assessment was performed to detect and measure ADA (antibodies to botencilimab and balstilimab) using a multi-stage strategy of screening, confirmation, and titer assays. Samples that tested positive by ADA assay were tested for neutralizing antibodies. Serum botencilimab and balstilimab immunogenicity / ADA blood samples were collected from patients at designated time points. Blood samples may be used for additional bioanalytical characterization.

[0213] F. Tumor Biomarkers Tumor tissue was collected during the study and biomarker measurements in the tumor tissue included, but were not limited to, PD-L1 expression, microsatellite instability status, and other markers deemed relevant to the current study.

[0214] G. Objectives and Endpoints [Table 5]

[0215] The primary analysis was performed approximately 6 months after the last patient was randomized and is considered the final analysis of the study. A supplemental analysis including updated efficacy and safety data was performed at study completion.

[0216] H. Initial results An initial analysis was conducted to evaluate the best overall response rate (BOR), objective response rate (ORR), and disease control rate (DCR) in patients without active liver metastases, including an efficacy-evaluable (EE) population (n=70) and an intention-to-treat (ITT) population (n=77). A total of 70 efficacy-evaluable patients received botencilimab (1 mg / kg or 2 mg / kg every 6 weeks) and balstilimab (3 mg / kg every 2 weeks). The change in total target lesions from the start of combination / salvage therapy in the EE population is shown in Figure 1, and the best change in total target lesions is shown in Figure 2. BOR assessment for patients in the EE population showed one patient (1%) had a complete response (CR), 16 patients (23%) had a partial response (PR), 39 patients (56%) had stable disease (SD), and 14 patients (20%) had progressive disease (PD). The DCR (defined as CR + PR + SD) in the EE population was 80% (95% CI 69%-89%). The confirmed ORR in the EE population was 24% (95% CI 15%-36%). The median follow-up period was 12.3 months (range 1.4 to 40.5 months), with 10 ongoing responses (59%). In the ITT population, the ORR was 22% and the DCR was 73%. The ORR reported for patients treated with current standard of care was 2.8%.

[0217] Current overall survival probabilities were also analyzed for the EE and ITT population groups, as shown in Figure 3 and summarized below in Table 6. Further analysis of study results is ongoing. [Table 6]

[0218] Incorporation by Reference All patent and non-patent literature cited above is incorporated herein by reference in its entirety.

Claims

1. A method for treating colorectal cancer in a subject in need of treatment, comprising administering to the subject an antibody that specifically binds to human cytotoxic T-lymphocyte antigen 4 (CTLA-4) at a dose of 25 mg to 200 mg, wherein the antibody comprises a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO:

8.

2. A method for enhancing colon cancer T cell activation in a subject with colon cancer, comprising administering to the subject an antibody that specifically binds to human CTLA-4 at a dose of 25 mg to 200 mg, wherein the antibody comprises a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO:

8.

3. 3. The method of claim 1 or 2, wherein the antibody is administered at a dose of 50 mg to 175 mg.

4. 3. The method of claim 1 or 2, wherein the antibody is administered at a dose of 75 mg to 150 mg.

5. 3. The method of claim 1 or 2, wherein the antibody is administered at a dose of 25 mg, 50 mg, 75 mg, 100 mg, or 150 mg.

6. The method of any one of claims 1 to 5, wherein the antibody is administered intravenously.

7. The method of any one of claims 1 to 6, wherein the antibody is administered by intravenous infusion over about 30 minutes.

8. The method of any one of claims 1 to 7, wherein the antibody is administered once a week.

9. The method of any one of claims 1 to 7, wherein the antibody is administered once every two weeks.

10. The method of any one of claims 1 to 7, wherein the antibody is administered once every three weeks.

11. The method of any one of claims 1 to 7, wherein the antibody is administered once every four weeks.

12. The method of any one of claims 1 to 7, wherein the antibody is administered once every five weeks.

13. The method of any one of claims 1 to 7, wherein the antibody is administered once every six weeks.

14. 8. The method of any one of claims 1 to 7, wherein the antibody is administered intravenously at a dose of 25 mg once every six weeks.

15. 8. The method of any one of claims 1 to 7, wherein the antibody is administered intravenously at a dose of 50 mg once every six weeks.

16. 8. The method of any one of claims 1 to 7, wherein the antibody is administered intravenously at a dose of 75 mg once every six weeks.

17. 8. The method of any one of claims 1 to 7, wherein the antibody is administered intravenously at a dose of 100 mg once every six weeks.

18. 8. The method of any one of claims 1 to 7, wherein the antibody is administered intravenously at a dose of 150 mg once every six weeks.

19. The method of any one of claims 1 to 18, wherein the dose is a therapeutically effective amount.

20. The method of any one of claims 1 to 19, wherein the colon cancer is colorectal adenocarcinoma.

21. The method of any one of claims 1 to 20, wherein the colon cancer is unresectable.

22. The method of any one of claims 1 to 20, wherein the antibody is administered to the subject prior to surgical resection of the primary tumor.

23. The method of any one of claims 1 to 22, wherein the colon cancer is metastatic.

24. The method of any one of claims 1 to 23, wherein the subject does not have liver metastases.

25. The method according to any one of claims 1 to 24, wherein the colorectal cancer is recurrent and / or refractory.

26. The method of any one of claims 1 to 25, wherein the subject has not previously received any chemotherapy.

27. The method of any one of claims 1 to 26, wherein the subject has not previously received any radiation therapy.

28. The method of any one of claims 1 to 25, wherein the subject has undergone at least one previous chemotherapy.

29. 29. The method of claim 28, wherein the at least one prior chemotherapy is a fluoropyrimidine, irinotecan, oxaliplatin, or an anti-EGFR antibody.

30. 30. The method of claim 29, wherein the anti-EGFR antibody is cetuximab or panitumumab.

31. 26. The method of any one of claims 1 to 25, wherein the subject has previously been treated with folinic acid, 5-fluorouracil, oxaliplatin, and irinotecan.

32. 32. The method of any one of claims 1 to 31, wherein the subject is unable to tolerate standard care treatment.

33. The method of any one of claims 1 to 32, wherein the subject has a RAS mutation.

34. 34. The method of claim 33, wherein the RAS mutation is a KRAS or NRAS mutation.

35. The method of any one of claims 1 to 34, wherein the colon cancer is not microsatellite instability-high (MSI-H).

36. The method of any one of claims 1 to 35, wherein the colon cancer is microsatellite stable (MSS).

37. The method of any one of claims 1 to 36, wherein the colorectal cancer is not mismatch repair deficient (dMMR).

38. 38. The method of any one of claims 1 to 37, wherein the subject has not previously been treated with an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

39. 39. The method of any one of claims 1 to 38, wherein the subject has not previously been treated with regorafenib, trifluridine, and / or tipiracil.

40. 40. The method of any one of claims 1 to 39, wherein the cancer is refractory to standard of care treatment.

41. 41. The method of claim 40, wherein the standard of care treatment is chemotherapy or radiation therapy.

42. 42. The method of claim 40 or 41, wherein the standard of care treatment is folinic acid, 5-fluorouracil, oxaliplatin, irinotecan, fluoropyrimidines, cetuximab, and / or panitumumab.

43. 43. The method of any one of claims 1 to 42, wherein administration of the antibody reduces tumor size in the subject.

44. 44. The method of any one of claims 1 to 43, wherein administration of the antibody increases activation of T cells, memory T cells, myeloid cells, and / or antigen-presenting cells in the subject.

45. The method of any one of claims 1 to 44, wherein administration of the antibody reduces the number of Treg cells in the subject.

46. 10. The method of any one of the preceding claims, wherein prior to administration of the antibody, the subject has measurable disease on baseline imaging according to RECIST 1.

1.

47. 10. The method of any one of the preceding claims, wherein prior to administration of the antibody, the subject has an European Cooperative Oncology Group performance status (PS) of 0-1.

48. 10. The method of any one of the preceding claims, wherein prior to administration of the antibody, the subject has a predicted life expectancy of 12 weeks or greater.

49. Prior to administration of the antibody, the subject: a) Neutrophils ≧1500 / μL, b) Platelets ≧100×103 / μL, c) hemoglobin ≥ 8.0 g / dL; d) Creatinine clearance ≥ 30 mL / min measured or calculated according to local institutional standards; e) AST / ALT≦2.5×upper limit of normal (ULN), f) total bilirubin ≦1.5×ULN (except for patients with Gilbert's syndrome, in which case the total bilirubin value must be ≦3.0×ULN), and / or 10. The method of any one of the preceding claims, wherein the patient has adequate organ function as defined by one or more of the following: g) albumin > 3.0 g / dL.

50. 10. The method of any one of the preceding claims, wherein the subject has no partial or complete bowel obstruction, signs / symptoms of bowel obstruction, or known radiological evidence of impending obstruction within the past three months.

51. 10. The method of any one of the preceding claims, wherein the subject does not have refractory ascites defined as requiring two or more therapeutic paracentesis within the past four weeks, or four or more therapeutic paracentesis within the past 90 days, or one or more therapeutic paracentesis within the past two weeks prior to administration of the antibody.

52. 10. The method of any one of the preceding claims, wherein the subject does not have clinically significant cardiovascular disease.

53. 10. The method of any one of the preceding claims, wherein the subject does not have active brain or leptomeningeal metastases.

54. 10. The method of any one of the preceding claims, wherein the subject does not have a history of a concurrent malignancy requiring treatment or a malignancy that was active within two years prior to administration of the antibody.

55. 10. The method of any one of the preceding claims, wherein the subject has not received a cytotoxic or targeted therapy within three weeks prior to administration of the antibody.

56. 10. The method of any one of the preceding claims, wherein the subject has not received any other monoclonal antibody therapy, antibody drug conjugate therapy, or radioimmunoconjugate therapy within four weeks prior to administration of the antibody.

57. 10. The method of any one of the preceding claims, wherein the subject has not received small molecule tyrosine kinase inhibitor therapy within two weeks prior to administration of the antibody.

58. 10. The method of any one of the preceding claims, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

59. 10. The method of any one of the preceding claims, wherein the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

8.

60. 10. The method of any one of the preceding claims, wherein the antibody comprises a human IgG1 heavy chain constant region comprising S239D / A330L / I332E mutations numbered according to the EU numbering system.

61. 10. The method of any one of the preceding claims, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

10.

62. 10. The method of any one of the preceding claims, wherein the antibody is botencilimab.

63. 10. The method of any one of the preceding claims, wherein the method further comprises administering to the subject an antibody that specifically binds to human PD-1.

64. 64. The method of claim 63, wherein the antibody that specifically binds to human PD-1 comprises a heavy chain variable region (VH) comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO:

18.

65. 65. The method of claim 63 or 64, wherein the antibody that specifically binds to human PD-1 comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 11, 12, 13, 14, 15, and 16, respectively.

66. The method of any one of claims 63 to 65, wherein the antibody that specifically binds to human PD-1 comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

18.

67. 67. The method of any one of claims 63 to 66, wherein the antibody that specifically binds to human PD-1 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

20.

68. The method of any one of claims 63 to 67, wherein the antibody that specifically binds to human PD-1 is balstilimab.

69. 69. The method of any one of claims 63-68, wherein the antibody that specifically binds to human PD-1 is administered at a dose of 200 mg to 300 mg.

70. 70. The method of any one of claims 63-69, wherein the antibody that specifically binds to human PD-1 is administered at a dose of 240 mg.

71. 71. The method of any one of claims 63-70, wherein the antibody that specifically binds to human PD-1 is administered once a week or once every two weeks.

72. 72. The method of any one of claims 63-71, wherein the antibody that specifically binds to human PD-1 is administered at a dose of 240 mg once every two weeks.

73. An antibody that specifically binds to human CTLA-4 for use in the treatment of colon cancer, said treatment being carried out according to the method of any one of the preceding claims.

74. 10. An antibody that specifically binds to human CTLA-4 for use in the manufacture of a medicament for the treatment of colon cancer, wherein said treatment is carried out according to a method according to any one of the preceding claims.

75. 10. Use of an antibody that specifically binds to human CTLA-4 for the treatment of colon cancer, said treatment being carried out according to the method of any one of the preceding claims.

76. 10. An antibody that specifically binds to human CTLA-4 and an antibody that specifically binds to human PD-1 for use in the treatment of colorectal cancer, wherein the treatment is carried out according to a method according to any one of the preceding claims.

77. 10. An antibody that specifically binds to human CTLA-4 and an antibody that specifically binds to human PD-1 for use in the manufacture of a medicament for the treatment of colorectal cancer, wherein said treatment is carried out according to the method of any one of the preceding claims.

78. 10. Use of an antibody that specifically binds to human CTLA-4 and an antibody that specifically binds to human PD-1 for the treatment of colorectal cancer, wherein said treatment is carried out according to the method of any one of the preceding claims.

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