Combination of PD-1 inhibitor and LAG-3 inhibitor for enhanced therapeutic effects in treatment of melanoma

Abstract

The present disclosure provides methods for treating or inhibiting melanoma growth comprising selecting a patient suffering from melanoma and administering a LAG-3 inhibitor in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof). In certain embodiments, the administration of the PD-1 inhibitor enhances the efficacy of a LAG-3 inhibitor, such as an anti-LAG-3 antibody or antigen-binding fragment thereof, in inhibiting melanoma growth.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 489,900, filed March 13, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure partially provides compositions (including inhibitors of LAG-3 and PD-1) and methods for treating melanoma.

[0004] sequence list

[0005] An official copy of the sequence list and the specification were submitted electronically through the Patent Center. The contents of the electronic sequence list (11445WO01_Sequence_Listing_ST26.xml; size: 28,672 bytes; and creation date: March 12, 2024) are incorporated herein by reference in their entirety. Background Technology

[0006] Programmed death-1 (PD-1) receptor signaling in the tumor microenvironment plays a crucial role in allowing tumor cells to evade immune surveillance by the host immune system. The PD-1 receptor has two ligands—PD-ligand-1 (PD-L1) and PD-L2. Blockade of the PD-1 signaling pathway has demonstrated clinical activity in patients with various tumor types, and antibody therapies that block PD-1 / PDL1 signaling (e.g., nivolumab, pembrolizumab, atezolizumab, durvalumab, and cimiprimab) have been approved for the treatment of various cancers, including metastatic melanoma and metastatic squamous non-small cell lung cancer.

[0007] Similar to PD-1, lymphocyte activation gene-3 (LAG-3) negatively regulates T-cell activity. LAG-3 (also known as CD223) is a 503-amino acid transmembrane protein receptor expressed on activated CD4 and CD8 T cells, γδ T cells, natural killer T cells, B cells, natural killer cells, plasmacytoid dendritic cells, and regulatory T cells. LAG-3 is a member of the immunoglobulin (Ig) superfamily. The primary function of LAG-3 is to attenuate immune responses. Binding of LAG-3 to MHC class II molecules leads to the delivery of negative signals to LAG-3-expressing cells and downregulation of antigen-dependent CD4 and CD8 T cell responses. LAG-3 negatively regulates the ability of T cells to proliferate, produce cytokines, and lyse target cells, a process known as T cell “exhaustion.” LAG-3 has also been reported to play a role in enhancing the function of regulatory T (Treg) cells (Pardoll 2012, Nature Reviews Cancer 12:252-264).

[0008] Because both PD-1 and LAG-3 play important roles in tumor immunity, they are ideal targets for immunotherapy. Simultaneous targeting of LAG-3 and PD-1 (including in anti-PD-1 resistant tumors) may lead to objective responses across several tumor types in patients. Summary of the Invention

[0009] This disclosure relates to methods for treating melanoma and methods for inhibiting tumor growth.

[0010] This document provides methods for treating, improving at least one symptom or indication in a subject, or inhibiting melanoma growth in a subject. A method according to this disclosure includes administering to a subject in need a therapeutically effective amount of an antibody specifically binding to programmed death 1 (PD-1) or an antigen-binding fragment thereof, in combination with a therapeutically effective amount of an antibody specifically binding to LAG-3 or an antigen-binding fragment thereof. In the methods disclosed herein, inhibition achieved with combination therapy is more effective than administration of either antibody as monotherapy.

[0011] This article provides methods for treating melanoma or inhibiting melanoma growth. In some embodiments, the method includes administering to a subject in need a therapeutically effective amount of (a) an antibody that specifically binds to programmed death 1 (PD-1) or an antigen-binding fragment thereof; and (b) an antibody that specifically binds to lymphocyte activation gene-3 (LAG-3) or an antigen-binding fragment thereof. In some embodiments, the melanoma is unresectable locally advanced melanoma, unresectable metastatic melanoma, or completely resected high-risk melanoma.

[0012] A therapeutically effective dose may be one or more doses of the antibody or its antigen-binding fragment. In some aspects, a dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 50 mg to 1500 mg. In some aspects, a dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 350 mg. In some aspects, a dose of the anti-LAG-3 antibody or its antigen-binding fragment is between 50 mg and 8000 mg. In some aspects, a dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 400 mg. In some aspects, a dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 1600 mg.

[0013] Anti-LAG-3 antibody or its antigen-binding fragment may be administered before, simultaneously with, or after anti-PD-1 antibody or its antigen-binding fragment. In some aspects, anti-LAG-3 antibody or its antigen-binding fragment is administered before anti-PD-1 antibody or its antigen-binding fragment. In some aspects, anti-LAG-3 antibody or its antigen-binding fragment is administered on the same day as anti-PD-1 antibody or its antigen-binding fragment.

[0014] In some embodiments, two or more doses of anti-LAG-3 antibody or its antigen-binding fragment are administered in combination with two or more doses of anti-PD-1 antibody or its antigen-binding fragment. In some aspects, each dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 350 mg. In some aspects, each dose of the anti-LAG-3 antibody or its antigen-binding fragment is between 50 mg and 8000 mg. In some aspects, each dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 1600 mg. In some aspects, each dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 400 mg. In some aspects, each dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 200 mg, 250 mg, or 350 mg, and each dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 400 mg, 800 mg, 1000 mg, 1400 mg, or 1600 mg. The antibody, such as a dose containing the antibody, can be administered intravenously, subcutaneously, or intraperitoneally.

[0015] When two or more doses of anti-LAG-3 antibody or its antigen-binding fragment are administered in combination with two or more doses of anti-PD-1 antibody or its antigen-binding fragment, each dose of anti-PD-1 antibody or its antigen-binding fragment may be administered 0.5 to 12 weeks immediately following the previous dose. In some aspects, each dose of anti-LAG-3 antibody or its antigen-binding fragment is administered 0.5 to 12 weeks immediately following the previous dose. In some aspects, each dose of anti-PD-1 antibody or its antigen-binding fragment is administered every six weeks. In some aspects, each dose of anti-LAG-3 antibody or its antigen-binding fragment is administered every six weeks. In some aspects, each dose of anti-PD-1 antibody or its antigen-binding fragment is administered every three weeks. In some aspects, each dose of anti-LAG-3 antibody or its antigen-binding fragment is administered every three weeks.

[0016] In some implementations, melanoma is unresectable locally advanced melanoma. In some aspects, melanoma is unresectable metastatic melanoma. In such implementations, patients may also be selected if they meet one or more of the following criteria: (i) are at least 12 years old on the date of informed consent; (ii) are in stage IIC, III, or IV according to AJCC 8th edition (Amin, 2017) and have histologically confirmed melanoma that has been completely surgically removed; (iii) patients with stage IIIA disease must have at least one lymph node micrometastasis with a maximum measured diameter >1 mm; (iv) a pathologically negative sentinel lymph node biopsy. (SLNB) Specimen confirmed stage IIC melanoma with no evidence of regional or distant metastasis; (v) Complete surgical resection performed within 12 weeks prior to treatment, with treatment only permitted after satisfactory wound healing; (vi) No disease status confirmed by comprehensive physical examination and imaging studies prior to treatment; (vii) Patient showing ≥1% LAG3 in tumor tissue as determined by IHC or iPET; and (viii) Patient must not have received systemic anticancer therapy or radiation therapy for melanoma within the past 5 years.

[0017] In some implementations, melanoma is defined as high-risk melanoma that has been completely resected. In such implementations, patients are further selected if they meet one or more of the following criteria: (1) ≥12 years of age; (2) stage IIc, III, or IV (all M stages) and histologically confirmed melanoma that has been completely resected <12 weeks prior to treatment; (3) no systemic anticancer therapy or radiation therapy for melanoma in the past 5 years; (4) ≥1% LAG3 in tumor tissue as determined by IHC or iPET; (5) no evidence of metastatic disease; and (6) an Eastern Oncology Cooperative Group performance status (PS) of 0 or 1 (for adult patients), Karnofsky PS >70 (for patients >16 years of age) or Lansky PS >70 (for patients <16 years of age).

[0018] This article provides methods for treating melanoma or inhibiting melanoma growth. In some embodiments, the method includes: (1) selecting a patient with melanoma, wherein the patient has unresectable locally advanced melanoma, unresectable metastatic melanoma, or completely resected high-risk melanoma; and (2) administering to the patient (a) 350 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:1 / 2; and (b) 400 mg or 1600 mg of an anti-LAG-3 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:11 / 12.

[0019] In some respects, patients were further selected if they met one or more of the following criteria: (1) ≥12 years of age; (2) stage IIc, III or IV (all M stages) and histologically confirmed melanoma that was completely resected <12 weeks prior to treatment; (3) no systemic anticancer therapy or radiation therapy for melanoma in the past 5 years; (4) no evidence of metastatic disease on staging; and (5) Eastern Oncology Cooperative Group Performance Status (PS) of 0 or 1 (for adult patients), Karnofsky PS >70 (for patients >16 years of age) or Lansky PS >70 (for patients <16 years of age).

[0020] In some respects, step (2) is administered every 3 weeks. In other respects, step (2) is administered every 6 weeks.

[0021] This article provides a method for treating or inhibiting melanoma growth, comprising: (1) selecting a patient with unresectable locally advanced melanoma, unresectable metastatic melanoma, or completely resected high-risk melanoma, wherein the selected patient has not previously received systemic treatment for advanced disease; and (2) administering to the patient (a) 350 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:1 / 2; and (b) 400 mg or 1600 mg of an anti-LAG-3 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:11 / 12.

[0022] In some respects, step (2) is administered every 3 weeks. In other respects, step (2) is administered every 6 weeks.

[0023] In some respects, patients were further selected if they met one or more of the following criteria: (1) ≥12 years of age; (2) stage IIc, III or IV (all M stages) and histologically confirmed melanoma that was completely resected <12 weeks prior to treatment; (3) no systemic anticancer therapy or radiation therapy for melanoma in the past 5 years; (4) no evidence of metastatic disease on staging; and (5) Eastern Oncology Cooperative Group Performance Status (PS) of 0 or 1 (for adult patients), Karnofsky PS >70 (for patients >16 years of age) or Lansky PS >70 (for patients <16 years of age).

[0024] This article provides a method for treating or inhibiting melanoma growth, the method comprising: (1) selecting a patient with melanoma; and (2) administering to the patient (a) 400 mg or 1600 mg of an anti-LAG-3 antibody or an antigen-binding fragment thereof, the anti-LAG-3 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:11 / 12; and (b) 350 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof, in combination with (a), the anti-PD-1 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:1 / 2.

[0025] The application procedure can be performed every 3 weeks. In some cases, the application procedure is performed every 6 weeks.

[0026] In some aspects, the treatment produced therapeutic effects selected from groups comprising delayed melanoma growth, reduced melanoma cell count, melanoma regression, prolonged survival, partial remission, and complete remission. In some aspects, melanoma growth was delayed by at least 10 days compared to untreated subjects. In some aspects, melanoma growth was inhibited by at least 50% compared to untreated subjects. In some aspects, melanoma growth was inhibited by at least 20% compared to subjects receiving either antibody as monotherapy.

[0027] In some embodiments, the method further includes administering an additional therapeutic agent or therapy to the subject, wherein the additional therapeutic agent or therapy is selected from the group consisting of: radiotherapy, surgery, chemotherapy agents, cancer vaccines, PD-L1 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, CD28 agonists, CD38 inhibitors, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, angiopoietin-2 (Ang2) inhibitors, and transforming growth factor β (TG). Fβ inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens, BCG, granulocyte-macrophage colony-stimulating factor, oncolytic viruses, cytotoxins, interleukin-6 receptor (IL-6R) inhibitors, interleukin-4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-21, IL-12, IL-15, antibody-drug conjugates, GITR agonists, 4-1BB agonists, CD20xCD3 bispecific antibodies, MUC16xCD3 bispecific antibodies, and anti-inflammatory drugs.

[0028] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain complementarity-determining region (HCDR1, HCDR2, and HCDR3) of the heavy chain variable region (HCVR) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) of the light chain variable region (LCVR), wherein HCDR1 contains the amino acid sequence of SEQ ID NO:3; HCDR2 contains the amino acid sequence of SEQ ID NO:4; HCDR3 contains the amino acid sequence of SEQ ID NO:5; LCDR1 contains the amino acid sequence of SEQ ID NO:6; LCDR2 contains the amino acid sequence of SEQ ID NO:7; and LCDR3 contains the amino acid sequence of SEQ ID NO:8. In some aspects, HCVR contains the amino acid sequence of SEQ ID NO:1, and LCVR contains the amino acid sequence of SEQ ID NO:2. In some aspects, the anti-PD-1 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:9 and a light chain containing the amino acid sequence of SEQ ID NO:10.

[0029] In some embodiments, the anti-LAG-3 antibody or its antigen-binding fragment comprises a heavy chain CDR (HCDR1, HCDR2, and HCDR3) of HCVR and three light chain CDRs (LCDR1, LCDR2, and LCDR3) of LCVR, wherein HCDR1 contains the amino acid sequence of SEQ ID NO:13; HCDR2 contains the amino acid sequence of SEQ ID NO:14; HCDR3 contains the amino acid sequence of SEQ ID NO:15; LCDR1 contains the amino acid sequence of SEQ ID NO:16; LCDR2 contains the amino acid sequence of SEQ ID NO:17; and LCDR3 contains the amino acid sequence of SEQ ID NO:18. In some aspects, HCVR contains the amino acid sequence of SEQ ID NO:11, and LCVR contains the amino acid sequence of SEQ ID NO:12. In some aspects, the anti-LAG-3 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:19 and a light chain containing the amino acid sequence of SEQ ID NO:20.

[0030] This article provides methods for treating melanoma or inhibiting melanoma growth. In some aspects, the method includes: (a) selecting a patient with melanoma who has undergone surgery to treat melanoma; and (b) administering to the patient: (1) an initial loading dose comprising an anti-PD-1 antibody or an antigen-binding fragment thereof and an anti-LAG-3 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:1 / 2 and the anti-LAG-3 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:11 / 12; and (2) one or more secondary doses, wherein the one or more secondary doses occur one to four weeks immediately following the previous dose. In some aspects, the one or more secondary doses occur three weeks immediately following the previous dose.

[0031] In some cases, the patient showed ≥1% more LAG3 in the melanoma tissue. In some cases, the patient was diagnosed with stage IV melanoma.

[0032] In some respects, patients were further selected if they met one or more of the following criteria: (1) ≥12 years of age; (2) stage IIc, III, or IV (all M stages) and histologically confirmed melanoma that had been completely resected <12 weeks prior to treatment; (3) no systemic anticancer therapy or radiation therapy for melanoma in the past 5 years; (4) ≥1% LAG3 in tumor tissue as determined by IHC or iPET; (5) no evidence of metastatic disease; and

[0033] (6) Eastern Oncology Collaboration Group Performance Status (PS) of 0 or 1 (applicable to adult patients), Karnofsky PS > 70 (patients > 16 years old) or Lansky PS > 70 (patients < 16 years old).

[0034] In some aspects, the method includes further administration to a patient in need of: (3) one or more three-dose administrations, wherein the one or more three-dose administrations occur three to twelve weeks immediately following the previous dose. In some aspects, the one or more three-dose administrations occur three or six weeks immediately following the previous dose.

[0035] In some aspects, the initial loading dose comprises (a) 50 mg to 1500 mg of anti-PD-1 antibody or its antigen-binding fragment and (b) 50 mg to 8000 mg of anti-LAG-3 antibody or its antigen-binding fragment. In some aspects, one or more secondary doses comprise: (a) 350 mg of anti-PD-1 antibody or its antigen-binding fragment and (b) 400 mg, 800 mg, 1000 mg, 1400 mg, 1600 mg, or 2000 mg of anti-LAG-3 antibody or its antigen-binding fragment. In some aspects, one or more tertiary doses comprise: (a) 350 mg of anti-PD-1 antibody or its antigen-binding fragment and (b) 400 mg, 800 mg, 1000 mg, 1400 mg, 1600 mg, or 2000 mg of anti-LAG-3 antibody or its antigen-binding fragment.

[0036] The various aspects provided herein can be applied to any or every other method provided herein. Exemplarily, in any of the foregoing methods, a dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 50 mg to 1500 mg or 350 mg. In any of the foregoing methods, a dose of the anti-LAG-3 antibody or its antigen-binding fragment is between 50 mg and 8000 mg, or comprises 400 mg or 1600 mg. In any of the foregoing methods, the anti-LAG-3 antibody or its antigen-binding fragment is administered before, simultaneously with, or after the anti-PD-1 antibody or its antigen-binding fragment. In any of the foregoing methods, the anti-LAG-3 antibody or its antigen-binding fragment is administered before the anti-PD-1 antibody or its antigen-binding fragment. In any of the foregoing methods, the anti-LAG-3 antibody or its antigen-binding fragment is administered on the same day as the anti-PD-1 antibody or its antigen-binding fragment. In any of the foregoing methods, two or more doses of the anti-LAG-3 antibody or its antigen-binding fragment are administered in combination with two or more doses of the anti-PD-1 antibody or its antigen-binding fragment. In some embodiments, each dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 350 mg. In some embodiments, each dose of the anti-LAG-3 antibody or its antigen-binding fragment is between 50 mg and 8000 mg. In some embodiments, each dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 1600 mg. In some embodiments, each dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 400 mg. In any of the foregoing methods, each dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 200 mg, 250 mg, or 350 mg, and each dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 400 mg, 800 mg, 1000 mg, 1400 mg, or 1600 mg. In some aspects, each dose of the anti-PD-1 antibody or its antigen-binding fragment is administered 0.5 to 12 weeks immediately following the previous dose. In some aspects, each dose of the anti-LAG-3 antibody or its antigen-binding fragment is administered 0.5 to 12 weeks immediately following the previous dose. 18. In some aspects, each dose of anti-PD-1 antibody or its antigen-binding fragment is administered every six weeks. In some aspects, each dose of anti-LAG-3 antibody or its antigen-binding fragment is administered every six weeks. In some aspects, each dose of anti-PD-1 antibody or its antigen-binding fragment is administered every three weeks. In some aspects, each dose of anti-LAG-3 antibody or its antigen-binding fragment is administered every three weeks. In any of the foregoing methods, the antibody is administered intravenously, subcutaneously, or intraperitoneally. In some aspects, the melanoma is unresectable locally advanced melanoma. In some aspects, the melanoma is unresectable metastatic melanoma. In some aspects, the melanoma is completely resected high-risk melanoma.

[0037] In some implementation schemes, patients are further selected based on meeting one or more of the following criteria:

[0038] (i) At least 12 years old on the date the informed consent form is provided;

[0039] (ii) According to AJCC 8th edition (Amin, 2017), a patient is in stage IIC, III or IV and has a histologically confirmed melanoma that has been completely surgically removed to qualify.

[0040] (iii) Patients with stage IIIA disease must have at least one lymph node micrometastasis with a maximum measured diameter >1 mm;

[0041] (iv) Stage IIC melanoma confirmed by a pathologically negative sentinel lymph node biopsy (SLNB) specimen, with no evidence of regional or distant metastasis;

[0042] (v) Complete surgical resection was performed within 12 weeks prior to randomization, and treatment was only permitted after satisfactory wound healing.

[0043] (vi) Before treatment, a comprehensive physical examination and imaging examination confirmed that there was no disease state;

[0044] (vii) As determined by IHC or iPET, the patient shows ≥1% LAG3 in tumor tissue; and

[0045] (viii) The patient must not have received systemic anticancer therapy or radiation therapy for melanoma in the past 5 years.

[0046] In some implementation schemes, patients are further selected based on meeting one or more of the following criteria:

[0047] (1) ≥12 years old;

[0048] (2) Stage IIc, III or IV (all M stages) and histologically confirmed melanoma that was completely resected <12 weeks before randomization;

[0049] (3) No systemic anticancer treatment or radiation therapy for melanoma in the past 5 years;

[0050] (4) LAG3 is identified as ≥1% in tumor tissue by IHC or iPET;

[0051] (5) No evidence of metastatic disease; and

[0052] (6) Eastern Oncology Collaboration Group Performance Status (PS) of 0 or 1 (applicable to adult patients), Karnofsky PS > 70 (patients > 16 years old) or Lansky PS > 70 (patients < 16 years old).

[0053] Treatment according to any of the methods provided herein produced a therapeutic effect selected from the group consisting of delayed melanoma growth, reduced melanoma cell number, melanoma regression, prolonged survival, partial remission, and complete remission. In some aspects, melanoma growth was delayed by at least 10 days compared to untreated subjects. In some aspects, melanoma growth was inhibited by at least 50% compared to untreated subjects. In some aspects, melanoma growth was inhibited by at least 20% compared to subjects treated with any antibody as monotherapy.

[0054] In some embodiments, the methods provided herein further include administering an additional therapeutic agent or therapy to the subject, wherein the additional therapeutic agent or therapy is selected from the group consisting of: radiotherapy, surgery, chemotherapy agents, cancer vaccines, PD-L1 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, CD28 agonists, CD38 inhibitors, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, angiopoietin-2 (Ang2) inhibitors, transforming growth factor β (… TGFβ inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens, BCG, granulocyte-macrophage colony-stimulating factor, oncolytic viruses, cytotoxins, interleukin-6 receptor (IL-6R) inhibitors, interleukin-4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-21, IL-12, IL-15, antibody-drug conjugates, GITR agonists, 4-1BB agonists, CD20xCD3 bispecific antibodies, MUC16xCD3 bispecific antibodies, and anti-inflammatory drugs.

[0055] In some embodiments, an additional therapeutic agent is an anti-PD-1 antibody or its antigen-binding fragment comprising a heavy chain complementarity-determining region (HCDR1, HCDR2, and HCDR3) of the heavy chain variable region (HCVR) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) of the light chain variable region (LCVR), wherein HCDR1 contains the amino acid sequence of SEQ ID NO:3; HCDR2 contains the amino acid sequence of SEQ ID NO:4; HCDR3 contains the amino acid sequence of SEQ ID NO:5; LCDR1 contains the amino acid sequence of SEQ ID NO:6; LCDR2 contains the amino acid sequence of SEQ ID NO:7; and LCDR3 contains the amino acid sequence of SEQ ID NO:8. In some aspects, HCVR contains the amino acid sequence of SEQ ID NO:1, and LCVR contains the amino acid sequence of SEQ ID NO:2. In some aspects, the anti-PD-1 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:9 and a light chain containing the amino acid sequence of SEQ ID NO:10.

[0056] In some embodiments, the anti-LAG-3 antibody or its antigen-binding fragment comprises a heavy chain CDR (HCDR1, HCDR2, and HCDR3) of HCVR and three light chain CDRs (LCDR1, LCDR2, and LCDR3) of LCVR, wherein HCDR1 contains the amino acid sequence of SEQ ID NO:13; HCDR2 contains the amino acid sequence of SEQ ID NO:14; HCDR3 contains the amino acid sequence of SEQ ID NO:15; LCDR1 contains the amino acid sequence of SEQ ID NO:16; LCDR2 contains the amino acid sequence of SEQ ID NO:17; and LCDR3 contains the amino acid sequence of SEQ ID NO:18. In some aspects, HCVR contains the amino acid sequence of SEQ ID NO:11, and LCVR contains the amino acid sequence of SEQ ID NO:12. In some aspects, the anti-LAG-3 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:19 and a light chain containing the amino acid sequence of SEQ ID NO:20.

[0057] According to any of the methods provided in this article, the inhibition is more effective than the administration of any antibody as monotherapy.

[0058] Other implementation methods will become apparent upon referring to the following detailed description. Attached Figure Description

[0059] Figure 1A and Figure 1B A flowchart of the research process for Example 1 is described.

[0060] Figure 2 A flowchart of the research process for Example 2 is described. Detailed Implementation

[0061] It should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. When referring to a specifically enumerated numerical value, the term “about” as used herein means that the value may vary from the enumerated value by no more than 1%. For example, the expression “about 100” as used herein includes 99 and 101 and all values ​​between them (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0063] As used herein, the term "antibody" includes immunoglobulin molecules and their multimers (e.g., IgM) comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) linked together by disulfide bonds. In a typical antibody, each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or V). H ) and the heavy-chain constant region. The heavy-chain constant region contains three structural domains: C H 1. C H 2 and C H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this document). L The light chain constant region contains a structural domain (C) and a light chain constant region. L 1). V H District and V L The region can be further subdivided into highly variable regions known as complementary determinant regions (CDRs), which are interspersed with more conservative regions known as frame regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of this disclosure, the FRs of the anti-IL-4R antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. The common amino acid sequence may be defined based on the parallel analysis of two or more CDRs.

[0064] The term "antibody" as used herein also includes the antigen-binding fragment of a complete antibody molecule. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from complete antibody molecules, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optional constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to align one or more variable and / or constant domains into suitable conformations, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0065] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) the smallest recognition unit consisting of amino acid residues of a hypervariable region of a mimicking antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. The term “antigen-binding fragment” as used herein also encompasses other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, dimers, triplets, tetramers, microsomes, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0066] Antigen-binding fragments of antibodies typically include at least one variable domain. Variable domains can have any size or amino acid composition and will generally include at least one CDR adjacent to or aligned with one or more frame sequences. In the presence of V... H Domain and V L In the antigen-binding fragment associated with the structural domain, V H and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V.H or V L Structural domain.

[0067] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be found within the antigen-binding fragment of the antibody disclosed herein include: (i) V H -C H 1; (ii)V H -C H 2; (iii)V H -C H 3; (iv)V H -C H 1-C H 2; (v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L (viii)V L -C H 1; (ix)V L -C H 2; (x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3; (xiii)V L -C H 2-C H 3; and (xiv)V L -C L In any configuration of the variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other or linked via complete or partial hinge regions or connecting regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, resulting in flexible or semi-flexible connections between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody disclosed herein may comprise each other and / or one or more monomers V H or V LHomodimers or heterodimers (or other polymers) of any of the above-described variable and constant domain configurations of non-covalently associated domains (e.g., via disulfide bonds).

[0068] The term "antibody" as used herein also includes multispecific (e.g., bispecific) antibodies. Multispecific antibodies or antigen-binding fragments of antibodies typically comprise at least two distinct variable domains, each capable of specifically binding to a single antigen or a different epitope on the same antigen. Any form of multispecific antibody can be applied to the cases of the antibodies or antigen-binding fragments of antibodies disclosed herein using conventional techniques available in the art. For example, this disclosure includes methods involving the use of bispecific antibodies, wherein one arm of an immunoglobulin is specific for PD-1 or LAG-3 or fragments thereof, and the other arm of the immunoglobulin is specific for or conjugated to a second therapeutic target. Exemplary bispecific forms that may be used in the context of this disclosure include, but are not limited to: for example, scFv-based or biantibody-based bispecific forms, IgG-scFv fusion proteins, dual variable region (DVD)-Ig, tetrageneous hybridomas, knock-in-hole structures, common light chains (e.g., common light chains with knock-in-hole structures, etc.), CrossMab, CrossFab fragments, chain exchange engineered domain (SEED) antibodies, leucine zippers, Duobody, IgG1 / IgG2, bifunctional Fab fragments (DAF)-IgG and Mab 2 Bispecific forms (for a review of the aforementioned forms, see, for example, Klein et al. 2012, mAbs4:6, 1-11 and the references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugations, for example, where site-specific antibody-oligonucleotide conjugates are generated using non-natural amino acids with orthogonal chemical reactivity, and then the antibody-oligonucleotide conjugates are self-assembled into multimeric complexes with defined composition, valence state, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. [Electronic publication date: December 4, 2012]).

[0069] The antibodies used in the methods of this disclosure may be human antibodies. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, the human antibodies of this disclosure may, for example, include amino acid residues in the CDR, and specifically in CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-specific mutagenesis, or by in vivo somatic mutations). However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human frame sequence.

[0070] The antibodies used in the methods disclosed herein may be recombinant human antibodies. The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells (further described below), antibodies isolated from recombinant, combined human antibody libraries (further described below), antibodies isolated from animals transfected against human immunoglobulin genes (e.g., mice) (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using animals transfected against human Ig sequences, in vivo somatic cell mutagenesis), and therefore the V of the recombinant antibody... H and V L The amino acid sequence of the region is, although it originates from human lineage V H and V L Sequences and related sequences that may not naturally exist within the human antibody germline library in vivo.

[0071] While any methods and materials similar to or equivalent to those described herein may be used in the practice of this disclosure, exemplary methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference in their entirety.

[0072] General Method

[0073] Standard methods in molecular biology are described in Sambrook, Fritsch, and Maniatis (1982 and 1989, 2nd ed.; 2001, 3rd ed.), *Molecular Cloning, A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001), *Molecular Cloning*, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Wu (1993), *Recombinant DNA*, Vol. 217, Academic Press, San Diego, Calif. Standard methods also appear in Ausbel et al. (2001), *Current Protocols in Molecular Biology*, Vols. 1–4, John Wiley and Sons, Inc., New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0074] Methods for protein purification include immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as described in (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Describe chemical analysis, chemical modification, post-translational modification, fusion protein production, and protein glycosylation (see, for example, Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo., pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391). The generation, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, ibid.). Standard techniques for characterizing ligand / receptor interactions are available (see, for example, Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0075] Monoclonal antibodies, polyclonal antibodies, and humanized antibodies can be prepared (see, for example, Sheperd and Dean, eds. (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel, eds. (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York, China). SpringHarbor, NY, pp. 139-243; Carpenter et al. (2000) J. Immunol. 165:6205; He et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Patent No. 6,329,511).

[0076] Alternatives to humanization include using human antibody libraries presented on bacteriophages or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol. 17:397-399). Describe single-chain antibodies and bifunctional antibodies (see, for example, Malekti et al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al. (2001) J. Biol. Chem. 276:7346-7350; Desmyter et al. (2001) J. Biol. Chem. 276:26285-26290; Hudson and Kortt (1999) J. Immunol. Methods 231:177-189; and U.S. Patent No. 4,946,778).Provide bifunctional antibodies (see, for example, Mack et al. (1995) Proc. Natl. Acad. Sci. USA 92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel et al. (2001) Protein Engineering 14:815-823; Segal et al. (2001) J. Immunol. Methods 248:1-6; Brennan et al. (1985) Science 229:81-83; Raso et al. (1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207; Traunecker et al. (1991) EMBO J.10:3655-3659; and U.S. Patent Nos. 5,932,448, 5,532,210, and 6,129,914. Fully human antibodies can also be developed in genetically engineered mice such as VelociMouse. See, for example, DeChiara et al., Producing fully ES cell-derived mice from eight-cell stage embryo injections, Methods Enzymol, 476:285-94 (2010); DeChiara et al., VelociMouse: fully ES cell-derived F0-generation mice obtained from the injection of ES cells into eight-cell-stage embryos. Methods Mol Biol, 530:311-24 (2009); U.S. Patent Nos. 7,576,259, 7,659,442, or 7,294,754, and US2008 / 0078000A1.

[0077] Antigen purification is not typically required for antibody production. Animals can be immunized with cells carrying the antigen of interest. Subsequently, spleen cells can be isolated from the immunized animal, and these spleen cells can be fused with myeloma cell lines to generate fusion tumors (see, for example, Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., ibid.; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

[0078] Antibodies can bind to, for example, small drug molecules, enzymes, liposomes, and polyethylene glycol (PEG). Antibodies are suitable for therapeutic, diagnostic, kit, or other purposes and include antibodies conjugated to, for example, dyes, radioisotopes, enzymes, or metals (e.g., colloidal gold) (see, for example, Le Doussal et al. (1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).

[0079] Methods for use in flow cytometry, including fluorescence-activated cell sorting (FACS), are available (see, for example, Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed., Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids (including nucleic acid primers and probes), peptides, and antibodies, for example as diagnostic reagents, are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

[0080] Describe the standard immune system histological methods (see, for example, Muller-Harmelink ed. (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.; Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0081] Software packages and databases for determining, for example, antigen fragments, leader sequences, protein folds, functional domains, glycosylation sites, and sequence alignment are available (see, for example, GenBank, Vector). Suite (Informax, Inc., Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); (TimeLogic Corp., Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16:741-742; Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren et al. (2002) Comput. Methods Programs Biomed. 68: 177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0082] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Canonical conventions for identifying CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences within antibodies.

[0083] PD-1 inhibitors

[0084] According to certain exemplary embodiments of this disclosure, the method includes administering a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof. The term "PD-1" refers to programmed death-1 protein, a T-cell co-inhibitory molecule, also known as CD279. The full-length amino acid sequence of PD-1 is available in GenBank under accession number NP_005009.2. PD-1 is a member of the CD28 / CTLA-4 / ICOS family of T-cell co-inhibitory molecules. PD-1 is a 288-amino acid protein with an extracellular N-terminal domain (which is IgV-like), a transmembrane domain, and an intracellular domain containing an immunoreceptor tyrosine inhibitory (ITIM) motif and an immunoreceptor tyrosine switching (ITSM) motif (Chattopadhyay et al., 2009, Immunol. Rev.). The PD-1 receptor has two ligands—PD-ligand-1 (PD-L1) and PD-L2.

[0085] PD-L1 is a 290-amino acid protein with an extracellular IgV-like domain, a transmembrane domain, and a highly conserved intracellular domain (approximately 30 amino acids). PD-L1 is constitutively expressed on a variety of cell types, including antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells) and on both hematopoietic and non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islets, and immune-immune sites). PD-L1 is also expressed in various tumors, virus-infected cells, and autoimmune tissues, and is a component of the immunosuppressive microenvironment (Ribas 2012, NEJM 366:2517-2519).

[0086] PD-1 inhibitors include antibodies and their antigen-binding fragments, as well as other substances (e.g., peptides and small molecules) that specifically bind to PD-1 and antagonize one or more biological activities of PD-1. Molecules that specifically bind to PD-1 may be referred to as "anti-PD-1". In embodiments of this disclosure, a PD-1 inhibitor is an antibody or its antigen-binding fragment that binds to PD-L1 or PD-L2.

[0087] In embodiments of this disclosure, the PD-1 inhibitor is an antibody or antigen-binding fragment thereof as described in US9,987,500.

[0088] According to certain embodiments, the antibodies used in the methods of this disclosure specifically bind to PD-1. The term "specific binding," etc., means that the antibody or its antigen-binding fragment forms a relatively stable complex with the antigen under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, as used in the context of this disclosure, antibodies that "specifically bind" PD-1 include, as measured in a surface plasmon resonance assay, at concentrations of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM. D Antibodies that bind to PD-1 or a portion thereof. However, isolated antibodies that specifically bind to human PD-1 may be cross-reactive with other antigens, such as PD-1 molecules from other (non-human) species.

[0089] According to certain exemplary embodiments of this disclosure, an anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity-determining region (CDR) containing any amino acid sequence of an anti-PD-1 antibody listed in U.S. Patent No. 9,987,500.

[0090] In some exemplary embodiments, the anti-PD-1 antibody or its antigen-binding fragment used in the methods of this disclosure comprises a heavy chain complementarity-determining region (HCDR) of a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO:1 and a light chain complementarity-determining region (LCDR) of a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO:2. According to some embodiments, the anti-PD-1 antibody or its antigen-binding fragment comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 contains the amino acid sequence of SEQ ID NO:3; HCDR2 contains the amino acid sequence of SEQ ID NO:4; HCDR3 contains the amino acid sequence of SEQ ID NO:5; LCDR1 contains the amino acid sequence of SEQ ID NO:6; LCDR2 contains the amino acid sequence of SEQ ID NO:7; and LCDR3 contains the amino acid sequence of SEQ ID NO:8. In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment comprises an HCVR containing SEQ ID NO:1 and an LCVR containing SEQ ID NO:2. In some embodiments, the method of this disclosure includes using an anti-PD-1 antibody, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:9. In some embodiments, the anti-PD-1 antibody comprises a light chain containing the amino acid sequence of SEQ ID NO:10. An exemplary antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO:1 and a light chain variable region containing the amino acid sequence of SEQ ID NO:2 is called REGN2810 (cimipril malabsorption lipoprotein antibody). The fully human anti-PD-1 antibody.

[0091] According to certain exemplary embodiments, the methods of this disclosure include the use of REGN2810 or a bioequivalent thereof. As used herein, the term "bioequivalent" means an anti-PD-1 antibody or PD-1 binding protein, or a fragment thereof that is a pharmaceutical equivalent or substitute, whose rate of absorption and / or extent of absorption does not show a significant difference from that of REGN2810 when administered at the same molar dose (single or multiple doses) under similar experimental conditions. In the context of this disclosure, the term refers to an antigen-binding protein that binds to PD-1 and does not have clinically significant differences from REGN2810 in terms of its safety, purity, and / or potency.

[0092] Other anti-PD-1 antibodies that may be used in the context of the methods disclosed herein include, for example, antibodies known in the art and referred to as nivolumab (US Patent No. 8,008,449), pembrolizumab (US Patent No. 8,354,509), MEDI0608 (US Patent No. 8,609,089), pidilizumab (US Patent No. 8,686,119), or any of the anti-PD-1 antibodies described in US Patent Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8,900,587. In embodiments of this disclosure, PD-1 inhibitors are described in any of the following: US20110008369, US20130017199, US20130022595, WO2006121168, WO20091154335, WO2012145493, WO2013014668, WO2009101611, EP2262837, and EP2504028.

[0093] The anti-PD-1 antibody used in the methods of this disclosure may have pH-dependent binding properties. For example, the anti-PD-1 antibody used in the methods of this disclosure may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, the anti-PD-1 antibody of this disclosure may exhibit enhanced binding to its antigen at acidic pH compared to neutral pH. The expression "acidic pH" includes pH values ​​less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or smaller. The expression "neutral pH" as used herein means a pH from about 7.0 to about 7.4. The term "neutral pH" includes pH values ​​of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0094] In some cases, "the binding of PD-1 is reduced at acidic pH compared to neutral pH" to describe the K+ of antibodies binding to PD-1 at acidic pH. D The K value of the antibody that binds to PD-1 at neutral pH D The ratio of values ​​is used to express this (or vice versa). For example, for the purposes of this disclosure, if the antibody or its antigen-binding fragment exhibits an acidic / neutral K value of about 3.0 or greater... DThe ratio of pH to pH indicates that the antibody or its antigen-binding fragment exhibits "a decreased binding to PD-1 at acidic pH compared to neutral pH." In some exemplary embodiments, the acidic / neutral pH of the antibody or antigen-binding fragment of this disclosure... D The ratio can be approximately 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.

[0095] Antibodies with pH-dependent binding properties can be obtained, for example, by screening antibody populations for reduced (or enhanced) binding to specific antigens at acidic pH compared to neutral pH. Additionally, modifying the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent properties. For example, by replacing one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, antibodies with reduced antigen binding at acidic pH relative to neutral pH can be obtained. As used herein, "acidic pH" refers to a pH of 6.0 or lower.

[0096] LAG-3 inhibitors

[0097] The term "LAG-3" refers to the lymphocyte activation gene-3 protein, an immune checkpoint receptor or T-cell co-inhibitory molecule, also known as CD223. The full-length amino acid sequence of LAG-3 is available in GenBank under accession number NP_002277.4. LAG-3 is a member of the immunoglobulin (Ig) superfamily. LAG-3 is a 503-amino acid type I transmembrane protein with four extracellular Ig-like domains D1 through D4, and is expressed on activated T cells, natural killer cells, B cells, plasmacytoid dendritic cells, and regulatory T cells. The LAG-3 receptor binds to MHC class II molecules present on antigen-presenting cells (APCs).

[0098] As used in this article, the term "T cell co-inhibitory molecule" refers to ligands and / or receptors that regulate immune responses through T cell activation or inhibition. The term "T cell co-inhibitory molecule" is also known as T cell co-signaling molecules, and includes, but is not limited to: programmed death-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuators (BTLA), CD-28, 2B4, LY108, T cell immunoglobulin and mucin 3 (TIM3), T cell immune receptor with immunoglobulin and ITIM (TIGIT, also known as VSIG9), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1, also known as CD305), inducible T cell co-stimulatory molecule (ICOS, also known as CD278), T cell activation V-domain Ig repressor (VISTA), and CD160.

[0099] LAG-3 inhibitors include antibodies and their antigen-binding fragments, as well as other substances (e.g., peptides and small molecules) that specifically bind to LAG-3 and antagonize one or more of LAG-3's biological activities. Molecules that specifically bind to LAG-3 may be referred to as "anti-LAG-3".

[0100] In embodiments of this disclosure, the LAG-3 inhibitor is an antibody or antigen-binding fragment thereof as described in US20170101472.

[0101] According to certain embodiments, the antibodies used in the methods of this disclosure specifically bind to LAG-3. The term "specific binding," etc., means that the antibody or its antigen-binding fragment forms a relatively stable complex with the antigen under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, as used in the context of this disclosure, antibodies that "specifically bind" to LAG-3 include those, as measured in a surface plasmon resonance assay, at concentrations of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM. D Antibodies that bind to LAG-3 or a portion thereof. However, isolated antibodies that specifically bind to human LAG-3 may be cross-reactive with other antigens, such as LAG-3 molecules from other (non-human) species.

[0102] According to certain exemplary embodiments of this disclosure, an anti-LAG-3 antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity-determining region (CDR), wherein the anti-LAG-3 antibody or its antigen-binding fragment comprises any amino acid sequence of an anti-LAG-3 antibody as set forth in US20170101472.

[0103] In some exemplary embodiments, the anti-LAG-3 antibody or its antigen-binding fragment used in the methods of this disclosure comprises a heavy chain complementarity-determining region (HCDR) of a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO:11 and a light chain complementarity-determining region (LCDR) of a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO:12. According to some embodiments, the anti-LAG-3 antibody or its antigen-binding fragment comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 contains the amino acid sequence of SEQ ID NO:13; HCDR2 contains the amino acid sequence of SEQ ID NO:14; HCDR3 contains the amino acid sequence of SEQ ID NO:15; LCDR1 contains the amino acid sequence of SEQ ID NO:16; LCDR2 contains the amino acid sequence of SEQ ID NO:17; and LCDR3 contains the amino acid sequence of SEQ ID NO:18. In other embodiments, the anti-LAG-3 antibody or its antigen-binding fragment comprises an HCVR containing SEQ ID NO:11 and an LCVR containing SEQ ID NO:12. In some embodiments, the method of this disclosure includes using an anti-LAG-3 antibody, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:19. In some embodiments, the anti-LAG-3 antibody comprises a light chain containing the amino acid sequence of SEQ ID NO:20. An exemplary antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO:11 and a light chain variable region containing the amino acid sequence of SEQ ID NO:12 is a fully human anti-LAG-3 antibody called REGN3767, i.e., fianlimab.

[0104] According to certain exemplary embodiments, the methods of this disclosure include the use of REGN3767 or a bioequivalence thereof. As used herein, the term "bioequivalence" refers to an anti-LAG-3 antibody or LAG-3 binding protein, or a fragment thereof that is a pharmaceutical equivalent or substitute, whose absorption rate and / or extent does not show significant difference from that of REGN3767 when administered at the same molar dose (single or multiple doses) under similar experimental conditions. In the context of this disclosure, the term refers to an antigen-binding protein that binds to LAG-3 and does not have clinically significant differences from REGN3767 in terms of safety, purity, and / or potency.

[0105] Other anti-LAG-3 antibodies that may be used in the context of the methods disclosed herein include, for example, antibodies known in the art and referred to as relatlimab (US20110150892), LAG525 (WO2017 / 037203), GSK2831781 (US2016 / 0017037), Sym022 (WO2018 / 069500), INCAGN02385 (US20180127499), or those described in U.S. Patent / Publication Nos. 5,976,877, 6,143,273, 6,197,524, 855,1481, 20110070238, and 20110150892. Any of the anti-LAG-3 antibodies described in 20130095114, 20140093511, 20140127226, 20140286935, WO95 / 30750, WO97 / 03695, WO98 / 58059, WO2004 / 078928, WO2008 / 132601, WO2010 / 019570, WO2014 / 008218, EP0510079B1, EP0758383B1, EP0843557B1, EP0977856B1, EP1897548B2, EP2142210A1 and EP2320940B1.

[0106] Methods to treat melanoma or inhibit melanoma tumor growth

[0107] This disclosure includes methods for treating, improving, or reducing the severity of at least one symptom or indication in a subject, or inhibiting melanoma growth in a subject. Methods according to this aspect include administering to a subject in need a combination of an antibody specifically binding to PD-1 or an antigen-binding fragment thereof and an antibody specifically binding to LAG-3 or an antigen-binding fragment thereof. In some aspects, the method includes administering to a subject in need a therapeutically effective amount of an antibody specifically binding to PD-1 or an antigen-binding fragment thereof and a therapeutically effective amount of an antibody specifically binding to LAG-3 or an antigen-binding fragment thereof. The terms “treat”, “treating,” etc., as used herein, mean relieving symptoms, temporarily or permanently eliminating the triggers for symptoms, delaying or inhibiting melanoma growth, reducing melanoma cell burden or tumor burden, promoting melanoma regression, causing melanoma shrinkage, necrosis, and / or disappearance, preventing melanoma recurrence, and / or prolonging the survival of the subject.

[0108] As used herein, the term "subject in need" refers to a human or non-human mammal exhibiting one or more symptoms or indications for melanoma and / or having been diagnosed with melanoma and requiring treatment for it. In many embodiments, the term "subject" may be used interchangeably with the term "patient." For example, a human subject may be diagnosed with primary or metastatic melanoma and / or have one or more symptoms or indications, including but not limited to lymphadenopathy, abdominal swelling, chest pain / pressure, unexplained weight loss, fever, night sweats, persistent fatigue, decreased appetite, splenomegaly, and pruritus. In specific embodiments, this expression includes human subjects with unresectable locally advanced or metastatic melanoma who require treatment. In some embodiments, the human subject has not previously received systemic treatment for advanced disease. In specific embodiments, this expression includes human subjects with completely resected high-risk melanoma who require treatment in the context of adjuvant therapy. In some embodiments, the phrase "subjects in need" includes patients with melanoma that is resistant, refractory, or poorly responsive to prior treatment (e.g., conventional anticancer agents or treatments such as radiotherapy, chemotherapy, or surgery, or anticancer biologics). For example, this phrase includes subjects who have been treated with PD-1 or PD-L1 inhibitors (e.g., anti-PD-1 antibodies). This phrase also includes subjects with melanoma who are unsuitable for conventional anticancer therapy due to toxic side effects, for example, patients who have received one or more cycles of chemotherapy with toxic side effects. In some embodiments, the phrase "subjects in need" includes patients with melanoma that has been treated but subsequently relapsed or metastasized. For example, a patient with melanoma treated with the methods provided herein may have received treatment with one or more anticancer agents that lead to melanoma regression but subsequently experienced melanoma relapse resistant to one or more anticancer agents (e.g., chemotherapy-resistant melanoma).

[0109] In some cases, patients with unresectable locally advanced melanoma or metastatic melanoma are further selected if they meet one or more of the following criteria:

[0110] (i) At least 12 years old on the date the informed consent form is provided;

[0111] (ii) According to AJCC 8th edition (Amin, 2017), a patient is in stage IIC, III or IV and has a histologically confirmed melanoma that has been completely surgically removed to qualify.

[0112] (iii) Patients with stage IIIA disease must have at least one lymph node micrometastasis with a maximum measured diameter >1 mm;

[0113] (iv) Stage IIC melanoma confirmed by a pathologically negative SLNB specimen, with no evidence of regional or distant metastasis.

[0114] (v) Complete surgical resection was performed within 12 weeks prior to treatment, and the patient was only enrolled after satisfactory wound healing.

[0115] (vi) A comprehensive physical examination and imaging studies within 4 weeks prior to treatment confirm the absence of any disease state;

[0116] (vii) For patients with abnormal or suspicious findings during screening scans, it is necessary to... 18 F-FDG PET-CT and / or biopsy to exclude residual malignancy and / or metastatic disease;

[0117] (viii) Patients must not have received systemic anticancer therapy or radiation therapy for melanoma within the past 5 years, except for stage IV M1d patients who may have received central nervous system (CNS) radiation therapy (RT) after radical resection. Adjuvant radiation therapy to the primary site and / or lymph node region is permitted after radical surgical resection, provided the institution's standard treatment guidelines are met.

[0118] In some cases, patients with completely resected high-risk melanoma in the context of adjuvant therapy are further selected based on meeting one or more of the following criteria:

[0119] (1) ≥12 years old;

[0120] (2) Stage IIc, III or IV (all M stages) and histologically confirmed melanoma that has been completely removed <12 weeks prior to treatment;

[0121] (3) No systemic anticancer treatment or radiation therapy for melanoma in the past 5 years;

[0122] (4) No evidence of metastatic disease was found in the staging examination; and

[0123] (5) The Eastern Oncology Collaboration Group's performance status (PS) is 0 or 1 (applicable to adult patients), Karnofsky PS > 70 (patients > 16 years old) or Lansky PS > 70 (patients < 16 years old).

[0124] The phrase "subjects in need" also includes subjects at risk of developing melanoma, such as those with a family history of melanoma, previous melanoma patients, or those with compromised immune system function. In some cases, subjects may have shown resistance or inadequate response to previous treatments, or have experienced relapse after previous treatments.

[0125] In some embodiments, the methods provided herein can be used to treat patients exhibiting elevated levels of one or more cancer-related biomarkers (e.g., PD-L1 or LAG-3). For example, the method of the present invention includes administering a therapeutically effective amount of a combination of an anti-LAG-3 antibody and an anti-PD-1 antibody to a patient with elevated LAG-3 and / or PD-L1 levels. In one embodiment, the method of the present invention is used for patients with melanoma selected based on LAG-3 expression in cancerous tissue comprising melanoma cells and tumor-infiltrating immune cells. In some embodiments, the method of the present invention is used to treat patients with melanoma, wherein the patients are selected based on ≥1% LAG-3 expression in cancerous tissue and / or immune cells. In one embodiment, the method of the present invention is used for patients with melanoma selected based on LAG-3 expression in cancerous tissue comprising melanoma cells and tumor-infiltrating immune cells. In some embodiments, the method of the present invention is used to treat patients with melanoma, wherein the patients are selected based on ≥1% LAG-3 expression in cancerous tissue and / or immune cells. Methods for determining LAG-3 or PD-L1 expression in cancer tissues and / or tumor-associated immune cells are well known in the art. In some embodiments, LAG-3 expression in tumor tissues is determined by any assay known in the art, such as by ELISA or by immunohistochemistry (IHC) (e.g., as described in He et al. 2017, J. Thoracic Oncol. 12:814-823; WO2016124558 or WO2016191751). In some embodiments, LAG-3 or PD-L1 expression is determined by quantitative RNA expression, for example by in situ hybridization or by RT-PCR. In some embodiments, LAG-3 expression is determined by imaging with labeled anti-LAG-3 antibodies, for example by immunopositron emission tomography or iPET [see, for example, The Oncologist, 12:1379 (2007); Journal of Nuclear Medicine, 52(8):1171 (2011); U.S. Patent Application Publication 2018 / 0228926]. In some embodiments, PD-L1 expression is determined by imaging with labeled anti-PD-L1 antibodies, for example by immunopositron emission tomography or iPET (U.S. Patent Application Publication 2018 / 0161464).

[0126] In some implementations, the methods provided herein are used on subjects with cancer. The terms “tumor,” “cancer,” and “malignant tumor” are used interchangeably herein.

[0127] In some implementations, cancer or tumor is melanoma. References to "tumor" or "cancer" throughout the text include melanoma; for example, a tumor or cancer is melanoma. In some aspects, melanoma is unresectable locally advanced melanoma. In some aspects, melanoma is metastatic melanoma. In some aspects, the patient has not previously received systemic treatment for advanced disease. In other aspects, melanoma is completely resected high-risk melanoma. In some implementations, melanoma is completely resected high-risk melanoma in the context of adjuvant therapy.

[0128] According to certain embodiments, this disclosure includes methods for treating or delaying or inhibiting melanoma tumor growth. In some embodiments, this includes methods for promoting melanoma regression. In some embodiments, this includes methods for reducing tumor cell burden or reducing tumor load. In some embodiments, this disclosure includes methods for preventing melanoma recurrence. According to this aspect, the method includes sequentially administering a therapeutically effective amount of a combination of an anti-PD-1 antibody and an anti-LAG-3 antibody to a subject in need, wherein each antibody is administered to the subject in multiple doses, for example as part of a specific therapeutic dosing regimen. For example, the therapeutic dosing regimen may include administering one or more doses of the anti-PD-1 antibody to the subject at a frequency of about once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every six weeks, once every two months, once every three months, once every four months, or less. In some implementations, the one or more doses of the anti-PD-1 antibody are administered in combination with one or more doses of a therapeutically effective amount of the anti-LAG-3 antibody, wherein the one or more doses of the anti-LAG-3 antibody are administered to the subject at a frequency of approximately once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every six weeks, once every two months, once every three months, once every four months, or less.

[0129] In some embodiments, this disclosure includes methods for inhibiting, delaying, or preventing the metastasis or invasion of melanoma into surrounding organs. According to this aspect, the method includes administering a therapeutically effective amount of an anti-PD-1 antibody to a subject in need. In some embodiments, the anti-PD-1 antibody is administered in combination with an anti-LAG-3 antibody.

[0130] In specific embodiments, this disclosure provides methods for enhancing antitumor efficacy or enhancing melanoma suppression. In some embodiments, the method provides increased melanoma suppression, for example, by about 20%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80%, compared to subjects receiving either antibody as monotherapy.

[0131] According to certain embodiments, the methods provided herein include administering a therapeutically effective amount of an anti-PD-1 antibody to a subject with melanoma before, simultaneously with, or after administration of a therapeutically effective amount of an anti-LAG-3 antibody. In some aspects, the anti-PD-1 antibody may be administered approximately 1 day, more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, or more than 8 days prior to the anti-LAG-3 antibody. In some aspects, the anti-PD-1 antibody and the anti-LAG-3 antibody may be administered simultaneously, or at intervals not exceeding 30 minutes, 60 minutes, 2 hours, 3 hours, or 1 day.

[0132] In some embodiments, the methods described herein include administering a therapeutically effective amount of an anti-PD-1 antibody to a subject with melanoma. In specific embodiments, the melanoma is indolent or aggressive. In some embodiments, the subject is unresponsive to prior treatment or has experienced recurrence after prior treatment. Prior treatment may include surgery, radiation therapy and / or chemotherapy, or treatment with a PD-1 inhibitor, a PD-L1 inhibitor, and / or any other anticancer biological agent.

[0133] In some embodiments, the method of this disclosure includes administering a combination of an anti-PD-1 antibody and an anti-LAG-3 antibody to a subject in need as "first-line" treatment (e.g., initial treatment). In other embodiments, the combination of the anti-PD-1 antibody and the anti-LAG-3 antibody is administered as "second-line" treatment (e.g., after prior treatment). For example, the combination of the anti-PD-1 antibody and the anti-LAG-3 antibody is administered as "second-line" treatment to a subject who has relapsed after prior treatment (e.g., chemotherapy or rituximab).

[0134] In some embodiments, the methods of this disclosure are used to treat patients with minimal residual disease (MRD). Minimal residual disease (MRD) refers to a small number of cancer cells remaining in a patient's body during or after treatment, where the patient may or may not show symptoms or signs of the disease. Such residual cancer cells, if not eliminated, often lead to disease recurrence. This disclosure includes methods for inhibiting and / or eliminating residual cancer cells in a patient after an MRD test. MRD can be determined according to methods known in the art (e.g., MRD flow cytometry). According to this aspect of the disclosure, the method includes administering a combination of an anti-PD-1 antibody and an anti-LAG-3 antibody to a subject in need.

[0135] According to certain embodiments, the method provided herein includes administering to a subject a therapeutically effective amount of each of an anti-PD-1 antibody and an anti-LAG-3 antibody in combination with a third therapeutic agent. The third therapeutic agent may be an agent selected from the group consisting of: for example, radiotherapy, chemotherapy, surgery, cancer vaccines, CAR-T, PD-L1 inhibitors (e.g., anti-PD-L1 antibodies), CD3 inhibitors, CD20 inhibitors, CTLA-4 inhibitors, CD38 inhibitors, TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, Ang2 inhibitors, transforming growth factor β (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, and anti-tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA)]. The following are listed as examples of drugs: BCG, granulocyte-macrophage colony-stimulating factor, oncolytic viruses, cytotoxins, CD28 agonists, GITR agonists, 4-1BB agonists, CD20xCD3 bispecific antibodies (e.g., REGN1979), MUC16xCD3 bispecific antibodies, vimentin, tumor M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9, vaccines (e.g., BCG), granulocyte-macrophage colony-stimulating factor, cytotoxins, chemotherapeutic agents, IL-6R inhibitors, IL-4R inhibitors, IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-12, IL-21, and IL-15, anti-inflammatory drugs such as corticosteroids, and nonsteroidal anti-inflammatory drugs.

[0136] In some embodiments, the antibody may be administered in combination with therapies including chemotherapy, radiation therapy, or surgery. The phrase "in combination with" as used herein means that the antibody is administered to the subject concurrently with, slightly earlier than, or slightly later than the administration of a third therapeutic agent. In some embodiments, the third therapeutic agent is administered as a co-preparation with the antibody. In relevant embodiments, this disclosure includes a method of administering a therapeutically effective amount of a combination of an anti-PD-1 antibody and an anti-LAG-3 antibody to a subject receiving a background anticancer treatment regimen. The background anticancer treatment regimen may include a course of treatment, such as chemotherapy or radiation therapy. The combination of the anti-PD-1 antibody and the anti-LAG-3 antibody may be added to the background anticancer treatment regimen. In some embodiments, the antibody is added as part of a "background tapering" regimen, wherein the background anticancer treatment is gradually withdrawn from the subject over time (e.g., using a step-down approach) while the antibody is administered to the subject at a constant, escalating, or decreasing dose over time.

[0137] In some embodiments, the method of this disclosure includes administering to a subject in need a therapeutically effective amount of a combination of an anti-PD-1 antibody and a therapeutically effective amount of an anti-LAG-3 antibody, wherein administration of the antibody results in increased inhibition of melanoma growth. In some embodiments, melanoma growth is inhibited by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% compared to untreated subjects or subjects receiving either antibody as monotherapy. In some embodiments, administration of the anti-PD-1 antibody and / or the anti-LAG-3 antibody to a subject results in increased melanoma regression, tumor shrinkage, and / or disappearance. In some implementations, administration of anti-PD-1 antibodies and / or anti-LAG-3 antibodies results in a delay in melanoma growth and progression, for example, melanoma growth may be delayed by approximately 3 days, more than 3 days, approximately 7 days, more than 7 days, at least 10 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years compared to untreated subjects or subjects treated with either antibody as monotherapy. In some implementations, administration of a combination of anti-PD-1 and anti-LAG-3 antibodies prevents melanoma recurrence and / or prolongs the survival duration of subjects, for example, by extending survival duration by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months. In some implementations, combined administration of antibodies prolongs progression-free survival or overall survival. In some embodiments, the combined administration of an anti-PD-1 antibody and an anti-LAG-3 antibody improves the response rate and prolongs the duration of response, for example, by more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50%, compared to treatment-naïve subjects or subjects who have received either antibody as monotherapy. In some embodiments, administration of the anti-PD-1 antibody and / or the anti-LAG-3 antibody to a subject with melanoma results in the complete disappearance of all evidence of melanoma cells (“complete response”). In some embodiments, administration of the anti-PD-1 antibody and / or the anti-LAG-3 antibody to a subject with melanoma results in a reduction of melanoma cells or tumor size of at least 30% or more (“partial response”). In some embodiments, administration of the anti-PD-1 antibody and / or the anti-LAG-3 antibody to a subject with melanoma results in the complete or partial disappearance of melanoma cells / lesions (including new measurable lesions). The reduction in melanoma tumor size can be measured by any of the methods known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytological, histological, or molecular genetic analysis.In some respects, administering the combination of anti-PD-1 and anti-LAG-3 antibodies to the patient population unexpectedly resulted in more patients responding to treatment, unexpectedly resulted in longer duration of treatment response even if the number of responding patients did not increase, and / or resulted in deeper responses in patients who responded to treatment.

[0138] In some implementations, the combination of antibodies administered is safe and well-tolerated by patients, with no increase or only a tolerable increase in adverse side effects compared to patients receiving either antibody as monotherapy.

[0139] Combination therapy

[0140] According to certain embodiments, the method of this disclosure includes administering a combination of an anti-LAG-3 antibody and an anti-PD-1 antibody to a subject. In some embodiments, the method of this disclosure includes administering antibodies to achieve additional or synergistic activity for the treatment of melanoma. The expression “combined with” as used herein means that the anti-LAG-3 antibody is administered before, after, or concurrently with the anti-PD-1 antibody. The term “combined with” also includes sequential or concurrent administration of the anti-PD-1 antibody and the anti-LAG-3 antibody. For example, when administered “before” the anti-LAG-3 antibody, the anti-PD-1 antibody may be administered more than 150 hours, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes prior to administration of the anti-LAG-3 antibody. When administered “following” the anti-LAG-3 antibody, the anti-PD-1 antibody may be administered approximately 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or more than 72 hours after administration of the anti-LAG-3 antibody. Administering “simultaneously” with the anti-LAG-3 antibody means administering the anti-PD-1 antibody to the subject in a separate dosage form or as a single combined dose formulation containing both the anti-PD-1 and anti-LAG-3 antibodies within 5 minutes (before, after, or simultaneously with) the administration of the anti-LAG-3 antibody (e.g., within 5 minutes after the completion of the anti-LAG-3 antibody infusion). In some respects, the anti-PD-1 antibody and the anti-LAG-3 antibody are administered on the same day. In some respects, anti-PD-1 antibodies and anti-LAG-3 antibodies are administered in separate formulations but at intervals not exceeding 8 hours (e.g., at intervals not exceeding 6 hours, or 5 hours, or 4 hours, or 3 hours, or 2 hours, or 60 minutes).

[0141] In some embodiments, the methods provided herein include administration of a third therapeutic agent, wherein the third therapeutic agent is an anticancer drug. As used herein, "anticancer drug" means any agent that can be used to treat cancer, including but not limited to cytotoxic agents and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radiopharmaceuticals. "Cytotoxic or cytotoxic agent" as used herein also refers to chemotherapeutic agents and means any agent that is harmful to cells. Examples include... (Paclitaxel), temozolomide, cytochalasin B, bacitracin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, procainox, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues.In some embodiments, the methods provided herein include administration of a third therapeutic agent selected from the group consisting of: radiation therapy, surgery, cancer vaccines, PD-L1 inhibitors (e.g., anti-PD-L1 antibodies), CD20 inhibitors, CD3 inhibitors, CTLA-4 inhibitors (e.g., ipilimumab), CD38 inhibitors, TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, antagonists of another T-cell co-inhibitory molecule or ligand (e.g., antibodies against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3-bis(t-gamma) Oxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., "VEGF-Trap", such as aflibercept; or other VEGF-inhibiting fusion proteins as described in U.S. Patent No. 7,087,411; or anti-VEGF antibodies or their antigen-binding fragments (e.g., bevacizumab or ranibizumab); or small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nevasumab), transforming growth factor β (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), targeting co-stimulation Agonists of stimulatory receptors (e.g., agonists targeting glucocorticoid-induced TNFR-related proteins), antibodies against tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA)], CD28 agonists, GITR agonists, 4-1BB agonists, CD20xCD3 bispecific antibodies (e.g., REGN1979), MUC16xCD3 bispecific antibodies, vimentin, tumor M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), vaccines (e.g., BCG, cancer vaccines), adjuvants that enhance antigen presentation Chemotherapy agents (e.g., granulocyte-macrophage colony-stimulating factor), oncolytic viruses, cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), radiation therapy, IL-6R inhibitors (e.g., sarrelumarab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-12, IL-21, and IL-15, antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADCs and anti-DS6-DM4 ADCs), chimeric antigen receptor T cells (e.g., CD19-targeting T cells) or other cell therapies, and anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs).

[0142] In some implementations, the methods provided herein include administering anti-PD-1 antibodies and anti-LAG-3 antibodies in combination with radiotherapy / chemotherapy to produce a long-lasting anti-tumor response and / or prolong the survival of patients with melanoma.

[0143] In some embodiments, the methods of this disclosure include administering radiotherapy before, concurrently with, or after administration of anti-PD-1 and anti-LAG-3 antibodies to a melanoma patient. For example, radiotherapy may be administered to the melanoma lesion in one or more doses after administration of one or more doses of the antibody. In some embodiments, radiotherapy may be administered locally to the melanoma lesion before or after systemic administration of anti-PD-1 and / or anti-LAG-3 antibodies, i.e., in the context of adjuvant therapy, to enhance the local immunogenicity of the patient's melanoma (adjuvant radiotherapy) and / or kill melanoma cells (ablative radiotherapy). In some embodiments, the antibody may be administered in combination with radiotherapy and a chemotherapeutic agent (e.g., temozolomide or cyclophosphamide) or a VEGF antagonist (e.g., aflibercept).

[0144] Pharmaceutical composition and administration

[0145] This document provides a method for administering a combination of an anti-PD-1 antibody and an anti-LAG-3 antibody to a subject, wherein the antibody is contained within a single or combined (single) pharmaceutical composition. The pharmaceutical compositions of this disclosure can be formulated using suitable carriers, excipients, and other agents that provide suitable delivery, tolerability, etc. Numerous suitable formulations are available in all formulation collections known to pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN). TM DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0146] Various delivery systems are known and can be used to administer the pharmaceutical compositions disclosed herein, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as by infusion or bolus, by absorption through epithelial or mucosal skin linings (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other bioactive agents.

[0147] The pharmaceutical compositions disclosed herein can be delivered subcutaneously or intravenously using standard needles and syringes. In one embodiment, the syringe is a pre-filled syringe. Alternatively, for subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the pharmaceutical compositions disclosed herein. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically utilize replaceable cartridges containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there are no replaceable cartridges. In fact, disposable pen-type delivery devices have a reservoir pre-filled with the pharmaceutical composition within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0148] In some cases, drug compositions can be delivered in controlled-release systems. In one embodiment, a pump can be used. In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise eds., 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115-138). Other controlled-release systems are discussed in a review in Langer, 1990, Science 249:1527-1533.

[0149] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, intravenous infusion, etc. These injectable formulations can be prepared by known methods. For example, they can be prepared, for instance, by dissolving, suspending, or emulsifying the antibodies or their salts described above in a conventional sterile aqueous or oily medium for injection. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other pharmaceutical excipients, which can be used in combination with suitable solubilizers (such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.). Oily media include, for example, sesame oil, soybean oil, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The resulting injectable formulation is preferably filled into suitable ampoules.

[0150] Advantageously, the pharmaceutical compositions described above for oral or parenteral use are formulated into unit dose dosage forms suitable for the appropriate dosage of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0151] Application plan

[0152] This disclosure includes methods of administering anti-PD-1 antibodies to subjects at a frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less (providing a therapeutic response is achieved). In some embodiments, this disclosure includes methods of administering anti-LAG-3 antibodies to subjects at a frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less (providing a therapeutic response is achieved). In some embodiments, the method involves administering a combination of anti-PD-1 and anti-LAG-3 antibodies at a frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every nine weeks, once every twelve weeks, or less (providing a therapeutic response is achieved).

[0153] According to certain embodiments of this disclosure, multiple doses of a combination of anti-PD-1 antibody and anti-LAG-3 antibody may be administered to a subject over a defined time period. A method according to this aspect of the disclosure includes sequentially administering one or more doses of a combination of anti-PD-1 antibody and one or more doses of anti-LAG-3 antibody to a subject. As used herein, "sequential administration" means administering each dose of antibody to a subject at different time points, such as on different dates separated by predetermined intervals (e.g., hours, days, weeks, or months). This disclosure includes a method comprising sequentially administering a single initial dose of anti-PD-1 antibody to a patient, followed by one or more second doses of anti-PD-1 antibody, and optionally subsequently administering one or more third doses of anti-PD-1 antibody. In some embodiments, the method further includes sequentially administering a single initial dose of anti-LAG-3 antibody to a patient, followed by one or more second doses of anti-LAG-3 antibody, and optionally subsequently administering one or more third doses of anti-LAG-3 antibody.

[0154] According to certain embodiments of this disclosure, multiple doses of anti-PD-1 antibody and anti-LAG-3 antibody may be administered to a subject over a defined time period. A method according to this aspect of the disclosure includes sequentially administering multiple doses of anti-PD-1 antibody and anti-LAG-3 antibody to a subject. As used herein, “sequential administration” means administering a combination of each dose of anti-PD-1 antibody and anti-LAG-3 antibody to a subject at different time points, such as on different dates separated by predetermined intervals (e.g., hours, days, weeks, or months).

[0155] According to certain embodiments of this disclosure, subjects may be administered multiple doses of anti-LAG-3 antibody for months or years, every 3 or 6 weeks, followed by administration of a combination of anti-PD-1 antibody and anti-LAG-3 antibody for months or years. In some aspects, the anti-LAG-3 antibody dose differs with respect to monotherapy versus combination therapy. In other aspects, the anti-LAG-3 antibody dose is the same regardless of whether it is administered as monotherapy or in combination with an anti-PD-1 antibody.

[0156] The terms “initial dose,” “second dose,” and “third dose” refer to the time sequence of administration. Therefore, the “initial dose” is the dose administered at the start of the treatment regimen (also known as the “baseline dose”); the “second dose” is the dose administered after the initial dose; and the “third dose” is the dose administered after the second dose. The initial, second, and third doses may all contain the same amount of antibody (anti-PD-1 antibody or anti-LAG-3 antibody). However, in some embodiments, the amounts contained in the initial, second, and / or third doses may differ from each other during treatment (e.g., up- or down-adjusted as appropriate). In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) doses are administered at the start of the treatment regimen as a “loading dose,” followed by subsequent doses administered at a lower frequency (e.g., a “maintenance dose”). For example, an anti-PD-1 antibody may be administered to a patient with melanoma at a loading dose of approximately 1 mg / kg patient body weight to 20 mg / kg patient body weight, followed by one or more maintenance doses of approximately 3 mg / kg patient body weight.

[0157] In one exemplary embodiment of this disclosure, each second and / or third dose is administered 1 / 2 to 14 weeks immediately following the previous dose (e.g., 1 / 2, 1, 1). 1 / 2、2、2 1 / 2, 3, 3 1 / 2、4、4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2、7、7 1 / 2、8、8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2 or more weeks) of administration. The phrase “immediately following the previous dose” as used in this article means, in a sequence of multiple administrations, the dose of anti-PD-1 antibody (and / or anti-LAG-3 antibody) administered to the patient when there is no intervention dose prior to the next dose in the sequence.

[0158] Methods according to some aspects may include administering any number of two- and / or three-dose doses of anti-PD-1 antibody (and / or anti-LAG-3 antibody) to a patient. For example, in some embodiments, only a single two-dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) two-dose doses are administered to the patient. Similarly, in some embodiments, only a single three-dose dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) three-dose doses are administered to the patient.

[0159] In implementations involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient one to two weeks immediately following the previous dose. Similarly, in implementations involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient two to four weeks immediately following the previous dose. Alternatively, the frequency of secondary and / or tertiary doses administered to the patient may vary during the treatment regimen. The physician may also adjust the frequency of administration during treatment as needed by each patient following clinical examination.

[0160] In some implementations, one or more doses of anti-PD-1 antibody and / or anti-LAG-3 antibody are administered at a higher frequency (twice a week, once a week, or once every 2 weeks) as an “induction dose” at the start of the treatment regimen, followed by subsequent doses (“consolidation dose” or “maintenance dose”) at a lower frequency (e.g., once every 4 to 12 weeks).

[0161] In some embodiments, this document covers the concurrent administration of anti-PD-1 antibody and anti-LAG-3 antibody, with the anti-LAG-3 antibody administered at a different dose at a similar or different frequency relative to the anti-PD-1 antibody. In some embodiments, the anti-LAG-3 antibody is administered before, after, or concurrently with the anti-PD-1 antibody. In some embodiments, the anti-LAG-3 antibody and anti-PD-1 antibody are administered as a single-dose formulation.

[0162] This disclosure includes methods for treating melanoma by sequentially administering a combination of an anti-PD-1 antibody and an anti-LAG-3 antibody to a patient. In some embodiments, the method of the present invention includes administering one or more doses of an anti-PD-1 antibody, followed by administering one or more doses of an anti-LAG-3 antibody. In some embodiments, the method of the present invention includes administering a single dose of an anti-PD-1 antibody, followed by administering one or more doses of an anti-LAG-3 antibody. In some embodiments, one or more doses of an anti-PD-1 antibody of about 0.1 mg / kg to about 20 mg / kg may be administered, followed by administering one or more doses of an anti-LAG-3 antibody of about 0.1 mg / kg to about 50 mg / kg to inhibit melanoma growth and / or prevent melanoma recurrence in subjects with melanoma. In some embodiments, administering one or more doses of an anti-PD-1 antibody, followed by administering one or more doses of an anti-LAG-3 antibody, results in enhanced antitumor efficacy (e.g., stronger inhibition of melanoma growth and enhanced prevention of melanoma recurrence compared to untreated subjects or subjects receiving either antibody as monotherapy).

[0163] This disclosure also includes methods for treating melanoma by sequentially administering a combination of anti-LAG-3 antibody and anti-PD-1 antibody to a patient. In some embodiments, the method of the present invention includes administering one or more doses of anti-LAG-3 antibody, followed by administering one or more doses of anti-PD-1 antibody. In some embodiments, the method of the present invention includes administering a single dose of anti-LAG-3 antibody, followed by administering one or more doses of anti-PD-1 antibody. In some embodiments, one or more doses of about 0.1 mg / kg to about 50 mg / kg of anti-LAG-3 antibody may be administered, followed by administering one or more doses of about 0.1 mg / kg to about 20 mg / kg of anti-PD-1 antibody to inhibit tumor growth and / or prevent melanoma recurrence in a subject with melanoma. In some embodiments, one or more doses of about 50 mg to about 8000 mg of anti-LAG-3 antibody may be administered, followed by administering one or more doses of about 50 mg to about 1500 mg of anti-PD-1 antibody to inhibit melanoma growth and / or prevent melanoma recurrence in a subject with melanoma. In some implementations, the anti-LAG-3 antibody is administered at one or more doses, followed by one or more doses of the anti-PD-1 antibody, resulting in enhanced antitumor efficacy (e.g., stronger inhibition of melanoma growth and enhanced prevention of melanoma recurrence compared to untreated subjects or subjects receiving either antibody as monotherapy).

[0164] dose

[0165] The amount of anti-PD-1 antibody and / or anti-LAG-3 antibody administered to a subject according to the methods of this disclosure is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to the amount of antibody (anti-PD-1 antibody or anti-LAG-3 antibody) that results in or has one or more of the following therapeutic effects: (a) reducing the severity of melanoma symptoms or shortening the duration of melanoma symptoms; (b) inhibiting melanoma growth or increasing melanoma cell necrosis, melanoma tumor shrinkage, and / or melanoma tumor disappearance; (c) delaying melanoma growth and progression; (d) inhibiting, delaying, or preventing melanoma metastasis; (e) preventing recurrence of melanoma growth; (f) prolonging the survival of a subject with melanoma; and / or (g) reducing the use or need for conventional anticancer therapies (e.g., reducing or eliminating the use of chemotherapeutic agents or cytotoxic agents) compared to untreated subjects or subjects receiving either antibody as monotherapy.

[0166] In the case of anti-PD-1 antibodies, the effective therapeutic dose can be from about 0.05 mg to about 1500 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 3... 20mg, approximately 330mg, approximately 340mg, approximately 350mg, approximately 360mg, approximately 370mg, approximately 380mg, approximately 390mg, approximately 400mg, approximately 410mg, approximately 420mg, approximately 430mg, approximately 440mg, approximately 450mg, approximately 460mg, approximately 470mg, approximately 480mg, approximately 490mg, approximately 500mg, approximately 510mg, approximately 520mg Anti-PD-1 antibody in doses of approximately 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1050 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, or 1500 mg. In some embodiments, 350 mg of anti-PD-1 antibody is administered. In some embodiments, 1050 mg of anti-PD-1 antibody is administered.

[0167] In the case of anti-LAG-3 antibodies, the effective therapeutic dose can be from approximately 10 mg to approximately 8000 mg, for example, approximately 10 mg, approximately 20 mg, approximately 50 mg, approximately 70 mg, approximately 100 mg, approximately 120 mg, approximately 150 mg, approximately 200 mg, approximately 250 mg, approximately 300 mg, approximately 350 mg, approximately 400 mg, approximately 450 mg, approximately 500 mg, approximately 550 mg, approximately 600 mg, approximately 700 mg, approximately 800 mg, approximately 900 mg, approximately 1000 mg, etc. Anti-LAG-3 antibody in doses of approximately 1050 mg, 1100 mg, 1500 mg, 1600 mg, 1700 mg, 2000 mg, 2050 mg, 2100 mg, 2200 mg, 2500 mg, 2700 mg, 2800 mg, 2900 mg, 3000 mg, 3200 mg, 4000 mg, 5000 mg, 6000 mg, 7000 mg, or 8000 mg.

[0168] The amount of anti-PD-1 antibody or anti-LAG-3 antibody contained in each dose can be expressed in milligrams of antibody per kilogram of subject body weight (i.e., mg / kg). In some embodiments, the anti-PD-1 antibody or anti-LAG-3 antibody used in the methods of this disclosure may be administered to the subject at a dose of about 1 mg / kg of subject body weight to about 50 mg / kg of subject body weight. For example, the anti-PD-1 antibody may be administered at a dose of about 0.1 mg / kg of patient body weight to about 20 mg / kg of patient body weight. The anti-LAG-3 antibody may be administered at a dose of about 0.1 mg / kg of patient body weight to about 50 mg / kg of patient body weight.

[0169] Example

[0170] The following examples are provided to provide a complete disclosure and description of how to prepare and use the methods and compositions of this disclosure to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their disclosure. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise specified, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are atmospheric pressure or near atmospheric pressure. The compositions and methods set forth in the examples form part of this disclosure.

[0171] Therapeutic monoclonal antibodies (mAbs) targeting immunosuppressive receptors (e.g., cytotoxic T-lymphocyte-associated protein 4 [CTLA-4] and programmed cell death 1 [PD-1]) have shown remarkable clinical activity in several tumor types with acceptable benefit-risk ratios (Topalian, 2014)(Wolchok, 2013)(Larkin, 2015a)(Baksh, 2015). However, sustained responses have only been achieved in a small number of patients, suggesting that combination approaches may be needed to overcome tumor immunosuppressive mechanisms (Topalian, 2012).

[0172] In the examples below, the combination of the antibody (REGN3767 (INN: fumarimab) and cimiprimab (REGN2810) was tested. REGN3767 is an antibody against the LAG-3 receptor that blocks LAG-3 / MHC II-mediated T-cell suppression, and REGN2810 is an antibody against the PD-1 receptor that blocks PD1 / programmed death-ligand 1 (PD-L1)-mediated T-cell suppression. (INN: Cimiprilmab, known in the United States as cimiprilmab rwlc) has been approved by several health authorities for the treatment of patients with different types of tumors.

[0173] Example 1: Phase 3 clinical trial of anti-LAG3 (REGN3767, ciampizumab) and anti-PD-1 (REGN2810, cimiprimab) in patients with completely resected high-risk melanoma in the context of adjuvant therapy, compared with pembrolizumab.

[0174] This current phase 3 study evaluated the combination of adjuvant fuamilumab and cimiprimab versus pembrolizumab in patients with fully resected high-risk melanoma.

[0175] Purpose

[0176] The primary objective of this study was to demonstrate the superiority of fumarlimab plus cimiprimab over pembrolizumab, as measured by relapse-free survival (RFS).

[0177] Secondary objectives include the following:

[0178] • Demonstrate the superiority of fumarazumab plus cimiprimab over pembrolizumab, as measured by overall survival (OS).

[0179] • Demonstrating the superiority of fumarazumab plus cimiprimab over pembrolizumab, as measured by melanoma-specific survival (MSS).

[0180] • Evaluate whether postoperative adjuvant therapy in stage IIC or III patients receiving fumbolizumab plus cimiprimab improves distant metastasis-free survival (DMFS) compared to pembrolizumab.

[0181] • Evaluate the impact of fumarazumab plus cimiprimab on adult quality of life compared to pembrolizumab.

[0182] • Evaluate the safety and tolerability of fumarazumab plus cimiprimab compared to pembrolizumab.

[0183] • Using sparse PK sampling, the pharmacokinetics (PK) of fumarazumab + cimiprimab were characterized in patients aged 12 years and older.

[0184] • Evaluate the immunogenicity of fumarazumab and cimiprimab.

[0185] Research Design

[0186] This randomized, double-blind, phase 3 study was conducted in patients aged 12 years or older with completely resected high-risk melanoma, and the study was divided into three groups in the context of adjuvant therapy:

[0187] Group A: Furamimab (1600 mg) + Cimiprimab (350 mg), administered intravenously every 3 weeks (Q3WIV).

[0188] Group B: Furamimab (400 mg) + Cimiprimab (350 mg), administered intravenously every 3 weeks.

[0189] Group C: Pembrolizumab (200 mg) + saline / glucose placebo, administered intravenously every 3 weeks.

[0190] Patients were randomly assigned to groups A, B, and C in a 1:1:1 ratio. Patients had not previously received systemic therapy for melanoma and had undergone complete resection of stage IIC, III, or IV melanoma (American Joint Committee on Cancer Staging Manual [AJCC] 8th edition).

[0191] The study population consisted of patients with high-risk stage IIC, III, or IV melanoma who underwent complete surgical resection within 12 weeks (American Joint Committee on Cancer Staging Manual for Melanoma, 8th Edition, 2017) (Amin, 2017).

[0192] Selection criteria

[0193] Patients must meet the following criteria to be eligible for inclusion in this study:

[0194] 1. Patients must be at least 12 years old on the date of informed consent. Note: Patients under the age of 18 years may only be enrolled in this study if approved by local laws, regulations, and ethics committees.

[0195] 2. According to AJCC 8th edition (Amin, 2017), all patients must be in stage IIC, III or IV and have histologically confirmed melanoma that has been completely surgically removed to be eligible.

[0196] • Patients with stage IIIA disease must have at least one lymph node micrometastasis with a maximum measured diameter >1 mm.

[0197] • The maximum number of patients in stage IIC, stage IIIA, and stage IV (M1c / d) is 10% of the total population (i.e., a total of 30%).

[0198] • Patients with acral melanoma and mucosal melanoma are permitted to have the tumor removed, provided they have undergone complete resection; the total limit for both types is 10%.

[0199] Patients with unexplained primary melanoma may be allowed to have it removed, provided they have undergone complete resection.

[0200] • Stage IIC melanoma must be confirmed by a pathologically negative sentinel lymph node biopsy (SLNB) specimen and there must be no evidence of regional or distant metastasis.

[0201] 3. Complete surgical resection must be performed within 12 weeks prior to treatment, and enrollment is only permitted after satisfactory wound healing.

[0202] Complete surgical resection is defined as resection of the primary tumor with negative margins on microscopy and negative SLNB (sinking lymph node dissection). In cases of positive SLNB, if the patient is clinically negative for lymph nodes and no tumor cells are seen on microscopy at the resection margins, routine complete lymph node dissection is not required. Before enrollment in the study, the investigator must review and sign the surgical / pathological report confirming complete resection for all patients.

[0203] • For isolated cutaneous melanoma lesions without epithelial components, they should be treated as primary tumors and SLNB must be performed.

[0204] • For all patients with resected stage IV M1d, the neurosurgical and pathological reports confirming complete resection and negative margins must be reviewed and signed by the investigator before enrollment.

[0205] 4. All patients must be free of disease through a comprehensive physical examination and imaging studies within 4 weeks prior to treatment. Imaging must include scans of the chest, abdomen, and pelvis (preferably enhanced X-ray CT), as well as scans of all relevant anatomical regions and lesion resection sites (applicable to stage III and IV patients). All patients must undergo enhanced brain MRI at the time of staging.

[0206] • Patients with abnormal or suspicious findings during screening scans must undergo [further treatment / treatment]. 18 F-FDG PET-CT and / or biopsy to rule out residual malignancy and / or metastatic disease.

[0207] • For patients with resected stage IV M1d disease, enhanced brain MRI must be performed at least 4 weeks after surgery / radiotherapy and before study enrollment to show no evidence of residual lesions or disease recurrence.

[0208] 5. Patients must not have received systemic anticancer therapy or radiation therapy for melanoma within the past 5 years, except for stage IV M1d patients who may have received central nervous system radiation therapy after radical resection. Adjuvant radiation therapy to the primary site and / or lymph node region is permitted after radical surgical resection, provided the institution's standard treatment guidelines are met.

[0209] 6. Physical condition:

[0210] • For adult patients: ECOGPS is 0 or 1.

[0211] • For patients <18 years of age: Karnofsky PS > 70 (patients > 16 years of age) or Lansky PS > 70 (patients < 16 years of age).

[0212] 7. Bone marrow function is adequate as determined by hematological parameters:

[0213] • Absolute neutrophil count (ANC) ≥ 1.5 × 10^9 / L (1500 / mm) 3 )

[0214] • Hemoglobin ≥ 9.0 g / dL (5.59 mmol / L)

[0215] • Platelet count ≥75,000 / mm 3

[0216] 8. Liver function is adequate as determined by the following indicators:

[0217] • Adult AST / ALT: Aspartate aminotransferase (AST) ≤ 3 times the upper limit of normal (ULN), alanine aminotransferase (ALT) ≤ 3 times the ULN

[0218] • Adolescent AST / ALT: Both AST and ALT < 2.5 times ULN

[0219] • Serum bilirubin ≤1.5 times ULN, but ≤3 times ULN is allowed for patients with clinically diagnosed Gilbert's syndrome.

[0220] 9. Sufficient kidney function:

[0221] • For adult patients: Determined by an estimated glomerular filtration rate (eGFR) ≥30 ml / min (using the creatine phosphokinase (CPK)-EPI equation).

[0222] For pediatric patients:

[0223] (i) Creatinine clearance or radioactive isotope glomerular filtration rate (GFR) > 70 mL / min / 1.73 m 2

[0224] ·or

[0225] (ii) Serum creatinine based on age / sex, as shown in Table 1:

[0226] Table 1: Serum creatinine based on age / sex

[0227]

[0228] 10. All patients must provide at least 25 slides or 125mm sections from the tissue block. 3 Fine-cut tumor tissue samples (FFPE) are used for biomarker analysis. Prepared slides must be sent to the testing laboratory within two weeks of slide preparation. Bone biopsy samples are not permitted.

[0229] 11. Women of childbearing potential (WOCBP*) must be negative for serum (β-human chorionic gonadotropin [β-hCG]) at screening and must agree not to become pregnant during the study treatment and for a maximum of 6 months after receiving the last study treatment dose.

[0230] *WOCBP is defined as a woman who is of reproductive capacity from menarche to menopause, excluding those who are permanently infertile. Permanent sterilization methods include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy.

[0231] Menopause is defined as the absence of menstruation for 12 consecutive months without other medical cause. For women not using hormonal contraception or hormone replacement therapy, high follicle-stimulating hormone (FSH) levels within the postmenopausal range can be used to confirm menopause. However, in the absence of a 12-month history of amenorrhea, a single FSH measurement is insufficient to determine menopause. The above definition is based on the Clinical Trials Facilitation Working Group (CTFG) guidelines. Women who have undergone a confirmed hysterectomy do not require pregnancy testing or contraception.

[0232] Male study participants with WOCBP partners were required to use condoms for at least 6 months after receiving their last dose, except for those who had undergone vasectomy or adhered to abstinence.

[0233] Partners who have undergone vasectomy or research participants who have undergone vasectomy must undergo a medical evaluation to assess the success of the procedure.

[0234] Periodic abstinence (natural rhythm method, sympathobasic temperature method, post-ovulation method), withdrawal (coitus interruptus), spermicide use alone, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female condoms and male condoms should not be used simultaneously.

[0235] 12. WOCBP must agree not to donate eggs (ovules, oocytes) for assisted reproductive purposes throughout the entire trial and for 6 months after the last treatment.

[0236] 13. All men must agree not to donate sperm during the trial and for 6 months after receiving the last treatment dose.

[0237] 14. Provide an informed consent form signed by the subject (for patients aged 12-17, provide an informed consent statement supplemented by the consent form from their parents or legal guardians).

[0238] 15. Willing and able to cooperate in completing outpatient visits and research-related procedures.

[0239] 16. Able to understand and complete research-related questionnaires.

[0240] Exclusion criteria

[0241] Patients meeting any of the following criteria were excluded from the study:

[0242] 1. Uveal melanoma.

[0243] 2. Evidence of residual lesions after surgery found through imaging, pathology, or cytology examination (including clinical or radiological suspicion of leptomeningeal metastasis).

[0244] 3. Evidence of a clinically significant autoimmune disease that is persistent or has recently (within 2 years) required for systemic treatment with immunosuppressants. The following are not excluded: vitiligo, cured childhood asthma, residual hypothyroidism requiring only hormone replacement therapy, and psoriasis not requiring systemic treatment.

[0245] 4. Uncontrolled HIV, HBV, or HCV infection; or a diagnosis of an immunodeficiency disorder associated with or leading to chronic infection.

[0246] Note:

[0247] • Patients with known HIV infection but controlled disease (spontaneous status or achieved under a stable antiretroviral regimen, undetectable viral load, and CD4 count greater than 350) were eligible for enrollment. Patients with controlled HIV infection were monitored according to local standards.

[0248] Patients with known hepatitis B infection (HepBsAg+) but with controlled disease (serum hepatitis B virus DNA PCR below the detection limit and currently receiving antiviral therapy for hepatitis B) are eligible for enrollment. Patients with controlled infection must undergo regular HBV DNA monitoring according to local standards and must continue antiviral therapy for at least 6 months after the last study drug dose.

[0249] Patients with known HCV Ab+ infection but whose condition is under control (either in a spontaneous state or after previous successful anti-HCV treatment, with no detectable HCV RNA by PCR) are allowed to enroll.

[0250] • Patients with HIV or hepatitis must be reviewed by a qualified specialist (such as an infectious disease specialist or hepatologist) who manages the disease before the trial begins and regularly during the trial.

[0251] 5. Another malignant tumor that is currently progressing or requires active treatment within the past 5 years, excluding malignant tumors with negligible risk of metastasis or death (such as adequately treated cervical carcinoma in situ, basal cell or squamous cell skin cancer, localized early prostate cancer, or ductal carcinoma in situ of the breast). Note: Any uncertain cases should be discussed with the medical monitor before enrollment.

[0252] 6. Pregnant or breastfeeding women.

[0253] 7. WOCBP patients who do not wish to use highly effective contraception before the initial dose / first treatment, during the study, and for at least 6 months after the last dose. Highly effective contraception includes:

[0254] • For two or more menstrual cycles prior to screening, candidates have been continuously using combined hormonal (including estrogen and progestin) contraceptive methods (oral, vaginal, transdermal) or progestin-only hormonal contraceptive methods (oral, injection, implantation) that inhibit ovulation.

[0255] • Intrauterine device (IUD); Intrauterine hormone-releasing system (IUS);

[0256] • Bilateral tubal ligation (blockage method);

[0257] • Partners who have undergone vasectomy (provided the male partner who has undergone vasectomy is the only sexual partner of the WOCBP study participant, and the partner who has undergone vasectomy has received a medical assessment of surgical success); and / or

[0258] Abstinence , .

[0259] (i) Abstinence is considered an effective method only when defined as avoiding heterosexual intercourse throughout the entire investigational drug risk period. The reliability of abstinence needs to be assessed in conjunction with the duration of the clinical trial and the participants' preferences and routine lifestyle habits.

[0260] (ii) Periodic abstinence (natural rhythm method, sympathobasic temperature method, post-ovulation method), withdrawal (coitus interruptus), spermicide use alone, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female condoms and male condoms should not be used simultaneously.

[0261] Previous / comorbid treatments:

[0262] 8. Systemic immunosuppression:

[0263] • Use immunosuppressive doses of corticosteroids (>10 mg prednisone or equivalent daily) within 14 days of the first study dose. Physiological alternative doses are permitted, up to and including 10 mg prednisone or equivalent daily. Inhaled or topical steroids are permitted.

[0264] • Other clinically relevant forms of systemic immunosuppression.

[0265] 9. Received any anticancer treatment for malignant tumors other than melanoma within 5 years prior to treatment, including immunotherapy, chemotherapy, radiation therapy, or biotherapy. Adjuvant hormone therapy is permitted during long-term remission of breast cancer or other hormone-sensitive cancers.

[0266] Other complications:

[0267] 10. A history or existing evidence of a significant (CTCAE ≥ 2) local or systemic infection requiring systemic antibiotic treatment (e.g., cellulitis, pneumonia, sepsis) within 2 weeks prior to the first dose of the test drug.

[0268] Other exclusion criteria:

[0269] 11. Known hypersensitivity to either the active substance or the excipients, or any contraindications to cimiprizumab or pembrolizumab according to local prescribing information.

[0270] 12. The presence of serious comorbidities or other conditions (e.g., psychological, family, social, or geographical factors) prevents adequate follow-up and protocol adherence.

[0271] 13. Cardiovascular diseases, defined as follows:

[0272] • New York Heart Association (NYHA) functional class II, III, or IV; or

[0273] • Myocardial infarction (MI) or acute coronary syndrome (ACS) within the past 6 months; or

[0274] • Transient ischemic attack (TIA) or stroke within 1 year.

[0275] 14. Received a live vaccine within 30 days prior to the planned start of investigational drug treatment. Note: This refers to a live vaccine or attenuated live vaccine with replication potential. If the patient intends to receive a COVID-19 vaccine before the start of the investigational drug, participation in the study should be postponed for at least one week after any COVID-19 vaccination. It is recommended to postpone COVID-19 vaccination during treatment until the patient is receiving and can tolerate the investigational drug. The interval between the booster vaccine dose and the administration of the investigational drug should be no less than 48 hours.

[0276] 15. Those who have undergone major surgery (i.e., requiring general anesthesia) or severe trauma within 4 weeks prior to screening.

[0277] 16. Exceptions: Minor surgeries / biopsies related to melanoma are permitted, provided that the wound has healed satisfactorily prior to receiving research treatment.

[0278] 17. Previous allogeneic stem cell transplantation or solid organ transplantation

[0279] 18. Any medical condition that the investigators believe is not in the best interests of the patient to participate in the study.

[0280] 19. Members of the research team at the clinical research center and / or their immediate family members (unless otherwise approved in advance by the sponsor).

[0281] 20. Patients admitted to medical facilities by order of judicial or administrative authorities were excluded from this study.

[0282] 21. Adolescent patients weighing <40kg (aged ≥12 to <18).

[0283] Dosage / Route of Administration / Dosage Regimen:

[0284] The combination of cimiprimab plus fosbuvir and the combination of pembrolizumab plus placebo were formulated by unblinded pharmacists at the research center and administered in a blinded manner in an outpatient setting. Adult and adolescent patients received the combination infusion of the investigational drugs every 3 weeks via intravenous infusion over 30 minutes (±10 minutes). Similarly, pembrolizumab and placebo were infused every three weeks via intravenous infusion over 30 minutes (±10 minutes).

[0285] Fumarimab and Cimiprimab (for combination infusion)

[0286] Fumariumab 1600 mg is provided as a sterile, single-use liquid vial. Dosage preparation instructions are available in the pharmacy manual.

[0287] Fumariumab 400 mg is provided as a sterile, single-use liquid vial. Dosage preparation instructions are available in the pharmacy manual.

[0288] Cimiprilmab 350 mg is provided as a sterile, single-use liquid vial. Dosage preparation instructions are available in the pharmacy manual.

[0289] Pembrolizumab

[0290] Pembrolizumab 200 mg is prepared as a sterile, single-use vial and administered vial in combination with saline / glucose placebo at the research center. Adolescent patients receive 2 mg / kg of pembrolizumab (total dose not exceeding 200 mg). Dosage preparation instructions are available in the pharmacy booklet.

[0291] end

[0292] The primary endpoint for all patients was investigator-assessed relapse rate (RFS), defined as the time from the start of treatment to the first recorded recurrence of disease at any site (excluding new-onset primary melanoma) or all-cause death, whichever occurred first.

[0293] The key secondary endpoint for efficacy was overall survival, defined as the time from the start of treatment to the date of death. Other efficacy endpoints included melanoma-specific survival, defined as death from melanoma (death from other causes or unknown causes was considered censored); and distant metastasis-free survival, defined as the time between the date of treatment and the date of first distant metastasis.

[0294] Patient-reported outcomes

[0295] Secondary endpoints of global health status include patient-reported outcomes (PROs) for adult patients, such as those measured by the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 (EORTC-QLQ-C30), the European Five-Dimensional Health Scale Level 5 (EQ-5D-5L), the Functional Assessment of Cancer Treatment (FACT) – Melanoma (Melanoma Subscale Only), the Patient Global Impression Scale (PGIS), and the Patient Global Impression Change Scale (PGIC).

[0296] Procedures and assessments

[0297] The primary endpoint was RFS, which is the recognized primary endpoint in this clinical context. Secondary efficacy endpoints included adult-only OS, MSS, DMFS, and PRO, as measured by the EORTC QLQ-C30, EQ-5D-5L, FACT-Melanoma (Melanoma Subscale Only), PRO CTCAE Fatigue Scale, PGIS, and PGIC. Adolescents were assessed using the Pediatric Quality of Life Scale (PEDSQL). RFS analysis was based on investigator-administered assessments using imaging or pathology at disease relapse or patient death.

[0298] Evaluation of potential disease recurrence detected for the first time by clinical examination is performed using CT or MRI. For solitary suspected lesions or lesions of unknown etiology detected by imaging, biopsy is required, if medically appropriate, to confirm recurrence histologically. For skin lesions, biopsy is also required, if medically appropriate, to confirm disease recurrence histologically. According to established dermatology practice guidelines, local pathologists differentiate between new primary melanoma and recurrent lesions by reviewing biopsy and excised samples of skin lesions to identify the presence of intraepidermal components and whether they are consistent with new primary melanoma (rather than regional or distant metastatic recurrence).

[0299] Blood samples were collected from all patients to measure serum levels of fumizumab and cimiprimab, as well as immunogenicity (the presence of ADA and NAb against fumizumab and cimiprimab in serum).

[0300] Blood samples were collected for analysis of additional biomarkers in plasma and serum. Potential pharmacodynamic, predictive, and prognostic biomarkers related to treatment exposure, clinical activity, and / or underlying disease were investigated in plasma, serum, and tumor tissue.

[0301] The safety and tolerability of the combination of fumarazumab and cimiprimab were monitored through clinical assessment of adverse events (TEAE) / immune-mediated adverse events (imAE), serious adverse events (SAE), and adverse events of particular concern (AESI) occurring during treatment, as well as through repeated measurements and clinical evaluation of vital signs (temperature, blood pressure, pulse, and respiration), physical examination, 12-lead electrocardiogram (ECG), and laboratory assessments (including standard hematological, chemical, urinalysis, and other laboratory tests, including blood cortisol and thyroid-stimulating hormone [TSH]).

[0302] result

[0303] Surprisingly, in patients with completely resected high-risk melanoma, the combination of frangipani (REGN3767, anti-LAG-3) and cimiprimab Q3W improved recurrence-free survival (RFS) compared to pembrolizumab in the context of adjuvant therapy.

[0304] Example 2: Phase 3 clinical trial of anti-LAG-3 antibody (REGN3767, ciampilimab) and anti-PD-1 antibody (REGN2810, cimiprimab) in patients with previously untreated unresectable locally advanced or metastatic melanoma.

[0305] This current phase 3 study evaluated the combination of fumarazumab and cimiprizumab versus pembrolizumab in patients with previously untreated, unresectable, locally advanced or metastatic melanoma.

[0306] Purpose

[0307] The primary objective of this study was to demonstrate the superiority of fuamilimab 1600 mg + cimiprimab and / or fuamilimab 400 mg + cimiprimab over pembrolizumab, as measured by progression-free survival (PFS).

[0308] Secondary objectives include the following:

[0309] • Demonstrate the superiority of fuamilimab 1600 mg + cimiprimab and / or fuamilimab 400 mg + cimiprimab over pembrolizumab, as measured by overall survival (OS).

[0310] • Demonstrate the superiority of fumarazumab 1600mg + cimiprimab and / or fumarazumab 400mg + cimiprimab over pembrolizumab in terms of ORR.

[0311] • Compared with cimiprimab, the ORR, PFS and OS of fumigumab 1600mg + cimiprimab and / or fumigumab 400mg + cimiprimab were characterized to clarify the contribution of each component.

[0312] • Evaluate the immunogenicity of cimetizumab 1600 mg and / or 400 mg and cimetizumab.

[0313] • Evaluate the effects of fuamilimab 1600 mg + cimiprimab and / or fuamilimab 400 mg + cimiprimab on physical and role functioning, as well as overall health status / quality of life in adults, compared with pembrolizumab.

[0314] • Characterize the safety and tolerability of treatment in patients aged 12 to <18 years.

[0315] • Characterize ORR, PFS, and OS in the treatment group of patients aged 12 to <18 years.

[0316] • Evaluate the safety and tolerability of fumarazumab plus cimiprimab compared to pembrolizumab and compared to cimiprimab alone.

[0317] • In patients aged ≥12 years, sparse PK sampling was used to characterize the pharmacokinetics (PK) of the treatment.

[0318] Research Design

[0319] This is a phase 3 study in patients aged ≥12 years with unresectable locally advanced or metastatic melanoma who have not previously received systemic treatment for advanced disease.

[0320] For adult and adolescent patients, this study was conducted in a randomized, double-blind manner, with four main groups:

[0321] Group A: Furamimab (1600 mg, every 3 weeks [Q3W], intravenous [IV]) + Cimiprimab (350 mg Q3W IV)

[0322] Group A1: Fumarimab (400 mg, every 3 weeks [Q3W], intravenous [IV]) + Cimiprimab (350 mg Q3W IV)

[0323] Group B: Pembrolizumab (200 mg Q3W IV) + saline / glucose placebo (placebo)

[0324] Group C: Cimiprilmab (350 mg Q3W IV) + Saline / Glucose Placebo (Placebo)

[0325] The study population included patients with unresectable stage III or IV (metastatic) melanoma who had not previously received systemic anticancer therapy for advanced unresectable and metastatic disease (American Joint Committee on Cancer Melanoma Staging, 8th edition). The patient population included male and female patients aged ≥12 years.

[0326] Selection criteria

[0327] Patients must meet the following criteria to be eligible for inclusion in this study:

[0328] 1. Patients must be ≥12 years old on the date of providing informed consent. Note: Patients <18 years of age were enrolled in this study subject to approval by local laws, regulations, and ethics committees.

[0329] 2. According to the eighth revision of AJCC (Amin, 2017), the patient has histologically confirmed unresectable stage III and IV (metastatic) melanoma and has not previously received systemic treatment for advanced unresectable disease.

[0330] Patients receiving adjuvant and / or neoadjuvant systemic therapy are eligible for inclusion if they do not have evidence of disease progression or relapse during such treatment and / or discontinue treatment due to a grade ≥3 immune-related adverse event (irAE) (excluding endocrine disorders completely controlled by hormone replacement). In addition, patients must have a treatment-free and disease-free interval of >6 months.

[0331] Patients with acral and mucosal melanoma are eligible for enrollment. Recruitment should be limited to approximately 10% of the total population.

[0332] 3. Measurable diseases according to RECIST version 1.1

[0333] • Lesions that have previously received radiotherapy can only be counted as target lesions if they have been confirmed to have progressed and no other target lesions are available.

[0334] • Skin lesions should be assessed as non-target lesions.

[0335] 4. Physical condition:

[0336] • For adult patients: Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1

[0337] • For pediatric patients: Karnofsky Performance Status ≥70 (patients ≥16 years old) or Lansky Performance Status ≥70 (patients <16 years old)

[0338] 5. The expected survival period is at least 3 months.

[0339] 6. Bone marrow function is adequate as determined by hematological parameters:

[0340] • Absolute neutrophil count (ANC) ≥ 1.5 × 10^9 / L (1500 / mm) 3 )

[0341] • Hemoglobin ≥ 9.0 g / dL (5.59 mmol / L).

[0342] • Platelet count ≥75,000 / mm 3 .

[0343] 7. Liver function is adequate as determined by the following indicators:

[0344] • Adult AST / ALT: Aspartate aminotransferase (AST) ≤ 3 times ULN, alanine aminotransferase (ALT) ≤ 3 times ULN, and alkaline phosphatase < 2.5 times ULN (or < 5 times ULN if liver or bone metastases are present).

[0345] • Adolescent AST / ALT: AST, ALT, and ALP < 2.5 times the ULN (and no tumor invasion into the liver) (or <5 times the ULN if liver or bone metastases are present)

[0346] • Serum bilirubin ≤1.5 times ULN, but ≤3 times ULN is allowed for patients with clinically diagnosed Gilbert's syndrome.

[0347] 8. Sufficient kidney function:

[0348] • For adult patients: Determined by an estimated glomerular filtration rate (eGFR) ≥30 mL / min (using the CPK-EPI equation).

[0349] For pediatric patients:

[0350] (i) Creatinine clearance or radioactive isotope glomerular filtration rate (GFR) ≥ 70 mL / min / 1.73 m 2

[0351] or

[0352] (ii) Serum creatinine based on age / sex, as shown in Table 2:

[0353] Table 2: Serum creatinine based on age and sex

[0354]

[0355] 9. LAG-3 IHC biopsy results requirements: Patients must have valid LAG-3 IHC results as determined by a central laboratory to be eligible for study enrollment. Any LAG-3 level (0-100% expression) is permitted.

[0356] Sample requirements:

[0357] Patients must submit tumor tissue samples (formalin-fixed and paraffin-embedded [FFPE]) from tissue blocks that have not been treated for more than one year prior to enrollment. Prepared slides must be sent to the testing laboratory within two weeks of slide preparation.

[0358] If archived tissue is unavailable or insufficient for testing, a new biopsy may be requested prior to enrollment, provided that the research center investigator assesses that the procedure will not pose a potentially serious risk to the health, safety, or well-being of the participant. If archived or new biopsy tissue is unavailable, the patient is ineligible for enrollment.

[0359] Lesions eligible for biopsy must be non-target lesions, must have never received radiotherapy, or must have histological evidence of viable tumor after the last radiotherapy session. Bone biopsy samples are not permitted.

[0360] 10. Women of childbearing potential (WOCBP*) must be negative for serum (β-human chorionic gonadotropin [β-hCG]) at the time of screening.

[0361] *WOCBP is defined as a woman who is of reproductive capacity from menarche to menopause, excluding those who are permanently infertile. Permanent sterilization methods include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy.

[0362] Menopause is defined as the absence of menstruation for 12 consecutive months without other medical cause. For women not using hormonal contraception or hormone replacement therapy, high follicle-stimulating hormone (FSH) levels within the postmenopausal range can be used to confirm menopause. However, in the absence of a 12-month history of amenorrhea, a single FSH measurement is insufficient to determine menopause. The above definition is based on the Clinical Trials Facilitation Working Group (CTFG) guidelines. Women who have undergone a confirmed hysterectomy do not require pregnancy testing or contraception.

[0363] Male study participants with WOCBP partners were required to use condoms for at least 6 months, except for those who had undergone vasectomy or adhered to abstinence.

[0364] • Partners who have undergone vasectomy or research participants who have undergone vasectomy must undergo a medical evaluation to determine the success of the procedure.

[0365] Periodic abstinence (natural rhythm method, sympathobiatic method, post-ovulation method), withdrawal (coitus interruptus), spermicide use alone, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female condoms and male condoms should not be used simultaneously.

[0366] 11. WOCBP members must agree not to donate eggs (ovules, oocytes) for assisted reproductive purposes throughout the entire trial and for 6 months after the last treatment.

[0367] 12. All men must agree not to donate sperm during the trial period and for 6 months after receiving the last treatment dose.

[0368] 13. Provide an informed consent form signed by the subject (for patients aged 12-17 who have not lost their capacity for behavior due to mental disability, an informed consent statement supplemented by the consent form from their parents or legal guardians is required).

[0369] 14. Willing and able to cooperate in completing outpatient visits and research-related procedures.

[0370] 15. Able to understand and complete research-related questionnaires.

[0371] Exclusion criteria

[0372] Patients meeting any of the following criteria were excluded from the study:

[0373] Medical condition

[0374] 1. Uveal melanoma

[0375] 2. Evidence of a persistent or recent (within 2 years) autoimmune disease requiring systemic treatment with immunosuppressants. The following are not excluded: vitiligo, cured childhood asthma, residual hypothyroidism requiring only hormone replacement therapy, and psoriasis not requiring systemic treatment.

[0376] 3. Uncontrolled infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV); or a diagnosis of immunodeficiency disease associated with or causing chronic infection.

[0377] Note:

[0378] • Patients with known HIV infection but controlled disease (spontaneous or achieved under a stable antiretroviral regimen, undetectable viral load, and CD4 count greater than 350) were eligible for enrollment. For patients with controlled HIV infection, monitoring was conducted according to local standards.

[0379] Patients with known hepatitis B infection (HepBsAg+) but with controlled disease (serum hepatitis B virus DNA PCR below the detection limit and currently receiving antiviral therapy for hepatitis B) are eligible for enrollment. Patients with controlled infection must undergo regular HBV DNA monitoring according to local standards and must continue antiviral therapy for at least 6 months after the last study drug dose.

[0380] Patients who are known to be positive for hepatitis C virus antibodies (HCV Ab+) but whose infection is under control (either in a spontaneous state or after previous successful anti-HCV treatment, with no HCV RNA detectable by PCR) are allowed to enroll.

[0381] • Patients with HIV or hepatitis must be reviewed by a qualified specialist (such as an infectious disease specialist or hepatologist) who manages the disease before the trial begins and regularly during the trial.

[0382] 4. BRAF V600 mutation status unknown. Patients with BRAF-mutant melanoma who present with rapidly progressing disease symptoms and who, according to investigator assessment, may benefit from prior BRAF / MEK inhibitor therapy should not be enrolled in the study.

[0383] 5. Another malignant tumor that is progressing or requires active treatment within the past 2 years, except for malignant tumors with negligible risk of metastasis or death (such as well-treated cervical carcinoma in situ, basal cell or squamous cell skin cancer, localized early prostate cancer, or ductal carcinoma in situ of the breast).

[0384] Note: Any uncertainties should be discussed with the medical monitor.

[0385] 6. Pregnant or breastfeeding women.

[0386] 7. WOCBP patients who do not wish to use highly effective contraception before the initial dose / first treatment, during the study, and for at least 6 months after the last dose. Highly effective contraception includes:

[0387] • For two or more menstrual cycles prior to screening, candidates have been continuously using combined hormonal (including estrogen and progestin) contraceptive methods (oral, vaginal, transdermal) or progestin-only hormonal contraceptive methods (oral, injection, implantation) that inhibit ovulation.

[0388] • Intrauterine device (IUD); Intrauterine hormone-releasing system (IUS);

[0389] Bilateral tubal ligation;

[0390] • Partners who have undergone vasectomy (provided the male partner who has undergone vasectomy is the only sexual partner of the WOCBP study participant, and the partner who has undergone vasectomy has received a medical assessment of surgical success); and / or

[0391] Abstinence , .

[0392] (i) Abstinence is considered an effective method only when defined as avoiding heterosexual intercourse throughout the entire investigational drug risk period. The reliability of sexual abstinence needs to be evaluated based on the duration of the clinical trial, patient preferences, and usual lifestyle.

[0393] (ii) Periodic abstinence (natural rhythm method, sympathobasic temperature method, post-ovulation method), withdrawal (coitus interruptus), spermicide use alone, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female condoms and male condoms should not be used simultaneously.

[0394] Previous / combined treatment

[0395] 8. Systemic immunosuppression:

[0396] • Use immunosuppressive doses of corticosteroids (≤10 mg prednisone or equivalent daily) within 14 days of the first study dose. Physiological alternative doses are permitted, up to and including 10 mg prednisone or equivalent daily. Inhaled or topical steroids are permitted if not for the treatment of an autoimmune disease.

[0397] • Other clinically relevant forms of systemic immunosuppression.

[0398] 9. Received other anticancer treatments, including immunotherapy, chemotherapy, major surgery, or biotherapy, within 21 days prior to the first trial treatment dose. Adjuvant hormone therapy is permitted during long-term remission in breast cancer or other hormone-sensitive cancers.

[0399] 10. Patients with a history of grade ≥3 immune-mediated adverse events (excluding endocrine disorders that are completely controlled by hormone replacement) during previous checkpoint inhibitor therapy should be excluded.

[0400] Other complications

[0401] 11. A history or existing evidence of a significant (CTCAE grade ≥2) local or systemic infection requiring systemic antibiotic treatment (e.g., cellulitis, pneumonia, sepsis) within 14 days prior to the first dose of the test drug.

[0402] 12. Active or untreated brain metastases or spinal cord compression. Patients with leptomeningeal diseases should be excluded. Patients with known brain metastases are eligible if they meet the following criteria:

[0403] • Received radiation therapy or another appropriate standard treatment for brain metastases.

[0404] • Neurological function must have recovered to baseline levels for at least 14 days prior to enrollment (excluding residual signs and symptoms related to CNS treatment).

[0405] • No immunosuppressive doses of corticosteroids (>10 mg prednisone or equivalent daily) were required within 14 days prior to enrollment.

[0406] Note: Asymptomatic patients with a single untreated brain metastasis <10 mm in size are eligible for inclusion.

[0407] Other exclusion criteria

[0408] 13. Known to exhibit hypersensitivity to active substances or any excipients.

[0409] 14. The presence of serious comorbidities or other conditions (e.g., psychological, family, social, or geographical factors) that preclude adequate follow-up and protocol adherence.

[0410] 15. A live vaccine (i.e., a live vaccine with replication potential or a live attenuated vaccine) must have been administered within 30 days prior to the planned start of investigational drug treatment. If the patient intends to receive a COVID-19 vaccine before the start of the investigational drug, participation in the study should be postponed for at least one week after any COVID-19 vaccination. It is recommended to postpone COVID-19 vaccination during treatment until the patient is receiving and can tolerate a stable dose of the investigational drug. The interval between vaccine dose and investigational drug administration should be no less than 48 hours.

[0411] 16. Those who have undergone major surgery, open biopsy, or severe traumatic injury within 4 weeks prior to screening.

[0412] 17. Previous allogeneic stem cell transplantation or solid organ transplantation.

[0413] 18. Any medical condition that the investigators believe is not in the best interests of the patient to participate in the study.

[0414] 19. Members of the research team at the clinical research center and / or their immediate family members (unless otherwise approved in advance by the sponsor).

[0415] 20. Patients admitted to medical facilities by order of judicial or administrative authorities were excluded from this study.

[0416] 21. Adolescent patients weighing <40kg (aged ≥12 to <18).

[0417] Dosage / Route of Administration / Dosage Regimen

[0418] The combination of cimiprimab plus fumarlimab, the combination of pembrolizumab plus placebo, and the combination of cimiprimab plus placebo were formulated by non-blinded pharmacists at the research center and administered to all patients in a blinded manner.

[0419] All infusions for adults and adolescents were administered in an outpatient setting as 30-minute (±10-minute) IV infusions every 3 weeks.

[0420] Cimipril

[0421] Cimiprimab 350 mg IV every three weeks is an approved treatment for squamous cell carcinoma of the skin (CSCC), basal cell carcinoma (BCC), and non-small cell lung cancer (NSCLC). Cimipril (Cimiprazan)

[0422] Cimiprimab is provided as a liquid in sterile, single-use vials. Dosage preparation instructions are available in the pharmacy manual.

[0423] Fumarimab and Cimiprimab (for combination / infusion administration)

[0424] Based on efficacy and safety data from the ongoing Phase 1 study, it is recommended to advance the dose of fumarazumab 1600 mg Q3W IV and cimiprimab 350 mg Q3W IV to Phase 3. The efficacy of fumarazumab 400 mg Q3W IV and cimiprimab 350 mg Q3W IV has not been investigated, but this dose is added because it aligns with the expectation of considering lower doses in the context of oncology treatment and is consistent with the doses of other anti-LAG-3 antibodies used in Phase 2 and 3 clinical trials for melanoma and other solid tumors.

[0425] Fumariumab 1600 mg and 400 mg Q3W IV are provided as a sterile, single-use liquid in vials. Dosage preparation instructions are available in the pharmacy manual.

[0426] Fumarimab and cimiprimab were administered simultaneously as a combined infusion.

[0427] Pembrolizumab

[0428] Pembrolizumab 200 mg Q3W IV is the approved dose for the treatment of advanced and metastatic melanoma.

[0429] For adolescent patients randomly assigned to group B, pembrolizumab was administered at 2.0 mg / kg every 3 weeks (up to 200 mg) based on body weight.

[0430] Pembrolizumab was prepared as a sterile liquid and administered in combination with saline placebo at the research center. Dosage preparation instructions are available in the pharmacy manual.

[0431] end

[0432] The primary endpoint was PFS (progression-free survival) (assessed based on blinded independent central review [BICR] according to RECIST version 1.1).

[0433] The key efficacy endpoints are overall survival and objective response rate (ORR), the latter defined as the proportion of patients achieving optimal overall response (CR, or PR) (based on BICR, according to RECIST 1.1). Other efficacy endpoints include disease control rate (DCR), defined as the proportion of patients achieving optimal overall response (CR, PR, or stable disease (SD)) (based on BICR, according to RECIST 1.1; SD is assessed at least 6 months after the first dose).

[0434] DoR (duration of remission) is defined as the time from the first remission (CR or PR according to RECIST 1.1) to the first occurrence of PD (disease progression) (based on BICR according to RECIST 1.1) or all-cause death, whichever occurs first.

[0435] PFS, ORR, DCR, and DoR are based on assessments conducted by investigators according to RECIST 1.1 and iRECIST (immune RECIST).

[0436] Patient-reported outcomes:

[0437] The secondary endpoints of global health status are:

[0438] • Patient-reported outcomes, such as those measured by EORTC QLQ-C30, EQ-5D-5L, FACT-Melanoma (Melanoma Subscale Only), PGIS, and PGIC.

[0439] • According to the EORTC QLQ-C30, changes in physical function from baseline at week 25

[0440] • Changes in role functionality from baseline in week 25, according to EORTC QLQ-C30

[0441] • According to EORTC QLQ-C30, the change in GHS / QoL from baseline at week 25

[0442] • According to the EORTC QLQ-C30, changes in physical function from baseline during the study period

[0443] • According to EORTC QLQ-C30, changes in role function from baseline during the study period

[0444] • According to EORTC QLQ-C30, the change in GHS / QoL from baseline during the study period.

[0445] Procedures and assessments

[0446] The efficacy endpoint included antitumor activity. This was assessed using computed tomography (CT) or magnetic resonance imaging (MRI) for all patients in the study. For skin lesions, digital radiography was used.

[0447] The safety and tolerability of the combination of fambalimab and cimiprimab will be monitored through clinical assessments of TEAE / immune-related adverse events (irAE), SAE, and AESI, as well as through repeated measurements and clinical evaluations of vital signs (temperature, blood pressure, pulse, and respiration), physical examination, 12-lead electrocardiogram (ECG), and laboratory assessments (including standard hematological, chemical, urinalysis, and other laboratory tests, including blood cortisol and thyroid-stimulating hormone [TSH]).

[0448] Blood samples were collected from all patients to measure the levels of functional fuanlimumab and functional cimiprimab in serum, and to measure immunogenicity (the presence of ADA and NAb against fuanlimumab and cimiprimab in serum).

[0449] Patient eligibility requires a centrally determined LAG-3 immunohistochemical (IHC) result. Patients must have a valid LAG-3 IHC result as determined by a central laboratory to be eligible for study enrollment. Any LAG-3 level (0-100% expression) is permitted. Patient samples will be collected for analysis of additional biomarkers. Investigative biomarkers may be included in relation to exposure to fumarlimab and cimiprimab treatments; predictive and prognostic biomarkers; clinical activity; and / or underlying disease. Serum, plasma, peripheral blood mononuclear cells (PBMCs), and tumor tissue will be collected.

[0450] result

[0451] Surprisingly, the combination of fambalizumab (REGN3767, anti-LAG-3) and cimiprimab improved progression-free survival (PFS) in patients with previously untreated, unresectable locally advanced or metastatic melanoma compared to pembrolizumab. Furthermore, the combination of fambalizumab and cimiprimab is expected to exhibit similar PK, safety, and efficacy characteristics in adolescent patients as observed in adult patients with previously untreated, unresectable locally advanced or metastatic melanoma.

[0452] Example 3: A Phase 3 clinical trial comparing anti-LAG-3 (furanlimab) plus anti-PD-1 (cimiprilmab) versus pembrolizumab in patients with completely resected high-risk melanomas.

[0453] Background: Melanoma accounts for the majority of skin cancer-related deaths. Most newly diagnosed melanoma patients have resectable disease and are potentially curable with surgery. However, regional lymph node and / or distant recurrence can occur after radical resection. Postoperative adjuvant therapy with immune checkpoint inhibitors can improve recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) in high-risk melanoma patients. Fumarimab (anti-LAG-3) and cimiprimab (anti-PD-1) are both high-affinity, fully human IgG4 monoclonal antibodies (MAb), and their combination showed high clinical activity in patients with advanced melanoma in a phase 1 study.

[0454] Methods: This study was a three-arm, double-blind phase 3 trial comparing the effects of fombrolizumab plus cimiprilmab versus pembrolizumab as adjuvant therapy for high-risk resected melanoma. The primary objective was recurrence-free survival, and secondary objectives were overall survival, safety, pharmacology, and immunogenicity.

[0455] Patient eligibility: (1) ≥12 years of age; (2) stage IIc, III or IV (all M stages) and histologically confirmed melanoma, which was not treated prior to the diagnosis of melanoma. < (3) Complete resection within 12 weeks; (4) No systemic anticancer therapy or radiation therapy for melanoma in the past 5 years; (5) No evidence of metastatic disease in the staging examination; and (6) Eastern Oncology Cooperative Group Performance Status (PS) of 0 or 1 (for adult patients), Karnofsky PS > 70 (patients > 16 years old) or Lansky PS > 70 (patients < 16 years old).

[0456] Study groups (all treatments administered intravenously every 3 weeks for one year): A. Fumizumab (1600 mg) + Cimiprimab (350 mg); B. Fumizumab (400 mg) + Cimiprimab (350 mg); C. Pembrolizumab (200 mg) + Saline / Glucose placebo. This placebo-controlled trial recruited patients and randomly assigned them to groups A, B, and C in a 1:1:1 ratio, with treatment lasting up to one year. The trial was stratified by disease stage (IIIA vs. IIC-IIIB-IIIC vs. IIID-IV [M1a / b] vs. IV [M1c / d]) and geographic region (North America vs. Europe vs. the rest of the world).

[0457] The primary endpoint was investigator-assessed recurrence-free survival. Secondary endpoints included efficacy (overall survival, distant metastasis-free survival, melanoma-specific survival), safety [treatment-interventional adverse events (TEAEs), dosing interruption or discontinuation due to TEAEs], pharmacokinetics (changes in serum concentrations of fumbramab and cimiprimab over time), immunogenicity (anti-drug antibodies and neutralizing antibodies against fumbramab or cimiprimab in serum), and patient-reported outcomes. Analysis was performed when 242 recurrence-free survival events were observed.

[0458] Results: The trial is currently recruiting participants. Surprisingly, in patients with completely resected high-risk melanoma, administration of the combination of fumarazab and cimiprimab resulted in enhanced tumor regression and improved disease control.

[0459] This disclosure is not limited in scope to the specific embodiments described herein. In fact, various modifications to this disclosure will be apparent to those skilled in the art from the foregoing description and accompanying drawings, in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims.

[0460] Table 3: Informal Sequence List

[0461]

[0462]

Claims

1. A method for treating melanoma or inhibiting melanoma growth, the method comprising administering to a subject in need a therapeutically effective amount of each of (a) an antibody specifically binding to programmed death 1 (PD-1) or an antigen-binding fragment thereof and (b) an antibody specifically binding to lymphocyte activation gene-3 (LAG-3) or an antigen-binding fragment thereof, The melanoma mentioned therein is an unresectable locally advanced melanoma, an unresectable metastatic melanoma, or a completely resectable high-risk melanoma.

2. The method of claim 1, wherein a dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 50 mg to 1500 mg.

3. The method of claim 1 or 2, wherein one dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 350 mg.

4. The method according to any one of claims 1 to 3, wherein a dose of the anti-LAG-3 antibody or its antigen-binding fragment is between 50 mg and 8000 mg.

5. The method according to any one of claims 1 to 4, wherein a dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 400 mg.

6. The method of any one of claims 1 to 4, wherein a dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 1600 mg.

7. The method of any one of claims 1 to 6, wherein the anti-LAG-3 antibody or its antigen-binding fragment is administered before, simultaneously with, or after the anti-PD-1 antibody or its antigen-binding fragment.

8. The method of claim 7, wherein the anti-LAG-3 antibody or its antigen-binding fragment is administered prior to the anti-PD-1 antibody or its antigen-binding fragment.

9. The method of claim 7, wherein the anti-LAG-3 antibody or its antigen-binding fragment is administered on the same day as the anti-PD-1 antibody or its antigen-binding fragment.

10. The method of any one of claims 1 to 9, wherein two or more doses of the anti-LAG-3 antibody or its antigen-binding fragment are administered in combination with two or more doses of the anti-PD-1 antibody or its antigen-binding fragment.

11. The method of claim 10, wherein each dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 350 mg.

12. The method of claim 10 or 11, wherein each dose of the anti-LAG-3 antibody or its antigen-binding fragment is between 50 mg and 8000 mg.

13. The method of any one of claims 10 to 12, wherein each dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 1600 mg.

14. The method of any one of claims 10 to 12, wherein each dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 400 mg.

15. The method of claim 10, wherein each dose of the anti-PD-1 antibody or its antigen-binding fragment comprises 200 mg, 250 mg or 350 mg, and each dose of the anti-LAG-3 antibody or its antigen-binding fragment comprises 400 mg, 800 mg, 1000 mg, 1400 mg or 1600 mg.

16. The method of any one of claims 10 to 15, wherein each dose of the anti-PD-1 antibody or its antigen-binding fragment is administered 0.5 to 12 weeks immediately following the previous dose.

17. The method of any one of claims 10 to 16, wherein each dose of the anti-LAG-3 antibody or its antigen-binding fragment is administered 0.5 to 12 weeks immediately following the previous dose.

18. The method of any one of claims 10 to 17, wherein each dose of the anti-PD-1 antibody or its antigen-binding fragment is administered once every six weeks.

19. The method of any one of claims 10 to 18, wherein each dose of the anti-LAG-3 antibody or its antigen-binding fragment is administered once every six weeks.

20. The method of any one of claims 10 to 17, wherein each dose of the anti-PD-1 antibody or its antigen-binding fragment is administered once every three weeks.

21. The method of any one of claims 10 to 18 or 20, wherein each dose of the anti-LAG-3 antibody or its antigen-binding fragment is administered once every three weeks.

22. The method of any one of claims 1 to 21, wherein the antibody is administered intravenously, subcutaneously, or intraperitoneally.

23. The method of any one of claims 1 to 22, wherein the melanoma is an unresectable locally advanced melanoma.

24. The method of any one of claims 1 to 22, wherein the melanoma is an unresectable metastatic melanoma.

25. The method of claim 23 or 24, wherein the patient is further selected as having one or more of the following criteria: (i) At least 12 years old on the date the informed consent form is provided; (ii) According to AJCC 8th edition (Amin, 2017), the patient is in stage IIC, III or IV and has a histologically confirmed melanoma that has been completely surgically removed; (iii) Patients with stage IIIA disease must have at least one lymph node micrometastasis with a maximum measured diameter >1 mm; (iv) Stage IIC melanoma confirmed by a pathologically negative sentinel lymph node biopsy (SLNB) specimen, with no evidence of regional or distant metastasis; (v) Complete surgical excision was performed within 12 weeks prior to treatment, and treatment was only permitted after satisfactory wound healing. (vi) Before treatment, a comprehensive physical examination and imaging examination confirmed that there was no disease state; (vii) The patient shows ≥1% LAG3 in tumor tissue as determined by IHC or iPET; as well as (viii) The patient must not have received systemic anticancer therapy or radiation therapy for melanoma in the past 5 years.

26. The method of any one of claims 1 to 22, wherein the melanoma is a high-risk melanoma that has been completely removed.

27. The method of claim 26, wherein the patient is further selected as having one or more of the following criteria: (1) ≥12 years old; (2) Stage IIc, III or IV (all M stages) and histologically confirmed melanoma, which has been completely removed <12 weeks prior to treatment; (3) No systemic anticancer treatment or radiation therapy for melanoma in the past 5 years; (4) LAG3 is identified as ≥1% in tumor tissue by IHC or iPET; (5) No evidence of metastatic disease; and (6) Eastern Oncology Collaboration Group Performance Status (PS) of 0 or 1 (applicable to adult patients), Karnofsky PS > 70 (patients > 16 years old) or Lansky PS > 70 (patients < 16 years old).

28. The method of any one of claims 1 to 27, wherein the treatment produces a therapeutic effect selected from the group consisting of delayed melanoma growth, reduced melanoma cell number, melanoma regression, prolonged survival, partial remission, and complete remission.

29. The method of claim 28, wherein melanoma growth is delayed by at least 10 days compared to an untreated subject.

30. The method of claim 28, wherein the melanoma growth is inhibited by at least 50% compared to an untreated subject.

31. The method of claim 28, wherein melanoma growth is inhibited by at least 20% compared to a subject treated with any of the antibodies as monotherapy.

32. The method of any one of claims 1 to 31, further comprising administering to the subject an additional therapeutic agent or therapy, wherein the additional therapeutic agent or therapy is selected from the group consisting of: radiotherapy, surgery, chemotherapy agents, cancer vaccines, PD-L1 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, CD28 agonists, CD38 inhibitors, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, angiopoietin-2 (Ang2) inhibitors, and conversion therapy. Growth factor β (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens, BCG, granulocyte-macrophage colony-stimulating factor, oncolytic viruses, cytotoxins, interleukin-6 receptor (IL-6R) inhibitors, interleukin-4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-21, IL-12, IL-15, antibody-drug conjugates, GITR agonists, 4-1BB agonists, CD20xCD3 bispecific antibodies, MUC16xCD3 bispecific antibodies, and anti-inflammatory drugs.

33. The method of any one of claims 1 to 32, wherein the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain complementarity-determining region (HCDR1, HCDR2, and HCDR3) of the heavy chain variable region (HCVR) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) of the light chain variable region (LCVR), wherein HCDR1 contains the amino acid sequence of SEQ ID NO:3; HCDR2 contains the amino acid sequence of SEQ ID NO:4; HCDR3 contains the amino acid sequence of SEQ ID NO:5; LCDR1 contains the amino acid sequence of SEQ ID NO:6; LCDR2 contains the amino acid sequence of SEQ ID NO:7; and LCDR3 contains the amino acid sequence of SEQ ID NO:

8.

34. The method of claim 33, wherein the HCVR contains the amino acid sequence of SEQ ID NO:1 and the LCVR contains the amino acid sequence of SEQ ID NO:

2.

35. The method of any one of claims 1 to 34, wherein the anti-PD-1 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:9 and a light chain containing the amino acid sequence of SEQ ID NO:

10.

36. The method of any one of claims 1 to 35, wherein the anti-LAG-3 antibody or its antigen-binding fragment comprises a heavy chain CDR (HCDR1, HCDR2, and HCDR3) of HCVR and three light chain CDRs (LCDR1, LCDR2, and LCDR3) of LCVR, wherein HCDR1 contains the amino acid sequence of SEQ ID NO:13; HCDR2 contains the amino acid sequence of SEQ ID NO:14; HCDR3 contains the amino acid sequence of SEQ ID NO:15; LCDR1 contains the amino acid sequence of SEQ ID NO:16; LCDR2 contains the amino acid sequence of SEQ ID NO:17; and LCDR3 contains the amino acid sequence of SEQ ID NO:

18.

37. The method of claim 36, wherein the HCVR contains the amino acid sequence of SEQ ID NO:11 and the LCVR contains the amino acid sequence of SEQ ID NO:

12.

38. The method of any one of claims 1 to 37, wherein the anti-LAG-3 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 19 and a light chain containing the amino acid sequence of SEQ ID NO:

20.

39. The method of any one of claims 1 to 38, wherein the inhibition is more effective than the administration of any antibody as a monotherapy.

40. A method for treating melanoma or inhibiting melanoma growth, the method comprising: (1) Select patients with melanoma, wherein the patients have unresectable locally advanced melanoma, unresectable metastatic melanoma, or completely resected high-risk melanoma; and (2) The patient is given (a) 350 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:1 / 2; and (b) 400 mg or 1600 mg of an anti-LAG-3 antibody or an antigen-binding fragment thereof, the anti-LAG-3 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:11 / 12.

41. The method of claim 40, wherein the application in step (2) is performed once every 3 weeks.

42. The method of claim 40, wherein the application in step (2) is performed once every 6 weeks.

43. The method of claim 40, wherein the patient is further selected as having one or more of the following criteria: (1) ≥12 years old; (2) Stage IIc, III or IV (all M stages) and histologically confirmed melanoma, which has been completely removed <12 weeks prior to treatment; (3) No systemic anticancer treatment or radiation therapy for melanoma in the past 5 years; (4) No evidence of metastatic disease; and (5) The Eastern Oncology Collaboration Group's performance status (PS) is 0 or 1 (applicable to adult patients), Karnofsky PS > 70 (patients > 16 years old) or Lansky PS > 70 (patients < 16 years old).

44. A method for treating melanoma or inhibiting melanoma growth, the method comprising: (1) Patients with unresectable locally advanced melanoma, unresectable metastatic melanoma, or completely resected high-risk melanoma were selected, provided that these patients had not previously received systemic treatment for advanced disease; and (2) The patient is given (a) 350 mg of an anti-PD-1 antibody or an antigen-binding fragment thereof, the anti-PD-1 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:1 / 2; and (b) 400 mg or 1600 mg of an anti-LAG-3 antibody or an antigen-binding fragment thereof, the anti-LAG-3 antibody or an antigen-binding fragment thereof containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:11 / 12.

45. The method of claim 44, wherein the application in step (2) is performed once every 3 weeks.

46. ​​The method of claim 44, wherein the application in step (2) is performed once every 6 weeks.

47. The method of claim 44, wherein the patient is further selected as having one or more of the following criteria: (1) ≥12 years old; (2) Stage IIc, III or IV (all M stages) and histologically confirmed melanoma, which has been completely removed <12 weeks prior to treatment; (3) No systemic anticancer treatment or radiation therapy for melanoma in the past 5 years; (4) No evidence of metastatic disease was found in the staging examination; and (5) The Eastern Oncology Collaboration Group's performance status (PS) is 0 or 1 (applicable to adult patients), Karnofsky PS > 70 (patients > 16 years old) or Lansky PS > 70 (patients < 16 years old).

48. A method for treating melanoma or inhibiting melanoma growth, the method comprising: (1) Select patients with melanoma; as well as (2) Administer to the patient (a) 400 mg or 1600 mg of an anti-LAG-3 antibody or its antigen-binding fragment thereof, the anti-LAG-3 antibody or its antigen-binding fragment containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:11 / 12; and (b) 350 mg of an anti-PD-1 antibody or its antigen-binding fragment thereof in combination with (a), the anti-PD-1 antibody or its antigen-binding fragment containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO:1 / 2.

49. The method of claim 48, wherein the application step is performed every 3 weeks.

50. The method of claim 48, wherein the application step is performed every 6 weeks.

51. A method for treating melanoma or inhibiting melanoma growth, the method comprising: (a) Select patients with melanoma, wherein the patients have undergone surgery to treat melanoma; as well as (b) Administer to the patient: (1) An initial loading dose comprising an anti-PD-1 antibody or its antigen-binding fragment and an anti-LAG-3 antibody or its antigen-binding fragment; wherein the anti-PD-1 antibody or its antigen-binding fragment contains the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 1 / 2, and the anti-LAG-3 antibody or its antigen-binding fragment contains the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 11 / 12; and (2) One or more secondary doses, wherein the one or more secondary doses occur one to four weeks immediately following the previous dose.

52. The method of claim 51, wherein the patient shows ≥1% more LAG3 in melanoma tissue.

53. The method of claim 51, wherein the patient is diagnosed with stage IV melanoma.

54. The method of claim 51, wherein the patient is further selected as having one or more of the following criteria: (1) ≥12 years old; (2) Stage IIc, III or IV (all M stages) and histologically confirmed melanoma, which has been completely removed <12 weeks prior to treatment; (3) No systemic anticancer treatment or radiation therapy for melanoma in the past 5 years; (4) LAG3 is identified as ≥1% in tumor tissue by IHC or iPET; (5) No evidence of metastatic disease; and (6) Eastern Oncology Collaboration Group Performance Status (PS) of 0 or 1 (applicable to adult patients), Karnofsky PS > 70 (patients > 16 years old) or Lansky PS > 70 (patients < 16 years old).

55. The method of claim 51, further comprising administering the following to a patient in need: (3) One or more three-dose administrations, wherein the one or more three-dose administrations occur three to twelve weeks immediately following the previous dose.

56. The method of claim 51, wherein the one or more secondary doses occur three weeks immediately following the previous dose.

57. The method of claim 55, wherein the one or more three doses occur three or six weeks immediately following the previous dose.

58. The method of claim 51, wherein the initial loading dose comprises (a) 500 mg to 1500 mg of anti-PD-1 antibody or an antigen-binding fragment thereof and (b) 50 mg to 8000 mg of anti-LAG-3 antibody or an antigen-binding fragment thereof.

59. The method of claim 51, wherein the one or more secondary doses comprise: (a) 350 mg of anti-PD-1 antibody or an antigen-binding fragment thereof and (b) 400 mg, 800 mg, 1000 mg, 1400 mg, 1600 mg or 2000 mg of anti-LAG-3 antibody or an antigen-binding fragment thereof.

60. The method of claim 55, wherein the one or more triple doses comprise: (a) 350 mg of anti-PD-1 antibody or an antigen-binding fragment thereof and (b) 400 mg, 800 mg, 1000 mg, 1400 mg, 1600 mg or 2000 mg of anti-LAG-3 antibody or an antigen-binding fragment thereof.

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