How to treat melanoma using anti-CTLA4 antibodies

Abstract

A method for treating melanoma is provided using an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4).

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 492,717, filed on 28 March 2023, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence listing reference This application includes a sequence listing, which has been filed electronically in ST.26 format and is incorporated herein by reference in its entirety (the ST.26 copy prepared on 27 March 2024 is referred to as "208835_seqlist.xml" and is 10,379 bytes in size). [Background technology]

[0003] Melanoma is a deadly form of skin cancer, and its incidence has increased rapidly in recent decades. Although melanoma accounts for only 1% of all skin cancers, it is the cause of most skin cancer deaths due to its metastatic ability.

[0004] Late-stage melanoma is difficult to treat, but in recent years, immunotherapy has shown promising results in patients with stage III or stage IV melanoma. Currently, anti-PD-1 antibody monotherapy or anti-PD-1 antibody / anti-CTLA-4 antibody combination therapy is approved for first-line treatment in patients with stage III or stage IV melanoma. However, approximately 50% of patients with metastatic melanoma do not achieve an objective response with these treatments, and many of those who do respond subsequently experience relapse.

[0005] Therefore, alternative immunotherapy treatment options are needed for patients whose disease progresses on primary anti-PD-1 antibody monotherapy or primary anti-PD-1 / anti-CTLA-4 combination therapy. [Overview of the project]

[0006] This disclosure relates to a method for treating melanoma using an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4). Also provided herein is a specific method of administering an antibody that specifically binds to human CTLA-4 to reduce the tumor burden in a subject. In one embodiment, the method includes a therapeutically effective amount for safely and effectively treating melanoma. The method disclosed herein is not limited to the treatment of stage III or stage IV melanoma and can therefore be used to treat subjects with melanoma of other stages, such as stage I or stage II.

[0007] In one embodiment, a method for treating melanoma in a subject requiring treatment is provided herein, the method 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 VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0008] In one embodiment, a method for enhancing T cell activation in a subject having melanoma is provided herein, the method 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 VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0009] In one embodiment, the antibody that specifically binds to human CTLA-4 is administered in doses of 50 mg to 175 mg. In another embodiment, the antibody that specifically binds to human CTLA-4 is administered in doses of 75 mg to 150 mg. In yet another embodiment, the antibody that specifically binds to human CTLA-4 is administered in doses of 25 mg, 50 mg, 75 mg, 100 mg, or 150 mg.

[0010] In one embodiment, the antibody that specifically binds to human CTLA-4 is administered intravenously. In one embodiment, the antibody that specifically binds to human CTLA-4 is administered by intravenous infusion over a period of 30 minutes.

[0011] In one embodiment, the antibody that specifically binds to human CTLA-4 is administered once a week. In another embodiment, the antibody that specifically binds to human CTLA-4 is administered once every two weeks. In another embodiment, the antibody that specifically binds to human CTLA-4 is administered once every three weeks. In another embodiment, the antibody that specifically binds to human CTLA-4 is administered once every four weeks. In another embodiment, the antibody that specifically binds to human CTLA-4 is administered once every five weeks. In another embodiment, the antibody that specifically binds to human CTLA-4 is administered once every six weeks.

[0012] In one embodiment, the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 25 mg once every three weeks. In another embodiment, the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 50 mg once every three weeks. In yet another embodiment, the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 75 mg once every three weeks. In yet another embodiment, the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 100 mg once every three weeks. In yet another embodiment, the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 150 mg once every three weeks.

[0013] In one embodiment, the dose is a therapeutically effective amount.

[0014] In one embodiment, the melanoma is a cutaneous melanoma. In one embodiment, the melanoma is unresectable. In one embodiment, the melanoma is metastatic. In one embodiment, the melanoma is an intraocular melanoma, uveal melanoma, or mucosal melanoma.

[0015] In one embodiment, melanoma is refractory to checkpoint inhibitor therapy. In one embodiment, the checkpoint inhibitor therapy is an anti-PD-1 antibody, an anti-PD-L1 antibody, ipilimumab, or tremelimumab.

[0016] In one embodiment, the subject has received at least one prior anticancer therapy. In one embodiment, the at least one prior anticancer therapy is an anti-PD-1 antibody, which is optionally valstirimab, nivolumab, or pembrolizumab, an anti-PD-L1 antibody, ipilimumab, or tremelimumab.

[0017] In one embodiment, at least one prior anticancer therapy is a BRAF inhibitor and / or a MEK inhibitor. In one embodiment, the BRAF inhibitor is selected from the group consisting of vemurafenib, dabrafenib, and encorafenib. In one embodiment, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, and binimetinib.

[0018] In one embodiment, an antibody that specifically binds to human CTLA-4 is administered to the subject before radiotherapy, chemotherapy, or surgical removal of a tumor.

[0019] In one embodiment, the subjects have not previously received anti-CTLA-4 antibody therapy.

[0020] In one embodiment, the subject has an unresectable stage III or stage IV cutaneous melanoma.

[0021] In one embodiment, the subject has a BRAF mutation. In one embodiment, the BRAF mutation is the BRAF V600 mutation.

[0022] In one embodiment, the subject is homozygous for phenylalanine at position 158 of FcγRIIIA. In one embodiment, the subject is homozygous for valine at position 158 of FcγRIIIA. In one embodiment, the subject is heterozygous for valine / phenylalanine at position 158 of FcγRIIIA.

[0023] In one embodiment, the melanoma is not an intraocular melanoma. In one embodiment, the melanoma is not a choroidal melanoma. In one embodiment, the melanoma is not a mucosal melanoma.

[0024] In one embodiment, the subject has no persistent toxicity (Common Terminology Criteria for Adverse Events [CTCAE] ≥ 2) from previous cancer therapy.

[0025] In one embodiment, the subject has no history of immune-mediated toxicity with CTCAE ≥ 3 from previous checkpoint inhibition (excluding endocrine disorders and non-necrotizing / vesicular rash).

[0026] In one embodiment, administration of the antibody reduces the tumor size of the subject. In one embodiment, administration of the antibody enhances T cell activation in the subject.

[0027] In one embodiment, prior to administration of the antibody, the subject has a measurable disease by baseline imaging according to RECIST 1.1. In one embodiment, prior to administration of the antibody, the subject has a Performance Status (PS) of 0-1 of the Eastern Cooperative Oncology Group. In one embodiment, prior to administration of the antibody, the subject has a predicted life expectancy of more than 3 months.

[0028] In one embodiment, prior to administration of the antibody, the subject has appropriate organ function defined by one or more of the following: a) neutrophils ≥ 1500 / μL, b) platelets ≥ 100 × 10 3 / μL, c) hemoglobin ≥ 8.0 g / dL, d) creatinine clearance ≥ 45 mL / min as measured or calculated according to local facility standards, e) AST / ALT ≤ 3 × upper limit of normal (ULN), f) total bilirubin ≤ 1.5 × ULN (except for patients with Gilbert's syndrome, where total bilirubin must be ≤ 3.0 × ULN), g) albumin ≥ 3.0 g / dL, and / or h) international normalized ratio or prothrombin time ≤ 1.5 × ULN and activated partial thromboplastin time ≤ 1.5 × ULN (except when the patient is receiving anticoagulant therapy).

[0029] In one embodiment, the subject does not have any known radiological evidence of partial or complete bowel obstruction, signs / symptoms of bowel obstruction, or impending obstruction within the past three months.

[0030] In one embodiment, the subjects do not have refractory ascites, which is defined as requiring two or more therapeutic paracentesis procedures within the past four weeks prior to antibody administration, or four or more within the past 90 days.

[0031] In one embodiment, the subjects do not have clinically significant cardiovascular disease.

[0032] In one embodiment, the subject does not have active brain metastases or leptomeningeal metastases. In one embodiment, the subject does not have a history of synchronous malignancies requiring treatment, or of previous malignancies that were active within two years prior to antibody administration.

[0033] In one embodiment, the subject has not received cytotoxic therapy or targeted therapy within three weeks prior to antibody administration. In another embodiment, the subject has not received other monoclonal antibody therapy, antibody-drug conjugate therapy, or radioimmune conjugate therapy within four weeks prior to antibody administration.

[0034] In one embodiment, a method for treating melanoma in a subject requiring treatment is provided herein, the method comprising administering to the subject a dose of 50 mg once every three weeks of an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4), wherein the subject has stage III or stage IV cutaneous melanoma, and the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0035] In one embodiment, a method for treating melanoma in a subject requiring treatment is provided herein, the method comprising administering to the subject a dose of 150 mg once every three weeks of an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4), wherein the subject has stage III or stage IV cutaneous melanoma, and the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0036] In one embodiment, an antibody that specifically binds to human CTLA-4 includes the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0037] In one embodiment, the antibody that specifically binds to human CTLA-4 includes VH, which contains the amino acid sequence described in SEQ ID NO: 7, and VL, which contains the amino acid sequence described in SEQ ID NO: 8.

[0038] In one embodiment, an antibody that specifically binds to human CTLA-4 comprises a human IgG1 heavy chain constant region containing the S239D / A330L / I332E mutation, numbered according to the EU numbering system.

[0039] In one embodiment, the antibody that specifically binds to human CTLA-4 comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 9 and a light chain containing the amino acid sequence described in SEQ ID NO: 10.

[0040] In one embodiment, the antibody that specifically binds to human CTLA-4 is botensilimab.

[0041] In one embodiment, an antibody that specifically binds to human CTLA-4 for use in the treatment of melanoma is provided herein, wherein the treatment is carried out according to the method described in any one of the prior claims.

[0042] In one embodiment, an antibody that specifically binds to human CTLA-4 for use in the manufacture of a pharmaceutical product for the treatment of melanoma is provided herein, wherein the treatment is carried out according to the method described in any one of the prior claims.

[0043] In one embodiment, the use of an antibody that specifically binds to human CTLA-4 for the treatment of melanoma is provided herein, wherein the treatment is carried out according to the method described in any one of the prior claims. [Brief explanation of the drawing]

[0044] [Figure 1A] The percentage change in tumor burden over time from baseline (A) and the best-case percentage change (B) are shown in 10 patients with cutaneous melanoma who received botensilimab as monotherapy or in combination with valstilimab, according to aspects of this disclosure. [Figure 1B] The percentage change in tumor burden over time from baseline (A) and the best-case percentage change (B) are shown in 10 patients with cutaneous melanoma who received botensilimab as monotherapy or in combination with valstilimab, according to aspects of this disclosure. [Modes for carrying out the invention]

[0045] This disclosure relates to a method for treating melanoma using an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4). Also provided herein is a specific method for administering an antibody that specifically binds to human CTLA-4 to reduce the tumor burden of the target. In one embodiment, the method includes a therapeutically effective amount for safely and effectively treating melanoma.

[0046] definition As used herein, the terms “antibody” and “antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing 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, heteroconjugated antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv(scFv), camelized antibodies, aphibodies, Fab fragments, F(ab')2 fragments, disulfide-bonded Fv(sdFv), anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies), and any of the antigen-binding fragments described above. In certain embodiments, the antibodies described herein refer to a population of polyclonal antibodies. The antibody may be any type of immunoglobulin molecule (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). In certain embodiments, the antibody described herein is an IgG antibody, or a class thereof (e.g., human IgG1 or IgG4), or a subclass thereof. In one embodiment, the antibody is a humanized monoclonal antibody. In one embodiment, the antibody is a human monoclonal antibody.

[0047] As used herein, the term “CDR” or “complementarity-determining region” refers to non-adjacent antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. These specific 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), by Chothia et al., J. Mol. Biol. 196: 901-917 (1987), all of which are incorporated herein by reference in their entirety, and the definition includes overlaps or subsets of amino acid residues when compared to one another (see Table 1 below). In certain embodiments, the term "CDR" refers to the CDR as defined by MacCallum et al., J.Mol.Biol.262:732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Duebel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" refers to the CDR as defined by 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 CDR and light chain CDR of an antibody are defined using different rules. In certain embodiments, heavy-chain CDRs and / or light-chain CDRs are defined by performing a structural analysis of the antibody and identifying residues in the variable region(s) 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]

[0048] As used herein, the terms “variable region” and “variable domain” are interchangeable and common in the art. A variable region refers to a portion of an antibody, generally a portion of the light chain or heavy chain, typically the nearly amino-terminus 110–120 or 110–125 amino acids in the mature heavy chain, and about 90–115 amino acids in the mature light chain, whose sequences differ significantly between antibodies and are used in the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in these regions, called complementarity-determining regions (CDRs), while more highly conserved regions within the variable domain are called framework regions (FRs). While we do not wish to be constrained by any particular mechanism or theory, the CDRs of the light and heavy chains are thought to be primarily involved in antibody-antigen interaction and specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes rodent or mouse 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 includes a rodent or mouse CDR and a primate (e.g., non-human primate) framework region (FR).

[0049] As used herein, the terms "VH" and "VL" refer to the heavy chain and light chain variable regions of an antibody, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), and are incorporated herein by reference in their entirety.

[0050] As used herein, the term “constant region” is common in the art. The constant region is the carboxyl-terminal portion of the light and / or heavy chain that does not directly participate in the binding of the antibody to the antigen, for example, but can exhibit various effector functions, such as interaction with Fc receptors (e.g., Fc gamma receptors).

[0051] As used herein, the term “heavy chain” may refer to any different type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which respectively give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0052] As used herein, the term “light chain,” when used in reference to an antibody, may refer to any different type, for example, 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.

[0053] 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 molecules that bind to an antigen (e.g., an epitope or immune complex) in such a way that such binding is understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides with generally lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA 3000 instrument (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 KA at least 2 log (e.g., 10 times), 2.5 log, 3 log, or 4 log greater than the KA when the molecule binds nonspecifically to another antigen.

[0054] As used herein, the term “EU numbering system” refers to the EU numbering rules for the constant region of antibodies, such as those described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969), and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein in whole by reference.

[0055] As used herein, the term “subject” includes any human or non-human animal. In one embodiment, the subject is a human.

[0056] As used herein, in the context of therapy, the term “effective dose” refers to the amount of therapy that achieves the desired preventive or therapeutic effect.

[0057] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to the therapeutic or preventive measures described herein. A “treatment” method involves administering antibodies to a subject with a disease or disorder, or a predisposition to such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or improve one or more symptoms of the disease or disorder or recurrent disease or disorder, or to extend the subject’s survival time beyond what would be expected in the absence of such treatment.

[0058] As used herein, the term “targeted therapy” refers to therapy that inhibits a specific protein. In one embodiment, targeted therapy inhibits a protein known to be important for the growth and / or survival of melanoma cells (e.g., BRAF).

[0059] As used herein, the term “cytotoxic therapy” refers to a therapy that inhibits or slows cell division. In one embodiment, cytotoxic therapy kills cancer cells. In one embodiment, cytotoxic therapy is fluorouracil, capecitabine, oxaliplatin, irinotecan, or triflurizine tipiracil.

[0060] As used herein, the term “tumor load” refers to the number of cancer cells, the size of a tumor, or the amount of cancer in the body of the subject.

[0061] As used herein, the term “about” refers to a measurable value, such as a dosage, and includes variations of ±20%, ±15%, ±10%, ±5%, ±1%, or ±0.1% of a given value or range, as is appropriate for carrying out the methods disclosed herein.

[0062] Anti-CTLA-4 antibody Antibodies that specifically bind to human CTLA-4 (i.e., anti-CTLA-4 antibodies) useful for the methods and uses described herein include, but are not limited to, those listed below.

[0063] In one embodiment, the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7. In another embodiment, the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0064] In one embodiment, the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences described in SEQ ID NOs: 1, 2, and 3, respectively. In another embodiment, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0065] In one embodiment, the antibody comprises VH containing the amino acid sequence described in SEQ ID NO: 7. In one embodiment, the antibody comprises VH containing the amino acid sequence described in SEQ ID NO: 7 and VL containing an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 8.

[0066] In one embodiment, the antibody comprises VH containing the amino acid sequence described in SEQ ID NO: 7 and VL containing the amino acid sequence described in SEQ ID NO: 8.

[0067] In one embodiment, the antibody includes 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 includes a light chain constant region selected from the group consisting of human kappa light chain constant regions and human lambda light chain constant regions.

[0068] In one embodiment, the antibody includes the IgG1 heavy chain constant region. In one embodiment, the amino acid sequence of the IgG1 heavy chain constant region includes the S239D / I332E mutation, numbered according to the EU numbering system. In one embodiment, the amino acid sequence of the IgG1 heavy chain constant region includes the S239D / A330L / I332E mutation, numbered according to the EU numbering system. In one embodiment, the amino acid sequence of the IgG1 heavy chain constant region includes the L235V / F243L / R292P / Y300L / P396L mutation, numbered according to the EU numbering system. In one embodiment, the IgG1 heavy chain constant region is afucosylated IgG1.

[0069] In one embodiment, the antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 9. In one embodiment, the antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 9 and a light chain containing an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 10.

[0070] In one embodiment, the antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 9 and a light chain containing the amino acid sequence described in SEQ ID NO: 10. In one embodiment, the amino acid sequence of the heavy chain consists of the amino acid sequence described in SEQ ID NO: 9, and the amino acid sequence of the light chain consists of the amino acid sequence described in SEQ ID NO: 10.

[0071] In one embodiment, the antibody is botensilimab (also known as AGEN1181), and its amino acid sequence is shown in Table 2 below. [Table 2]

[0072] Treatment methods This disclosure demonstrates that antibodies that specifically bind to human CTLA-4 (e.g., botensilimab) are highly effective in treating melanoma. This disclosure also demonstrates that antibodies that specifically bind to human CTLA-4 (e.g., botensilimab) are highly effective in treating stage III or stage IV cutaneous melanoma that is refractory to checkpoint inhibitor therapy (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies). Therefore, this disclosure relates to a method for treating melanoma using antibodies that specifically bind to human CTLA-4.

[0073] In one embodiment, a method for treating melanoma in a subject requiring treatment is provided herein, the method comprising administering an effective amount of an antibody specifically bound to human CTLA-4 to the subject, wherein the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0074] In one embodiment, a method for enhancing T cell activation in a subject having melanoma is provided herein, the method comprising administering an effective amount of an antibody that specifically binds to human CTLA-4 to the subject, wherein the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0075] In one embodiment, a method for treating melanoma in a subject requiring treatment is provided herein, the method comprising administering to the subject an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4) in a dose of about 5 mg to about 200 mg, wherein the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0076] In one embodiment, a method for enhancing T cell activation in a subject having melanoma is provided herein, the method comprising administering to the subject an antibody that specifically binds to human CTLA-4 at a dose of about 5 mg to about 200 mg, wherein the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0077] In one embodiment, a method for treating melanoma in a subject requiring treatment is provided herein, the method comprising administering to the subject an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4) at a dose of 5 mg to 200 mg, wherein the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0078] In one embodiment, a method for enhancing T cell activation in a subject having melanoma is provided herein, the method comprising administering to the subject an antibody that specifically binds to human CTLA-4 at a dose of 5 mg to 200 mg, wherein the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0079] In one embodiment, the antibody is administered in a dose of approximately 25 mg to approximately 200 mg. In one embodiment, the antibody is administered in a dose of approximately 25 mg to approximately 150 mg. In one embodiment, the antibody is administered in a dose of approximately 50 mg to approximately 150 mg. In one embodiment, the antibody is administered in a dose of approximately 75 mg to approximately 150 mg.

[0080] In one embodiment, the antibody is administered in a dose of 25 mg to 200 mg. In one embodiment, the antibody is administered in a dose of 50 mg to 175 mg. In one embodiment, the antibody is administered in a dose of 25 mg to 150 mg. In one embodiment, the antibody is administered in a dose of 50 mg to 150 mg. In one embodiment, the antibody is administered in a dose of 75 mg to 150 mg.

[0081] In one embodiment, the antibody is administered in doses of approximately 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, 150 mg, 175 mg, or 200 mg.

[0082] In one embodiment, the antibody is administered in doses 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, 150 mg, 175 mg, or 200 mg.

[0083] In one embodiment, the antibody is administered intravenously. In another embodiment, the antibody is administered into the tumor.

[0084] In one embodiment, the antibody is administered by intravenous infusion over approximately 30 minutes. In another embodiment, the antibody is administered by intravenous infusion over approximately 45 minutes. In yet another embodiment, the antibody is administered by intravenous infusion over approximately 60 minutes. In yet another embodiment, the antibody is administered by intravenous infusion over approximately 90 minutes.

[0085] In one embodiment, the antibody is administered approximately once a week. In one embodiment, the antibody is administered approximately once every two weeks. In one embodiment, the antibody is administered approximately once every three weeks. In one embodiment, the antibody is administered approximately once every four weeks. In one embodiment, the antibody is administered approximately once every five weeks. In one embodiment, the antibody is administered approximately once every six weeks. In one embodiment, the antibody is administered approximately once every seven weeks. In one embodiment, the antibody is administered approximately once every eight weeks.

[0086] In one embodiment, the antibody is administered once a 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. In one embodiment, the antibody is administered once every seven weeks. In one embodiment, the antibody is administered once every eight weeks.

[0087] In one embodiment, the antibody is administered intravenously at a dose of approximately 25 mg once every three weeks. In another embodiment, the antibody is administered intravenously at a dose of approximately 50 mg once every three weeks. In yet another embodiment, the antibody is administered intravenously at a dose of approximately 75 mg once every three weeks. In yet another embodiment, the antibody is administered intravenously at a dose of approximately 100 mg once every three weeks. In yet another embodiment, the antibody is administered intravenously at a dose of approximately 150 mg once every three weeks.

[0088] In one embodiment, the antibody is administered intravenously at a dose of 25 mg once every three weeks. In another embodiment, the antibody is administered intravenously at a dose of 50 mg once every three weeks. In yet another embodiment, the antibody is administered intravenously at a dose of 75 mg once every three weeks. In yet another embodiment, the antibody is administered intravenously at a dose of 100 mg once every three weeks. In yet another embodiment, the antibody is administered intravenously at a dose of 150 mg once every three weeks.

[0089] In one embodiment, the dose is a therapeutically effective amount.

[0090] In one embodiment, the melanoma is a cutaneous melanoma. In one embodiment, the melanoma is an intraocular melanoma, uveal melanoma, or mucosal melanoma. In one embodiment, the melanoma is unresectable. In one embodiment, the melanoma is metastatic. In one embodiment, the melanoma is recurrent and / or refractory.

[0091] In one embodiment, melanoma is refractory to checkpoint inhibitor therapy. In one embodiment, the checkpoint inhibitor therapy is an anti-PD-1 antibody, an anti-PD-L1 antibody, ipilimumab, or tremelimumab.

[0092] In one embodiment, the subject has received at least one prior anticancer therapy. In one embodiment, the at least one prior anticancer therapy is an anti-PD-1 antibody, which is optionally valstirimab, nivolumab, or pembrolizumab, an anti-PD-L1 antibody, ipilimumab, or tremelimumab. In one embodiment, the at least one prior anticancer therapy is a BRAF inhibitor and / or a MEK inhibitor. In one embodiment, the BRAF inhibitor is selected from the group consisting of vemurafenib, dabrafenib, and encorafenib. In one embodiment, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, and binimetinib. In one embodiment, the subject has received at least one prior anticancer therapy selected from the group consisting of an anti-PD-1 antibody (optionally selected, the anti-PD-1 antibody is valstirimab, nivolumab, or pembrolizumab), an anti-PD-L1 antibody, ipilimumab, tremelimumab, a BRAF inhibitor, or a MEK inhibitor.

[0093] In one embodiment, an antibody that specifically binds to human CTLA-4 is administered to the subject before radiotherapy, chemotherapy, or surgical removal of a tumor.

[0094] In one embodiment, the subject has not previously received anti-CTLA-4 antibody therapy. In another embodiment, the subject has not previously been treated with an anti-CTLA-4 antibody having a modified Fc (e.g., BMS-96218, BMS-986288, HBM4003, XTX101).

[0095] In one embodiment, the subject has an unresectable stage III or stage IV cutaneous melanoma.

[0096] In one embodiment, the subject has a BRAF mutation. In one embodiment, the BRAF mutation is the BRAF V600 mutation.

[0097] In one embodiment, the subject is homozygous for phenylalanine at position 158 of FcγRIIIA. In another embodiment, the subject is homozygous for valine at position 158 of FcγRIIIA. In yet another embodiment, the subject is heterozygous for valine / phenylalanine at position 158 of FcγRIIIA. Methods for evaluating the allele status of the FcγRIIIA gene are readily available and known to those skilled in the art. For example, but not limited to, high-speed sequencing, DNA microarraying, and PCR amplification specific to the FcγRIIIA gene can be performed on tissue and / or blood samples taken from the subject.

[0098] In one embodiment, the melanoma is not intraocular melanoma. In one embodiment, the melanoma is not uveal melanoma. In one embodiment, the melanoma is not mucosal melanoma.

[0099] In one embodiment, a method for treating melanoma in a subject requiring treatment is provided herein, the method comprising administering to the subject a dose of 50 mg once every three weeks of an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4), wherein the subject has stage III or stage IV cutaneous melanoma, and the antibody comprises H, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0100] In one embodiment, a method for treating melanoma in a subject requiring treatment is provided herein, the method comprising administering to the subject a dose of 150 mg once every three weeks of an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4), wherein the subject has stage III or stage IV cutaneous melanoma, and the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO: 8.

[0101] In one embodiment, the subject has no persistent toxicity (Common Terminology Criteria for Adverse Events [CTCAE] ≥ 2) from previous cancer therapies.

[0102] In one embodiment, the subjects have no prior history of any immunomediated toxicity with CTCAE ≥ 3 from previous checkpoint inhibition (excluding endocrine disorders and non-necrotizing / bullous rashes).

[0103] In one embodiment, prior to antibody administration, the subject has a disease measurable by baseline imaging according to RECIST 1.1. In one embodiment, prior to antibody administration, the subject has a performance status (PS) of 0-1 in the US East Coast Cancer Clinical Trials Group. In one embodiment, prior to antibody administration, the subject has a predicted life expectancy of 12 weeks or more.

[0104] In one embodiment, prior to antibody administration, the subject has appropriate organ function defined by one or more of the following: a) neutrophils ≥ 1500 / μL, b) platelets ≥ 100 × 10⁴ 3 / μL, c) hemoglobin ≥ 8.0 g / dL, d) creatinine clearance ≥ 45 mL / min as measured or calculated according to local facility standards, e) AST / ALT ≤ 3 × upper limit of normal (ULN), f) total bilirubin ≤ 1.5 × ULN (except for patients with Gilbert's syndrome, where total bilirubin must be ≤ 3.0 × ULN), g) albumin ≥ 3.0 g / dL, and / or h) international normalized ratio or prothrombin time ≤ 1.5 × ULN and activated partial thromboplastin time ≤ 1.5 × ULN (except when the patient is receiving anticoagulant therapy).

[0105] In one embodiment, the subject does not have any known radiological evidence of partial or complete bowel obstruction, signs / symptoms of bowel obstruction, or impending obstruction within the past three months.

[0106] In one embodiment, the subject has not received immune checkpoint inhibitor therapy prior to antibody administration. In one embodiment, the subject has not received multiple chemotherapy regimens prior to antibody administration. In one embodiment, the subject does not have active brain metastases. In one embodiment, the subject has no history of synchronous malignancies requiring treatment, or of previous malignancies that were active within two years prior to antibody administration. In one embodiment, the subject has not received cytotoxic therapy or targeted therapy within three weeks prior to antibody administration. In one embodiment, the subject has not received other monoclonal antibody therapy, antibody-drug conjugate therapy, or radioimmune conjugate therapy within four weeks prior to antibody administration.

[0107] In one embodiment, the subjects do not have refractory ascites requiring two or more therapeutic paracentesis procedures within the past four weeks prior to antibody administration, four or more within the past 90 days, or one or more within the past two weeks. In one embodiment, the subjects do not have clinically significant cardiovascular disease.

[0108] In one embodiment, objective response rate (ORR), duration of response (DOR), disease control rate (DCR), and progression-free survival (PFS) are evaluated for subjects according to the Response Evaluation Criteria for Solid Tumors version 1.1 (RECIST 1.1).

[0109] In one embodiment, the method results in a complete response as defined in RECIST 1.1. In another embodiment, the method results in a partial response as defined in RECIST 1.1. In yet another embodiment, the method results in a stable disease as defined in RECIST 1.1.

[0110] In one embodiment, the method results in a reduction of approximately 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the tumor burden in the subject. In one embodiment, the method results in no change in the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 1% of the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 5% of the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 10% of the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 20% of the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 30% of the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 40% of the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 50% of the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 60% of the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 70% of the tumor burden in the subject. In one embodiment, the method results in a reduction of approximately 80% of the tumor burden in the subject. In one embodiment, this method results in a reduction of approximately 90% of the tumor burden in the subject. In another embodiment, this method results in a reduction of approximately 100% of the tumor burden in the subject.

[0111] In one embodiment, this method results in a reduction of tumor burden. In one embodiment, this method results in an increase in survival rate. In one embodiment, this method results in an increase in overall survival. In one embodiment, this method results in an increase in progression-free survival.

[0112] In one embodiment, any one of the methods disclosed herein further comprises administering an additional therapeutic agent to a subject. In one embodiment, the additional therapeutic agent is an antibody that specifically binds to human PD-1. In one embodiment, the additional therapeutic agent is valstirimab.

[0113] In one embodiment, an antibody that specifically binds to human CTLA-4 for use in the treatment of melanoma is provided herein, wherein the treatment is carried out according to the method described in any one of the prior claims.

[0114] In one embodiment, an antibody that specifically binds to human CTLA-4 for use in the manufacture of a pharmaceutical product for the treatment of melanoma is provided herein, wherein the treatment is carried out according to the method described in any one of the prior claims.

[0115] In one embodiment, the use of an antibody that specifically binds to human CTLA-4 for the treatment of melanoma is provided herein, wherein the treatment is carried out according to the method described in any one of the prior claims. [Examples]

[0116] Example 1 - A multi-cohort, open-label phase 2 study of botensilimab (AGEN1181) for the treatment of advanced melanoma refractory to previous checkpoint inhibitor therapy. This Phase 2 study evaluated the clinical efficacy and safety of botensilimab as monotherapy and in combination with valstilimab in patients with advanced melanoma refractory to anti-PD-(L)1-based therapy, as well as in patients with advanced melanoma refractory to combination therapy with anti-PD-(L)1 and anti-CTLA-4.

[0117] A. Research Design Overall design This was a multicenter, multi-cohort, open-label phase 2 trial of botensilimab in patients with advanced melanoma refractory to checkpoint inhibitor therapy.

[0118] Patients had histologically confirmed stage III (unresectable) or stage IV cutaneous melanoma according to the American Joint Committee on Cancer (AJCC) 8th edition staging system. Baseline study procedures included cross-sectional images of the chest, abdomen, and pelvis (C / A / P), magnetic resonance imaging (MRI) of the brain, routine clinical examinations, and tumor markers, namely lactate dehydrogenase (LDH). Archived tumor tissue and / or fresh tissue, as well as whole blood, were collected at baseline for correlational studies.

[0119] Approximately 220 evaluable patients were enrolled. This study consisted of two parts.

[0120] In Part 1, at least 60 patients from each of Cohort A and Cohort B were randomized in a 1:1 ratio to one of two dose levels of botensilimab (Group 1 and Group 2), as described below (except in cases where a group was terminated early due to futility / toxicity or where the number of enrolled patients exceeded 30). Patients in Part 1 (all groups) received botensilimab for up to four cycles, or for the period of death, unacceptable toxicity, loss of follow-up, disease progression, withdrawal from the study, or termination of the study by the sponsor, whichever came first. ● Cohort A (patients refractory to PD-(L)1). Group 1: Botensilimab 50 mg IV Q3W for up to 4 cycles; Group 2: Botensilimab 150 mg IV Q3W for up to 4 cycles. ● Cohort B (patients refractory to PD-(L)1 and CTLA-4). Group 1: Botensilimab 50 mg IV Q3W for up to 4 cycles; Group 2: Botensilimab 150 mg IV Q3W for up to 4 cycles.

[0121] In Part 2, at least 50 additional patients from each of Cohort A and Cohort B were administered botensilimab in combination with valstilimab, as described below. ● Cohort A (patients refractory to PD-(L)1). Group 3: Botensilimab 75mg Q3W + Valstilimab 450mg Q3W, Group 4: Botensilimab 75mg Q3W + Valstilimab 450mg Q3W.

[0122] Part 1 patients were stratified by prior BRAF therapy and LDH levels. Based on new data, the sponsor may, at their discretion, establish additional dose cohorts. [Table 3]

[0123] During the treatment period, patients received routine clinical care for administration of the research treatment and monitoring of its safety.

[0124] Antitumor efficacy was assessed by imaging evaluations performed every 9 weeks (Q9W) for the first year (up to week 54), and then every 12 weeks (Q12W). Disease response assessments were performed by the principal investigator, but may have been evaluated by an independent central review at the discretion of the sponsor.

[0125] Safety was assessed by monitoring for adverse events (AEs), serious adverse events (SAEs), treatment discontinuation due to AEs, physical examination, vital signs, hematology, and chemical tests. Patients who discontinued study treatment for reasons other than progressive disease continued imaging assessments. For survival follow-up, patients who initiated additional anti-cancer therapy or discontinued the study for other reasons were followed up by telephone at Q12W to check their survival status.

[0126] The two cohorts were evaluated separately in the statistical plan.

[0127] Administration of test drug Botensilimab was administered via IV infusion over a period of 30 (±5) minutes. Patients were observed for 1 hour after the completion of infusions for infusion-related reactions during the first two cycles, and for 30 minutes after the completion of each subsequent infusion. Vital signs were measured before the start of each infusion and at the end of each infusion in each cycle. Immediately after the completion of infusions, the IV line was flushed with saline in accordance with institutional guidelines.

[0128] In the combination therapy group, valstirimab was administered before botensilimab. Both valstirimab and botensilimab were administered via IV infusion over a period of 30 (±5) minutes.

[0129] Number of patients Approximately 220 evaluable patients were enrolled.

[0130] Treatment after disease progression Patients who tolerate the drug and are deemed to be receiving a clinical benefit by the principal investigator may be permitted to continue treatment beyond the initial progressive disease as defined in RECIST 1.1, with the sponsor's approval, provided they meet the following clinical stability criteria: - The patient is not clinically stable, meaning there are no clinical symptoms or signs of disease progression (including worsening of clinical laboratory values), as determined by the principal investigator. - No decline in performance status in the East Coast Clinical Oncology Group (ECOG) due to underlying malignancies. - No rapidly progressing disease or progressive tumors in critical anatomical sites (such as spinal cord compression) requiring urgent alternative medical intervention.

[0131] New lesions were considered measurable during the initial stages of progression if their longest diameter was 10 mm or more (excluding pathological lymph nodes, the short axis of which had to be 15 mm or more). New lesions that were considered unmeasurable during the initial stages of progression became measurable and were included in the tumor volume measurement when their longest diameter increased to 10 mm or more (excluding pathological lymph nodes, the short axis of which had to have increased to 15 mm or more).

[0132] Treatment period Patients received research treatment until any disease progression occurred (with the exceptions mentioned above), unacceptable toxicity occurred, or the patient wished to withdraw consent for any reason.

[0133] In Part 1, all patients in cohorts A and B received botensilimab at Q3W for up to four cycles (1 cycle = 21 days) or approximately three months, or until death, unacceptable toxicity, loss of follow-up, disease progression, withdrawal from the study, or termination of the study by the sponsor, whichever came first.

[0134] In Part 2, all patients in cohorts A and B received botensilimab at Q3W for up to four cycles (1 cycle = 21 days) or approximately three months in combination with valstilimab for up to two years (24 months), or until death, unacceptable toxicity, loss of follow-up, disease progression, withdrawal from the study, or termination of the study by the sponsor, whichever came first.

[0135] Following completion / discontinuation of treatment (e.g., due to toxicity), patients were followed up for safety at 30 and 90 days and underwent long-term follow-up every 3 months for at least 12 months from the last dose. Despite completion or discontinuation of study treatment, patients continued scheduled imaging assessments and concurrent outpatient visits if they remained eligible to continue the study.

[0136] Prohibited drugs and treatments Drugs or vaccines specifically prohibited by the exclusion criteria were not permitted during the ongoing trial. If there was a clinical indication for a specifically prohibited drug or vaccine during the trial, discontinuation of the study therapy or vaccination may have been necessary. The final decision regarding any supportive care or vaccination rested with the principal investigator and / or the patient's attending physician. However, the decision by the patient to continue the study therapy required mutual agreement between the principal investigator, the sponsor, and the patient.

[0137] The specific restrictions on combination therapy during the trial period are as follows: - Systemic antitumor chemotherapy or biological therapy. - Immuno-oncological therapies not specified in this protocol. - Investigational drugs other than botensilimab and valstilimab. - Live vaccine. - Systemic glucocorticoids (prednisone equivalent doses exceeding 10 mg for more than one week) for purposes other than the treatment of immune-related adverse events (irAEs). Note: Prophylactic use of corticosteroids to avoid allergic reactions (e.g., intravenous contrast agents or blood transfusions) was permitted, as was the use of inhaled steroids or intranasal or local injections of corticosteroids.

[0138] Patients treated with any prohibited drug (with the exceptions listed above) for clinical management were excluded from the study. All treatments that the principal investigator considered necessary for the patient's welfare may have been administered at the principal investigator's discretion in accordance with local medical standards. All concomitant medications were documented in electronic case report forms (eCRFs), including all prescriptions, commercial medications, herbal supplements, and IV drugs and infusions. If any changes occurred during the study period, documentation of drug dosage, frequency, route, and date was included in the eCRF. All concomitant medications administered within 30 days prior to the first dose of the study treatment and within 30 days of the last dose of the study treatment were documented. Concomitant medications administered 30 days or more after the last dose of the study treatment should have been documented for SAEs, as further defined below.

[0139] Concomitant medications During the study, patients' medications were recorded at safety follow-up visits 30 days after the date of consent and again at the 90-day safety follow-up visit.

[0140] Surgery and radiation therapy If patients required surgery to manage a progressive malignant disease, they were discontinued from the study treatment. In the case of surgery for bowel obstruction, patients may have continued treatment in the study if PD was not confirmed. Palliative radiotherapy for non-targeted lesions may have been permitted in consultation with the sponsor based on specific clinical scenarios.

[0141] Permanent discontinuation of investigational drug treatment Administration of the investigational drug (i.e., botensilimab) was permanently discontinued in the patient for one of the following reasons: - Occurrence of an immune-related adverse event (irAE) that meets the criteria for discontinuation. - Unless the principal investigator determines that the patient is clinically benefiting from the treatment, the patient is clinically stable, and the sponsor has approved the diagnosis of a progressive disease. - If there is no radiological progression based on RECIST 1.1 as suggested by one or more of the following items, then clinical progression: ○ Signs and / or symptoms consistent with the progression of a clinically significant disease (e.g., worsening of clinical laboratory values, appearance of new lesions / worsening of the most commonly observed lesion) ○ Decreased ECOG performance status due to worsening of malignant tumor ○ Tumor progression to critical anatomical sites requiring urgent medical intervention (e.g., CNS metastasis with the potential for spinal cord progression) - If, unless specifically approved by the clinical trial sponsor, the research therapy is not administered two or more times consecutively due to a non-compliance. -pregnancy

[0142] Tumor flare phenomena, defined as localized pain, inflammation, or rash limited to a known or suspected tumor site, did not require discontinuation of treatment.

[0143] Definition of research completion If the study did not terminate for the reasons stated above, the entire trial terminated 24 months after the last patient completed their 90-day safety follow-up visit. If the last patient dropped out of the trial, or if follow-up was not possible before the 90-day safety follow-up visit (i.e., the principal investigator was unable to contact the patient), the study terminated approximately 24 months after the date of that event.

[0144] B. Research Group Inclusion Criteria To participate in the trial, patients had to meet all of the following inclusion criteria:

[0145] Cohort A only: 1. At least six weeks had passed since previous treatment with anti-PD-(L)1, and radiographic progression had been confirmed by two scans at least four weeks apart. In cases where symptoms of progressive malignancy were present, one scan showing progression was sufficient. 2. The previous progression had to have occurred during treatment with an anti-PD-(L)1 regimen, within 12 weeks of the last anti-PD-(L)1 dose in the metastatic setting, or within 24 weeks of completion of therapy in the adjuvant / neoadjuvant setting. 3. For Part 2 only, there should be no interventional anticancer therapies between the last course of anti-PD-(L)1 treatment and the first dose of the study treatment, except for local measures (e.g., surgical resection, biopsy, local radiotherapy) or, if applicable to patients with BRAF mutations, BRAF±MEK inhibition.

[0146] Cohort B only: 1. Previous treatment involved first-generation anti-CTLA-4 (e.g., ipilimumab and tremelimumab), and previous treatment involved anti-PD-(L)1 for at least 6 weeks. 2. Progression during the latest antitumor therapy. 3. For Part 2 only, prior therapy in the BRAF mutation progression setting must be 3 lines or less, and prior therapy in the BRAF wild-type progression setting must be 2 lines or less.

[0147] Cohorts A and B: 1. Voluntarily consent to participate by providing a signed, dated, and written informed consent prior to any research-specific procedure. 2.18 years of age or older. 3. Histological confirmation of cutaneous melanoma of stage III (unresectable) or stage IV according to the AJCC 8th edition staging system. 4. Diseases measurable by baseline imaging according to RECIST 1.1 criteria. 5. Consent to undergo BRAF V600 mutation testing based on BRAF V600 mutation status during the screening period or local facility standards. 6. Life expectancy is ≥ 3 months. 7. ECOG performance status is 0 or 1. 8. Within 21 days from day 1 of cycle 1 (C1D1), appropriate organ function, as defined by the following clinical laboratory values: a. Neutrophils > 1500 / μL (stable with discontinuation of any growth factor within 4 weeks of the initial study treatment dose). b. Platelets>100×10 3 / μL (Blood transfusions to achieve this level were not permitted within two weeks of the initial study treatment dose). c. Hemoglobin > 8.0 g / dL (Blood transfusions to achieve this level were not permitted within two weeks of the initial study treatment dose). d. Creatinine clearance ≥ 45 mL / min (measured or calculated using the Dietary Modification of Renal Disease [MDRD]). e. Aspartate aminotransferase (AST) / alanine aminotransferase (ALT) < 3.0 × upper limit of normal (ULN). f. Total bilirubin <1.5 × ULN or <3.0 × ULN in patients with Gilbert's syndrome. g. Albumin ≥ 3.0 g / dL. h. International normalized ratio or prothrombin time ≤ 1.5 × ULN and activated partial thromboplastin time ≤ 1.5 × ULN (except when the patient was receiving anticoagulant therapy). 9. Patients must provide a sufficient and adequate formalin-fixed paraffin-embedded (FFPE) tumor tissue sample from the most recent biopsy of the tumor lesion, obtained within 90 days of signing the Informed Consent Form (ICF). If recent tumor tissue was unavailable or insufficient, a new biopsy was required unless the sponsor agreed that it was unsafe / unfeasible. 10. Women of childbearing potential (WOCBP) must have negative urine or serum pregnancy tests at the time of screening (within 72 hours of the first dose of the study drug) and before administration of the study drug. Non-fertility was defined as follows: a. You are 50 years of age or older and have not had a menstrual period for more than one year. b. Having experienced amenorrhea for more than two years without hysterectomy or bilateral oophorectomy, and having follicle-stimulating hormone levels within the postmenopausal range at the time of pre-study (screening) evaluation. c. The condition is post-hysterectomy, bilateral oophorectomy, or tubal ligation. In Part 1, WOCBPs must agree to use highly effective contraception from their screening visit until 90 days after the last dose of the study treatment. In Part 2, WOCBPs must agree to use highly effective contraception from their screening visit until 5 months after the last dose of the study treatment. Highly effective contraception is defined in Appendix B, "Guidance on Contraception," or specified in national or local guidelines. Note: Abstinence was permitted if it was an established and preferred form of contraception for the patient. WOCBP had to agree not to donate eggs (oocytes, oocytes) during the treatment period and for at least 90 days after the last dose of the study drug. 11. In Part 1, male patients with one or more childbearing female partners were required to agree to use highly effective contraception throughout the study, from their screening visit until 90 days after receiving their final dose of the study drug. In Part 2, male patients with one or more childbearing female partners were required to agree to use highly effective contraception throughout the study, from their screening visit until 5 months after receiving their final dose of the study drug. Men with pregnant partners were required to agree to use condoms. Pregnant partners were not required to use additional contraception. 12. The party had the willingness and ability to comply with the protocol requirements.

[0148] Exclusion criteria Patients were not enrolled in this study if any of the following criteria were met.

[0149] Cohort A only: 1. The patient had previously received anti-CTLA-4 therapy.

[0150] Cohort B only: 1. The patient had undergone Fc manipulation or Fc-intensified anti-CTLA-4 therapy (e.g., BMS-96218, BMS-986288, HBM4003, XTX101, CTLA-4 targeted bispecific therapy, or other approaches such as ONC-392).

[0151] Cohorts A and B: 1. Melanoma of the eye, uvea, or mucosal melanoma. 2. Persistent toxicity from previous cancer therapy (Common Terminology Criteria for Adverse Events [CTCAE] ≥ 2), excluding stable endocrine disorders, stable neuropathy, and alopecia caused by medication. 3. A history of immune-mediated toxicity with CTCAE ≥ 3 from previous checkpoint inhibitors (excluding endocrine disorders and non-necrotizing / bullous rashes). 4. Refractory ascites defined as requiring two or more therapeutic paracentesis within the four weeks prior to participation in the study, or four or more therapeutic paracentesis within the past 90 days. 5. Intestinal obstruction or impending intestinal obstruction within the past three months. 6. Clinically significant (i.e., active) cardiovascular disease: cerebrovascular event / stroke or myocardial infarction within 6 months of registration, unstable angina, congestive heart failure (New York Heart Association class ≥ III), or severe, uncontrolled arrhythmia requiring medication. 7. Active brain metastases or leptomeningeal metastases, with the following exceptions: a. Treated brain metastases required either a) surgical resection or b) stereotactic radiosurgery. These patients had to discontinue steroid therapy at least 10 days prior to randomization for the purpose of managing brain metastases. If the last brain MRI was within the screening window, follow-up brain imaging after surgical resection or stereotactic radiosurgery was not required. Whole-brain radiotherapy was not permitted. b. Solitary brain metastases that were too small (e.g., 1-2 mm) and / or of unknown cause and untreated were potentially eligible but required consultation with the study's medical monitors for approval. 8. Patients with a history of synchronous malignancies requiring treatment (present during screening) or a history of a previous malignancy that was active within two years prior to the first dose of the study treatment were eligible if treatment was completed at least two years prior to the first dose of the study treatment and the patient had no evidence of disease. Patients with a history of a previous early basal cell / squamous cell carcinoma, low-risk prostate cancer under active surveillance, or non-invasive or in-situ carcinoma that had received curative treatment at any time were also eligible. 9. Clinically significant adverse events related to the most recent treatment / intervention prior to enrollment have not been fully resolved. 10. You have received a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine or booster shot within 7 days prior to C1D1. For vaccines requiring more than one dose, the entire series should be completed before C1D1 if feasible. A booster shot is not required, but it should be administered more than 7 days after C1D1, or more than 7 days from a future cycle of the study. 11. Known allergies or hypersensitivity to any of the test drugs or any of the test drug excipients. 12. You currently have evidence of interstitial lung disease (ILD) or pneumonia, or a history of ILD or non-infectious pneumonia requiring high doses of glucocorticoids. 13. History of allogeneic organ transplantation. 14. A mental illness or substance abuse disorder that would interfere with participation in the requirements of this study. 15. Patients requiring systemic treatment with corticosteroids (more than 10 mg / day in prednisone equivalent) within 14 days of the first dose of the study treatment, or treatment with another immunosuppressant within 30 days. Inhaled or topical steroids, and adrenal replacement steroid doses of >10 mg / day in prednisone equivalent were tolerated in the absence of active autoimmune disease. 16. Active autoimmune disease, or a history of autoimmune disease requiring systemic treatment within two years prior to the start of treatment, i.e., with the use of disease-modifying agents or immunosuppressants. 17. Any history or current evidence of any condition, comorbidity, therapy, any active infection, or abnormal clinical laboratory values ​​that the principal investigator performing the procedure believes may interfere with the results of the study, may prevent the patient from participating throughout the entire duration of the study, or may not be in the patient's best interest to participate. 18. WOCBP during pregnancy or breastfeeding. 19. Previous SARS-CoV-2 infection within 10 days prior to C1D1, in the case of mild or asymptomatic infection, or within 20 days prior to severe / critical illness. 20. Uncontrolled human immunodeficiency virus (HIV) infection. Patients who have been receiving stable, highly active antiretroviral therapy (HAART) with undetectable viral load and normal CD4 count for at least 6 months prior to study participation are eligible. Serological testing for HIV is not required at screening. 21. Patients who are positive for hepatitis B virus (HBV) surface antigen, or who have tested positive for any other HBV test indicating acute or chronic infection, are eligible. Patients who are currently receiving or have previously received anti-HBV therapy and who have not had HBV DNA detected for at least 6 months prior to participating in the study are eligible. Serological testing for HBV at screening is not required. 22. Known active hepatitis C virus (HCV) confirmed by serological testing and polymerase chain reaction (PCR). Patients receiving or previously receiving antiretroviral therapy were eligible if the virus was not detected by PCR testing for at least 6 months prior to study participation. Serological testing for HCV at screening was not mandatory. 23. Dependence on central venous nutrition.

[0152] C. Evaluation and Procedures for Research These procedures may have had to be performed at unscheduled times if the principal investigator deemed it clinically necessary. Furthermore, the principal investigator and / or sponsor may have deemed additional evaluations / tests necessary for reasons related to patient safety. In these cases, such evaluations / tests were conducted in accordance with those rules.

[0153] Adherence to the research design requirements was essential and necessary for conducting the research.

[0154] All screening evaluations had to be completed and reviewed to ensure that potential patients met all eligibility criteria. The principal investigator maintained a screening log to record details of all screened patients, verify eligibility, or, where applicable, record reasons for screening failure.

[0155] Procedures performed as part of a patient's routine clinical management (e.g., blood cell count) and obtained before signing an Informed Consent Form (ICF) may have been used for screening or baseline purposes if the procedure met the protocol-specified criteria and was performed within the defined time frame of other baseline assessments.

[0156] Due to safety reasons or technical problems with the sample, repeated or unplanned samples and images may have been taken.

[0157] screening Prior to participating in this clinical study, the principal investigator or a qualified nominee had to obtain documented informed consent from each candidate patient for optional genetic research.

[0158] After a patient signs an Informed Consent Form (ICF), they are assigned a unique, sequential patient number. Once a number is assigned, it cannot be reassigned, even if the original patient is found to be ineligible or withdraws their consent.

[0159] Patients who met all inclusion criteria and none of the exclusion criteria were enrolled in the trial. Patients who did not meet either the inclusion or exclusion criteria were considered screening failures, and their demographic information and reasons for screening failure were documented.

[0160] During the screening period, attention was paid to prior treatment and washout periods for prohibited substances.

[0161] Treatment and evaluation period The patient continued to receive all treatments while actively undergoing treatment.

[0162] Visit when treatment is discontinued A discontinuation visit (within 7 days of meeting the criteria for permanent discontinuation) should only be made if the investigational drug is discontinued for any reason other than completion of the full study therapy period (four planned cycles of botensilimab and / or up to two years of valstilimab treatment). If the discontinuation visit was made approximately 30 days after the last dose of the investigational drug, the procedure did not need to be repeated at the time of the mandatory 30-day safety follow-up visit.

[0163] 30-day follow-up visit for safety checkup. A mandatory 30-day safety follow-up visit must be made for all patients 30 days (±7 days) after the last dose of the study drug, or before the initiation of new antitumor therapy, whichever comes first. Patients with grade 1 or higher adverse events (AEs) will be further followed until the AE resolves to grade 0 or grade 1, or until new antitumor therapy is initiated, whichever comes first. Patients with persistent treatment-related adverse events (TRAEs) at the time of the safety visit will be followed until the TRAE resolves, stabilizes, or is deemed clinically minor by the principal investigator.

[0164] 90-day follow-up visit for safety All patients who discontinue treatment for any reason will also undergo a safety follow-up visit 90 days later (±7 days).

[0165] Efficacy follow-up (treatment discontinued, research ongoing) Starting at week 18, patients will have follow-up visits in accordance with their imaging evaluation schedule, at Q9W until week 54, and then at Q12W thereafter. Patients who discontinue the study treatment for reasons other than progressive disease may continue their follow-up visits / imaging evaluations as scheduled.

[0166] Survival follow-up (off-study) Patients who were diagnosed with progressive disease, those who initiated additional antitumor therapy, or those who discontinued the trial for other reasons were followed up on their survival status by phone at Q12W until the study ended. For patients who were unable to be followed up, survival data was obtained from public records.

[0167] Post-research anti-cancer therapy The principal investigator or a qualified designated investigator will review all new anticancer therapies initiated after the last dose of the study drug. If a patient initiates a new anticancer therapy within four weeks of the last dose of the study drug, a 30-day safety follow-up visit must be scheduled before the first dose of the new therapy. Once a new anticancer therapy is initiated, the patient will transition to survival follow-up.

[0168] Effectiveness evaluation The response evaluation will be conducted according to RECIST 1.1 [Eisenhauer 2009].

[0169] For all patients, tumor response assessment was performed by radiographic imaging of the chest / abdomen / pelvis (and other areas as required by the specific tumor type or medical history) using computed tomography (CT [preferred]) or MRI (if CT was contraindicated). In general, lesions detected at baseline were followed up at subsequent tumor evaluation visits using the same imaging method, preferably the same imaging equipment.

[0170] The tumor response to treatment was assigned based on the assessment of the response to target lesions, non-target lesions, and new lesions according to RECIST 1.1 (all measurements must be recorded in metric units) [Eisenhauer 2009]. To assess objective response, baseline tumor volume was estimated and compared with subsequent measurements. At baseline, tumor lesions were classified into target lesions and non-target lesions [Eisenhauer 2009].

[0171] These evaluation results were recorded with as much specificity as possible to provide the best opportunity to accurately assess the tumor response before and after treatment. Complete response (CR) or partial response (PR) was confirmed at the next scheduled scan (Eisenhauer 2009). If a patient discontinued treatment but continued the trial, scans were required according to the treatment schedule. Additional imaging was performed at the discretion of the investigator if clinically necessary.

[0172] Tumor imaging Early tumor imaging was performed during the screening period to establish a baseline of disease burden. Scans performed as part of routine clinical management were acceptable as screening scans if they were of sufficient quality and performed up to 21 days prior to the first dose.

[0173] The principal investigator performed additional scans beyond the scheduled trial scans if clinically necessary, at their discretion.

[0174] The timing of imaging during the study followed calendar days and was not adjusted for delays in treatment administration or hospital visits. For consistency, the same imaging technique was used for the same patients throughout the trial.

[0175] Brain imaging Regardless of the presence or absence of contrast agents, MRI was the preferred brain imaging method. However, when MRI was clinically contraindicated, CT was acceptable.

[0176] Patients with a history of central nervous system (CNS) metastases underwent brain imaging on the same schedule as C / A / P imaging.

[0177] During the trial, brain CT / MRI scans were performed if clinically necessary due to the onset of new symptoms.

[0178] Safety evaluation The safety profile of the study treatment was assessed through the recording, reporting, and analysis of baseline medical conditions, adverse events (AEs), physical examination findings including vital signs, and laboratory tests. A comprehensive assessment of any apparent toxicity experienced by patients was conducted from the time of signing informed consent through the course of the study. Research facility staff reported any adverse events, whether observed by the principal investigator or reported by the patient.

[0179] Primary effectiveness analysis Objective response rate (ORR) ORR is defined as the proportion of patients who achieved the best overall response rate (BOR) among objective response rates (CR or PR). BOR is defined as the best response rate recorded from randomization (or initiation of first dose in patients in Cohort B) to data cutoff, disease progression, or initiation of new anticancer therapy. Patients who did not have a response assessment after baseline were considered non-responders for BOR. Verified ORRs, assessed by the principal investigator according to RECIST 1.1, were reported for each group along with the corresponding Clopper-Pearson 95% CI. Furthermore, differences in ORR between groups were calculated using the Miettinen-Nurminen method (Miettinen 1985) at 95% CI. This trial lacked the power to test hypotheses. Therefore, the lack of statistical significance cannot be interpreted as evidence of no difference.

[0180] The percentage of each response category (e.g., CR, PR, SD, PD, NE, and NA) is presented.

[0181] ORR does not require verification, and the ORR for each protocol analysis was calculated in the sensitivity analysis.

[0182] The primary analysis will be conducted approximately 6 months after the last patient was randomized (or the initiation of the first dose in Cohort B patients) and will be considered the final analysis of the study.

[0183] Secondary effectiveness analysis Progression-free survival (PFS) PFS is defined as the earlier of the time from randomization (or initiation of first dose in Cohort B patients) to the time when progression is assessed by the investigator according to RECIST 1.1, or to death. Patients without an event (death or progressive disease) on the analysis cutoff date are censored on the date of the last tumor assessment or initiation of new anticancer therapy. For each treatment group, the median PFS and the cumulative probabilities of PFS at 6 and 12 months are calculated using the Kaplan-Meier method and presented as two-sided 95% CIs. The PFS censoring rules follow the FDA industry guidance for clinical trial endpoints for approval of anticancer drugs and biologics (U.S. Food and Drug Administration, 2007). Data from patients without disease progression or death at the time of analysis are censored at the time of the last appropriate tumor assessment. Data from patients who lost follow-up before recorded disease progression are censored on the last appropriate tumor assessment date on which the patient was found to be progression-free. Data from patients who started new anticancer therapy are censored on the last appropriate tumor assessment date before the introduction of the new therapy.

[0184] In the final analysis, the PFS distributions between treatment groups in Cohort A are descriptively compared using a log-rank test. The hazard ratios between groups are estimated from a Cox regression model.

[0185] Duration of response (DOR) DOR is analyzed as a time-to-event variable only in the subset of patients who respond. All censoring rules for PFS analysis should also be applied to DOR. DOR is summarized using the Kaplan-Meier method. The median event time and the 95% confidence interval of the median are provided using the Brookmeyer and Crowley methods (Brookmeyer 1982).

[0186] Overall survival (OS) Overall survival (OS) is defined as the time to death from any cause. Patients are censored on the earlier of either the last confirmed day of survival or the data cutoff date. The median OS and the estimated cumulative probability of OS at 6-month intervals are calculated for each treatment group using Kaplan-Meier estimation and presented with two-sided 95% confidence intervals.

[0187] In the final analysis, a descriptive comparison of OS between the two groups in Cohort A is performed, similar to the PFS analysis. Median OS time and the 95% confidence interval of the median are provided.

[0188] Safety analysis All safety endpoints were analyzed in the safety analysis set using actual treatment assignments, and data from two cohorts were pooled in the primary safety analysis.

[0189] Administration of test drug Exposure to each treatment group was descriptively summarized as the number of doses administered, duration of exposure, dose intensity, and relative dose intensity. Missed doses and interruptions were also summarized.

[0190] Adverse events (AEs) All adverse events (AEs) were descriptively summarized by number (n) and percentage (%) for each treatment group, from the perspective of both current MedDRA recommended terminology and NCI CTCAE 5.0 grade. AEs observed up to 90 days after discontinuation of the study treatment (i.e., the last dose of randomized treatment) or up to the initiation of the first subsequent anticancer therapy (including radiotherapy excluding palliative radiotherapy) after discontinuation of the study treatment (whichever came first) were used to report all AE summaries. This more accurately depicts AEs attributable solely to the study treatment, as the number of AEs up to 90 days after discontinuation of the study treatment is likely to be attributable to subsequent therapy.

[0191] The incidence of treatment-emergent adverse events (TEAEs) was reported as the number (percentage) of patients in whom TEAEs were detected by system organ class, organ, class, and preferred term. The number (percentage) of patients in whom TEAEs were detected was also summarized by relationship to the investigational medicinal product. TRAEs included AEs for which the investigator responsible for the trial judged to be related to the investigational medicinal product or for which the causal relationship assessment was missing. SAE, death, TEAEs of severity grade 3 or higher, irAEs, TRAEs, and TEAEs leading to treatment discontinuation, dose interruption, or dose delay were summarized.

[0192] D. Pharmacokinetics (PK) Serum botensilimab and balstilimab PK parameters may include, but are not limited to, Cmax ss , Cmin ss , area under the plasma concentration-time curve over the time interval t1 to t2 at steady state (AUC (t1-t2)-ss ), area under the plasma concentration-time curve from time zero to time t (AUC (0-t) ), area under the plasma concentration-time curve from time zero to infinity (AUC (0-∞) ), time to the observed maximum plasma concentration (tmax), elimination rate constant (λz), t 1 / 2 , total body clearance, and Vd.

[0193] For PK analysis, both non-compartmental analysis (NCA) and compartmental modeling, e.g., population PK (PopPK), may be used. Additional PK exposure metrics for serum botensilimab and balstilimab are evaluated via PopPK.

[0194] E. Immunogenicity Immunogenicity assessments were performed to detect and measure anti-drug antibodies (ADA) to botensilimab and balstilimab using a multi-step strategy of screening, confirmation, and titer assays. Samples that were positive in the ADA assay were tested for neutralizing antibodies. Serum samples for botensilimab and balstilimab immunogenicity / ADA were collected from patients at specified time points. Blood samples may be used for additional bioanalytical characterization assessments.

[0195] F. Exploratory Biomarkers Exploratory biomarkers may be evaluated in fresh tumor biopsy samples.

[0196] Appropriate FFPE tumor tissue samples, preferably from the most recent biopsy of the tumor lesion, collected at the time of or after a diagnosis of progressive or metastatic disease, should be available for biomarker evaluation.

[0197] If tumor tissue is unavailable within 90 days prior to signing the Informed Consent Form (ICF), a fresh biopsy was required (if safe / available). If a fresh biopsy is not safe / feasible and tumor tissue is unavailable within 90 days prior to signing the ICF, the sponsor may request archived tissue older than 90 days for research purposes.

[0198] If a tumor biopsy was obtained from a target lesion during eligibility assessment, it was preferable to obtain a new baseline scan. Biopsies of lesions in the study were limited to non-target lesions or new lesions where the pathological etiology was ambiguous.

[0199] Tissue preparation: Tumor biopsy specimens should be fixed and embedded in 10% neutral buffered formalin and regularly treated for histological evaluation. Formalin substitutes are not suitable fixatives.

[0200] Sample Provision: If it is not possible to provide the tumor-containing FFPE tissue block in total, sections freshly cut from this block to a thickness of 5 μm (during the previous month) and mounted on positively charged slides must be provided. Preferably, 25 slides must be provided. If this is not possible, a minimum of 15 slides is required. SuperFrost Plus microscope slides and slide containers will be provided by the central laboratory.

[0201] The objective of this exploratory biomarker study is to identify and / or evaluate the following biomarkers: ● Predictive biomarkers for the response to botensilimab ± valstilimab therapy. ● It is associated with progression to more serious diseases, i.e., it is a prognostic biomarker. ● Associated with congenital or acquired resistance to botensilimab ± valstilimab therapy. ● A safety biomarker that is associated with susceptibility to adverse events or may lead to improvements in the monitoring or investigation of adverse events. ● Indicators of therapeutic activity (i.e., pharmacodynamic biomarkers) and supporting mechanisms of action of botensilimab ± valstilimab. ● Helps improve knowledge and understanding of disease biology and drug safety.

[0202] Both tissue and blood samples are collected at screening and treatment. Measurements may include, but are not limited to, CD16a allele polymorphism (FcγRIIIa status), ctDNA (target panel), phosphorylated proteins, and IHC staining of PD-L1, as well as other markers, to characterize the immune population in TME. G. Purpose and Endpoints [Table 4-1] [Table 4-2]

[0203] Abbreviations: ADA: Anti-drug antibody; AE: Adverse event; ctDNA: Circulating tumor deoxyribonucleic acid; DOR: Duration of response; FcγRIIIA: Crystallizable gamma receptor IIIA fragment; IHC: Immunohistochemistry; NK: Natural killer; ORR: Objective response rate; OS: Overall survival; PFS: Progression-free survival; PK: Pharmacokinetics (multiple possible); RECIST: Criteria for evaluating response in solid tumors; RNA-seq: Ribonucleic acid sequencing; SAE: Serious adverse event; TME: Tumor microenvironment.

[0204] Cohort A (Part 1) included at least 60 patients with advanced cutaneous melanoma (30 per group). Patients were randomized in a 1:1 ratio between Group 1 and Group 2 (50 mg and 150 mg of botensilimab, respectively). No formal statistical comparisons were made between the groups. The sample size of 30 patients per group allowed for 84% power, assuming a target ORR of 30% for each treatment group, and excluding 10% of the ORR by the lower limit of the accurate 90% CI (see, for example, Pires et al. Lancet Oncol. 22(6):836-847 (2021), Zimmer et al. Eur. J. Cancer. 75:47-55 (2017), Idera Pharmaceuticals, retrieved from “ir.iderapharma.com / news-releases / news-release-details / idera-pharmaceuticals-announces-results-illuminate-301-trial” (2021)). Approximately 9 weeks after the enrollment of the first 12 patients in each treatment group, an unrestricted intermediate futility analysis of efficacy with safety assessment was performed for Cohort A without stopping enrollment. Given the limited amount of data available at the time of the interim futility analysis, it was based on unconfirmed responses. If there was 1 or fewer unconfirmed objective responses among the first 12 patients in the 50 mg dose group, the group could be stopped. The criterion of response ≤1 among the first 12 patients in the 50 mg dose group corresponds to a predicted probability of ≤6% of observing at least 25% ORR at the end of the study. If the true ORRs were 5%, 10%, 25%, and 30%, the probabilities of response ≤1 among the 12 patients were 88%, 66%, 28%, and 9%, respectively.

[0205] Cohort B (Part 1) also included a minimum of 60 patients with advanced cutaneous melanoma (30 per group). Patients were randomized in a 1:1 ratio between Group 1 and Group 2 (50 mg and 150 mg of botensilimab, respectively). No formal comparison was made between the two groups. This sample size allowed for 80% power, excluding 10% of unacceptable ORRs by an accurate lower limit of 80% CI, assuming a 25% target ORR for each treatment group. An unrestricted intermediate futility analysis of efficacy with safety assessment was planned for Cohort B approximately 9 weeks after the enrollment of the first 10 patients in each treatment group. Given the limited amount of data available at the time of the intermediate futility analysis, it is based on unconfirmed responses. Any group may be terminated if there is no unconfirmed objective response among the first 10 patients. The criterion for an objective response <1 among 10 patients corresponds to a predicted probability of ≤6% of observing an ORR of at least 20% at the end of the study. If the true ORR is 5%, 10%, 20%, and 25%, the probabilities of no response among the first 10 patients are 60%, 35%, 11%, and 6%, respectively.

[0206] In Part 2, both Cohorts A and B included 50 patients assigned to each cohort. No formal statistical trials were planned. For the 50 patients, with observed ORRs of 20%, 25%, 30%, 35%, 40%, and 50% respectively, the estimated 95% CIs are (10.0%, 33.7%), (13.8%, 39.2%), (17.9%, 44.6%), (22.1%, 49.8%), (26.4%, 54.8%), and (35.5%, 64.5%). If the observed ORR in the combination group is 15% or more higher (e.g., 35%-40%), the lower limit is to exclude the target ORR in Part 1 (e.g., 20%-25%). If the observed ORR is around the target ORR in Part 1 (e.g., 20%-25%), the lower limit is to exclude 10% of the ORR.

[0207] 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 10 efficacy-evaluable cutaneous melanoma patients who received botensilimab as monotherapy or in combination with valstilimab, as described above. Specifically, eight patients received botensilimab as monotherapy, and two patients received botensilimab in combination with valstilimab. The 10 patients were patients with anti-PD-1 (e.g., nivolumab) and / or anti-CTLA-4 (e.g., ipilimumab) relapsed / refractory (R / R) cutaneous melanoma, specifically two patients who were anti-PD-1 R / R and eight patients who were anti-PD-1 and anti-CTLA-4 R / R.

[0208] Figure 1A shows the percentage change in tumor burden from baseline over time in 10 patients, and Figure 1B shows the best percentage change in tumor burden from baseline over time. The BOR (Body-Oriented Recovery) assessment for these patients showed 0 patients with complete response (CR), 3 patients (30%) with partial response (PR), 3 patients (30%) with stable disease (SD), and 4 patients (40%) with progressive disease (PD). The disease-complete response rate (DCR), defined as CR+PR+SD, was 60%. The overall response rate (ORR) was 30%. Of the 10 patients, one of the two anti-PD-1 R / R patients showed a partial response with a 50% ORR. Of the eight anti-PD-1 and anti-CTLA-4 R / R patients, two showed a partial response with a 25% ORR.

[0209] Botensilimab monotherapy has been found to be more effective than conventional IgG1 (ipilimumab analog) in patients expressing the low-affinity FcγRIIIA V158F allele. See, for example, Levey et al. (2022, November 8-12). Botensilimab as a novel Fc-enhanced anti-CTLA-4 antibody-enhanced T cell: Enhancement of APC function and superior antitumor immunity [Poster presentation]. Society for Immunotherapy of Cancer, Boston, MA, United States, “agenusbio.com / wp-content / uploads / 2022 / 11 / Bot-preclinical_Levey_Chand_FINAL.pdf” (Its full disclosure is incorporated herein by reference). Based on the literature, the response to ipilimumab depends on the FcγRIIIA allele status and tumor mutational burden. Approximately 40% of patients (i.e., homozygous for the low-affinity FcγRIIIA allele) exhibit a limited response to anti-CTLA-4 monotherapy (e.g., ipilimumab). See, for example, Arce-Vargas et al. Cancer Cell. 33(4):649-663.e4 (2018). Unlike what has been reported for ipilimumab, the response to botensilimab was independent of FcγRIIIA allele status. Botensilimab monotherapy demonstrated differentiation profiles in cutaneous melanoma patients who were homozygous for low (V / V) and high-affinity FcγRIIIA alleles (F / F), as well as in patients who were heterozygous (V / F) (Table 5). Table 5 shows BORR and BORR% data in phase 1 and phase 2 cutaneous melanoma patients who received botensilimab monotherapy and were ipilimumab and nivolumab R / R. [Table 5]

[0210] Built-in by reference All patent and non-patent literature references cited above are incorporated herein by reference in their entirety.

Claims

1. A method for treating melanoma in a subject requiring 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 VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO:

8.

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

8.

3. The method according to claim 1 or 2, wherein the antibody that specifically binds to human CTLA-4 is administered in a dose of 50 mg to 175 mg.

4. The method according to claim 1 or 2, wherein the antibody that specifically binds to human CTLA-4 is administered in a dose of 75 mg to 150 mg.

5. The method according to claim 1 or 2, wherein the antibody that specifically binds to human CTLA-4 is administered in a dose of 25 mg, 50 mg, 75 mg, 100 mg, or 150 mg.

6. The method according to any one of claims 1 to 5, wherein the antibody that specifically binds to human CTLA-4 is administered intravenously.

7. The method according to any one of claims 1 to 6, wherein the antibody that specifically binds to human CTLA-4 is administered by intravenous infusion over a period of 30 minutes.

8. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered once a week.

9. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered once every two weeks.

10. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered once every three weeks.

11. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered once every four weeks.

12. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered once every five weeks.

13. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered once every six weeks.

14. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 25 mg once every three weeks.

15. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 50 mg once every three weeks.

16. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 75 mg once every three weeks.

17. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 100 mg once every three weeks.

18. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to human CTLA-4 is administered intravenously at a dose of 150 mg once every three weeks.

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

20. The method according to any one of the prior claims, wherein the melanoma is a cutaneous melanoma.

21. The method according to any one of the prior claims, wherein the melanoma is unresectable.

22. The method according to any one of the prior claims, wherein the melanoma is metastatic.

23. The method according to any one of the prior claims, wherein the melanoma is refractory to checkpoint inhibitor therapy.

24. The method according to claim 23, wherein the checkpoint inhibitor therapy is an anti-PD-1 antibody, an anti-PD-L1 antibody, ipilimumab, or tremelimumab.

25. The method according to any one of the prior claims, wherein the subject has received at least one prior anti-cancer therapy.

26. The method according to claim 25, wherein the at least one prior anticancer therapy is an anti-PD-1 antibody, and optionally the anti-PD-1 antibody is valstirimab, nivolumab, or pembrolizumab, an anti-PD-L1 antibody, ipilimumab, or tremelimumab.

27. The method according to claim 25, wherein the at least one prior anticancer therapy is a BRAF inhibitor and / or a MEK inhibitor.

28. The method according to claim 27, wherein the BRAF inhibitor is selected from the group consisting of vemurafenib, dabrafenib, and encorafenib.

29. The method according to claim 27 or 28, wherein the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, and binimetinib.

30. The method according to any one of claims 1 to 22, wherein the antibody that specifically binds to human CTLA-4 is administered to the subject before radiotherapy, chemotherapy, or surgical removal of the tumor.

31. The method according to any one of the prior claims, wherein the subject has not previously received anti-CTLA-4 antibody therapy.

32. The method according to any one of the prior claims, wherein the subject has an unresectable stage III or stage IV cutaneous melanoma.

33. The method according to any one of the prior claims, wherein the subject has a BRAF mutation.

34. The method according to claim 33, wherein the BRAF mutation is a BRAF V600 mutation.

35. The method according to any one of claims 1 to 34, wherein the subject is homozygous to phenylalanine at position 158 of FcγRIIIIA.

36. The method according to any one of claims 1 to 34, wherein the subject is homozygous to valine at position 158 of FcγRIIIIA.

37. The method according to any one of claims 1 to 34, wherein the subject is heterozygous to valine / phenylalanine at position 158 of FcγRIIIIA.

38. The method according to any one of the prior claims, wherein the melanoma is not an intraocular melanoma.

39. The method according to any one of the prior claims, wherein the melanoma is not uveal melanoma.

40. The method according to any one of the prior claims, wherein the melanoma is not a mucosal melanoma.

41. The method according to any one of the prior claims, wherein the subject does not have any persistent toxicity (Common Terminology Criteria for Adverse Events [CTCAE] ≥ 2) from previous cancer therapy.

42. The method according to any one of the prior claims, wherein the subject has no prior history of any immune-mediated toxicity (excluding endocrine disorders and non-necrotizing / bullous rash) with a CTCAE ≥ 3 from prior checkpoint inhibition.

43. The method according to any one of the prior claims, wherein the administration of the antibody reduces the size of the target tumor.

44. The method according to any one of the prior claims, wherein the administration of the antibody enhances T cell activation in the subject.

45. The method according to any one of the prior claims, wherein, prior to administration of the antibody, the subject has a disease measurable by baseline imaging using RECIST 1.

1.

46. The method according to any one of the prior claims, wherein, prior to administration of the antibody, the subject has a performance status (PS) of 0 to 1 in the US East Coast Cancer Clinical Trials Group.

47. The method according to any one of the prior claims, wherein, prior to the administration of the antibody, the subject has a predicted life expectancy of three months or more.

48. Before the administration of the antibody, the subject, a) Neutrophils ≧1500 / μL, b) Platelet count ≥ 100 × 10⁻⁶ ３ / μL, c) Hemoglobin ≥ 8.0 g / dL, d) Creatinine clearance ≥ 45 mL / min, as measured or calculated according to local facility standards. e) AST / ALT≦3×upper limit of normal (ULN), f) Total bilirubin ≤ 1.5 × ULN (excluding patients with Gilbert's syndrome, where the total bilirubin value must be ≤ 3.0 × ULN), g) Albumin ≥ 3.0 g / dL, and / or h) International normalized ratio or prothrombin time ≤ 1.5 × ULN and activated partial thromboplastin time ≤ 1.5 × ULN (except when the patient is receiving anticoagulant therapy), The method according to any one of the prior claims, having appropriate organ function as defined by one or more of the above.

49. The method according to any one of the prior claims, wherein the subject does not have known radiological evidence of partial or complete intestinal obstruction, signs / symptoms of intestinal obstruction, or impending obstruction within the past three months.

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

51. The method according to any one of the prior claims, wherein the subject does not have a clinically significant cardiovascular disease.

52. The method according to any one of the prior claims, wherein the subject does not have active brain metastases or leptomeningeal metastases.

53. The method according to any one of the prior claims, wherein the subject does not have a history of synchronous malignant tumors requiring treatment, or a history of previous malignant tumors that were active within two years prior to the administration of the antibody.

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

55. The method according to any one of the prior claims, wherein the subject has not received any other monoclonal antibody therapy, antibody-drug conjugate therapy, or radioimmune conjugate therapy within four weeks prior to the administration of the antibody.

56. A method for treating melanoma in a subject requiring treatment, the method comprising administering to the subject a dose of 50 mg once every three weeks of an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4), wherein the subject has stage III or stage IV cutaneous melanoma, and the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO:

8.

57. A method for treating melanoma in a subject requiring treatment, the method comprising administering to the subject a dose of 150 mg once every three weeks of an antibody that specifically binds to human cytotoxic T lymphocyte antigen 4 (CTLA-4), wherein the subject has stage III or stage IV cutaneous melanoma, and the antibody comprises VH, which includes the CDRH1, CDRH2, and CDRH3 amino acid sequences of the heavy chain variable region (VH) amino acid sequence described in SEQ ID NO: 7, and VL, which includes the CDRL1, CDRL2, and CDRL3 amino acid sequences of the light chain variable region (VL) amino acid sequence described in SEQ ID NO:

8.

58. The method according to any one of the prior claims, wherein the antibody that specifically binds to human CTLA-4 comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

59. The method according to any one of the prior claims, wherein the antibody that specifically binds to human CTLA-4 comprises VH having the amino acid sequence described in SEQ ID NO: 7 and VL having the amino acid sequence described in SEQ ID NO:

8.

60. The method according to any one of the prior claims, wherein the antibody that specifically binds to human CTLA-4 comprises a human IgG1 heavy chain constant region containing the S239D / A330L / I332E mutation, numbered according to the EU numbering system.

61. The method according to any one of the prior claims, wherein the antibody that specifically binds to human CTLA-4 comprises a heavy chain having the amino acid sequence described in SEQ ID NO: 9 and a light chain having the amino acid sequence described in SEQ ID NO:

10.

62. The method according to any one of the prior claims, wherein the antibody that specifically binds to human CTLA-4 is botensilimab.

63. An antibody that specifically binds to human CTLA-4 for use in the treatment of melanoma, wherein the treatment is carried out according to the method described in any one of the prior claims.

64. An antibody that specifically binds to human CTLA-4, for use in the manufacture of a pharmaceutical product for the treatment of melanoma, wherein the treatment is carried out according to the method of any one of the prior claims.

65. Use of an antibody that specifically binds to human CTLA-4 for the treatment of melanoma, wherein the treatment is carried out according to the method of any one of the prior claims.

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