Combining PD-1 and LAG-3 inhibitors for enhanced efficacy in the treatment of melanoma

Abstract

The present disclosure provides methods for treating or inhibiting the growth of melanoma, comprising selecting a patient with 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, administration of the PD-1 inhibitor enhances the effectiveness of the LAG-3 inhibitor (e.g., an anti-LAG-3 antibody or antigen-binding fragment thereof) in inhibiting the growth of melanoma.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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.

[0002] Technical Field This disclosure provides, in part, compositions comprising inhibitors of LAG-3 and PD-1, and methods for treating melanoma.

[0003] Sequence Listing An official copy of the Sequence Listing will be submitted electronically via the Patent Center contemporaneously with the present specification. The contents of the electronic Sequence Listing (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]

[0004] background Programmed death 1 (PD-1) receptor signaling in the tumor microenvironment plays a key role in enabling tumor cells to escape immunosurveillance 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 shown clinical activity in patients with multiple tumor types, and antibody therapeutics that block PD-1 / PDL1 signaling (e.g., nivolumab, pembrolizumab, atezolizumab, durvalumab, and cemiplimab) have been approved for the treatment of various cancers, including metastatic melanoma and metastatic squamous non-small cell lung cancer.

[0005] 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, gamma-delta 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. Its primary function is to attenuate immune responses. LAG-3 binding to MHC class II molecules results in the delivery of a negative signal to LAG-3-expressing cells, downregulating antigen-dependent CD4 and CD8 T cell responses. LAG-3 also 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 T regulatory (Treg) cell function (Pardoll 2012, Nature Reviews Cancer 12:252-264 (Non-Patent Document 1)).

[0006] Both PD-1 and LAG-3 play important roles in tumor immunity, making them ideal targets for immunotherapy. Targeting both LAG-3 and PD-1, including in anti-PD-1-resistant tumors, can result in objective responses in patients across several tumor types. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Pardoll 2012, Nature Reviews Cancer 12:252-264 Summary of the Invention

[0008] overview The present disclosure relates to methods for treating melanoma and for inhibiting tumor growth.

[0009] Provided herein are methods for treating or ameliorating at least one symptom or sign, or inhibiting the growth of melanoma in a subject. The method according to this aspect of the disclosure includes administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1), in combination with a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to LAG-3. In the methods disclosed herein, the inhibition achieved using the combination therapy is more effective than administration of either antibody as a monotherapy.

[0010] Methods for treating melanoma or inhibiting the growth of melanoma are provided herein. In some embodiments, the methods comprise administering to a subject in need thereof a therapeutically effective amount of each of (a) an antibody or antigen-binding fragment thereof that specifically binds to programmed death 1 (PD-1), and (b) an antibody or antigen-binding fragment thereof that specifically binds to lymphocyte activation gene-3 (LAG-3). In some aspects, the melanoma is unresectable locally advanced melanoma, unresectable metastatic melanoma, or completely resected high-risk melanoma.

[0011] A therapeutically effective amount can be one or more doses of the antibody or antigen-binding fragment thereof. In some embodiments, a single dose of the anti-PD-1 antibody or antigen-binding fragment thereof comprises 50-1500 mg. In some embodiments, a single dose of the anti-PD-1 antibody or antigen-binding fragment thereof comprises 350 mg. In some embodiments, a single dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 50-8000 mg. In some embodiments, a single dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 400 mg. In some embodiments, a single dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 1600 mg.

[0012] The anti-LAG-3 antibody or antigen-binding fragment thereof can be administered before, simultaneously with, or after the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof is administered before the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof is administered on the same day as the anti-PD-1 antibody or antigen-binding fragment thereof.

[0013] In some embodiments, two or more doses of an anti-LAG-3 antibody or antigen-binding fragment thereof are administered in combination with two or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof. In some aspects, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof comprises 350 mg. In some aspects, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 50-8000 mg. In some aspects, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 1600 mg. In some aspects, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 400 mg. In some aspects, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof comprises 200 mg, 250 mg, or 350 mg, and each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 400 mg, 800 mg, 1000 mg, 1400 mg, or 1600 mg. The antibody, eg, a dosage comprising an antibody, can be administered intravenously, subcutaneously, or intraperitoneally.

[0014] When two or more doses of an anti-LAG-3 antibody or antigen-binding fragment thereof are administered in combination with two or more doses of an anti-PD-1 antibody or antigen-binding fragment thereof, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof can be administered 0.5 to 12 weeks after the immediately preceding dose. In some embodiments, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof is administered 0.5 to 12 weeks after the immediately preceding dose. In some embodiments, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof is administered once every six weeks. In some embodiments, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof is administered once every six weeks. In some embodiments, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof is administered once every three weeks. In some embodiments, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof is administered once every three weeks.

[0015] In some embodiments, the melanoma is unresectable locally advanced melanoma. In some aspects, the melanoma is unresectable metastatic melanoma. In such embodiments, patients must meet the following criteria: (i) at least 12 years old on the date of providing informed consent; (ii) have histologically confirmed melanoma that is either Stage IIC, III, or IV according to the AJCC 8th edition (Amin, 2017) and has been completely surgically resected to be eligible; (iii) patients with Stage IIIA disease must have at least one lymph node micrometastasis measuring greater than 1 mm in greatest diameter; (iv) have a pathology-negative sentinel lymph node biopsy (SLNB) specimen and no local or distant metastases. (v) complete surgical resection performed within 12 weeks prior to treatment, and treatment may be administered only after satisfactory wound healing from surgery; (vi) disease-free status as demonstrated by whole-body physical examination and imaging studies prior to treatment; (vii) patients exhibit 1% or more LAG3 in tumor tissue as determined by IHC or iPET; and (viii) patients must not have received systemic anticancer therapy or radiation therapy for melanoma within the past 5 years.

[0016] In some embodiments, the melanoma is a completely resected high-risk melanoma. In such embodiments, the patient is further selected as having one or more of the following criteria: (1) age 12 years or older, (2) stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected less than 12 weeks prior to treatment, (3) no prior systemic anticancer therapy or radiation therapy for melanoma within the past 5 years, (4) tumor tissue showing 1% or more LAG3 as determined by IHC or iPET, (5) no evidence of metastatic disease, and (6) an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), a Karnofsky PS of greater than 70 (for patients over 16 years of age), or a Lansky PS of greater than 70 (for patients under 16 years of age).

[0017] Methods for treating or inhibiting the growth of melanoma are provided herein. In some embodiments, the methods include: (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 antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 / 2, and (b) 400 mg or 1600 mg of an anti-LAG-3 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 11 / 12.

[0018] In some embodiments, patients are further selected as having one or more of the following criteria: (1) age 12 years or older; (2) stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected less than 12 weeks prior to treatment; (3) no prior systemic anti-cancer therapy or radiation therapy for melanoma within the past five years; (4) no evidence of metastatic disease on staging studies; and (5) an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), a Karnofsky PS of greater than 70 (for patients over 16 years of age), or a Lansky PS of greater than 70 (for patients under 16 years of age).

[0019] In some embodiments, the administration of step (2) occurs once every three weeks. In some embodiments, the administration of step (2) occurs once every six weeks.

[0020] Provided herein are methods for treating or inhibiting the growth of melanoma, comprising: (1) selecting a patient with unresectable locally advanced melanoma, unresectable metastatic melanoma, or completely resected high-risk melanoma, wherein the patient has not received prior systemic treatment for progressive disease; and (2) administering to the patient (a) 350 mg of an anti-PD-1 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 / 2, and (b) 400 mg or 1600 mg of an anti-LAG-3 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 11 / 12.

[0021] In some embodiments, the administration of step (2) is once every three weeks. In some embodiments, the administration of step (2) is once every six weeks.

[0022] In some embodiments, patients are further selected as having one or more of the following criteria: (1) age 12 years or older; (2) stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected less than 12 weeks prior to treatment; (3) no prior systemic anti-cancer therapy or radiation therapy for melanoma within the past five years; (4) no evidence of metastatic disease on staging studies; and (5) an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), a Karnofsky PS of greater than 70 (for patients over 16 years of age), or a Lansky PS of greater than 70 (for patients under 16 years of age).

[0023] Provided herein are methods for treating or inhibiting the growth of melanoma, the methods 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 antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 11 / 12, and, in combination with (a), (b) 350 mg of an anti-PD-1 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 / 2.

[0024] The administering step may occur once every three weeks. In some embodiments, the administering step occurs once every six weeks.

[0025] In some embodiments, the treatment provides a therapeutic effect selected from the group consisting of delayed melanoma growth, reduced melanoma cell count, melanoma regression, increased survival, partial response, and complete response. In some embodiments, melanoma growth may be delayed by at least 10 days compared to untreated subjects. In some embodiments, melanoma growth is inhibited by at least 50% compared to untreated subjects. In some embodiments, melanoma growth is inhibited by at least 20% compared to subjects administered either antibody as monotherapy.

[0026] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent or therapy, wherein the additional therapeutic agent or therapy is selected from the group consisting of radiation, surgery, a chemotherapeutic agent, a cancer vaccine, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a CD28 agonist, a CD38 inhibitor, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an angiopoietin-2 (Ang2) inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an antibody against a tumor-specific antigen, a Bacillus subtilisin, a steroid hormone receptor (SHR), ... The antibody is selected from the group consisting of Calmette-Guerin vaccine, granulocyte-macrophage colony-stimulating factor, oncolytic virus, cytotoxin, interleukin-6 receptor (IL-6R) inhibitor, interleukin-4 receptor (IL-4R) inhibitor, IL-10 inhibitor, IL-2, IL-7, IL-21, IL-12, IL-15, antibody-drug conjugate, GITR agonist, 4-1BB agonist, CD20xCD3 bispecific antibody, MUC16xCD3 bispecific antibody, and anti-inflammatory drug.

[0027] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (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 comprises the amino acid sequence of SEQ ID NO: 3, HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 6, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8. In some aspects, the HCVR comprises the amino acid sequence of SEQ ID NO: 1, and the LCVR comprises the amino acid sequence of SEQ ID NO: 2. In some aspects, the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0028] In some embodiments, an anti-LAG-3 antibody or antigen-binding fragment thereof comprises the heavy chain CDRs of an HCVR (HCDR1, HCDR2, and HCDR3) and the three light chain CDRs of an LCVR (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18. In some aspects, the HCVR comprises the amino acid sequence of SEQ ID NO: 11, and the LCVR comprises the amino acid sequence of SEQ ID NO: 12. In some aspects, the anti-LAG-3 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0029] Methods for treating or inhibiting the growth of melanoma are provided herein. In some embodiments, the methods include: (a) selecting a patient with melanoma, where the patient has completed surgery to treat the melanoma; and (b) administering to the patient: (1) a loading dose comprising an anti-PD-1 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 / 2 and an anti-LAG-3 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 11 / 12; and (2) one or more secondary doses, the one or more secondary doses administered 1 to 4 weeks after the immediately preceding administration. In some embodiments, the one or more secondary doses administered 3 weeks after the immediately preceding administration.

[0030] In some embodiments, the patient exhibits greater than 1% or greater LAG3 in the melanoma tissue. In some embodiments, the patient has been diagnosed with stage IV melanoma.

[0031] In some embodiments, patients are further selected as having one or more of the following criteria: (1) age 12 years or older; (2) stage IIc, III, or IV (all M stage) and histologically confirmed melanoma that was completely resected less than 12 weeks prior to treatment; (3) no prior systemic anti-cancer therapy or radiation therapy for melanoma within the past 5 years; (4) tumor tissue exhibiting 1% or more LAG3 as determined by IHC or iPET; (5) no evidence of metastatic disease; and (6) an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), a Karnofsky PS of greater than 70 (for patients over 16 years of age), or a Lansky PS of greater than 70 (for patients under 16 years of age).

[0032] In some embodiments, the method further comprises administering to a patient in need thereof (3) one or more tertiary doses, wherein the one or more tertiary doses are administered 3 to 12 weeks after the immediately preceding dose, hi some embodiments, the one or more tertiary doses are administered 3 weeks or 6 weeks after the immediately preceding dose.

[0033] In some embodiments, the initial loading dose comprises (a) 50 mg to 1500 mg of an anti-PD-1 antibody or antigen-binding fragment thereof, and (b) 50 mg to 8000 mg of an anti-LAG-3 antibody or antigen-binding fragment thereof. In some embodiments, one or more secondary doses comprise (a) 350 mg of an anti-PD-1 antibody or antigen-binding fragment thereof, and (b) 400 mg, 800 mg, 1000 mg, 1400 mg, 1600 mg, or 2000 mg of an anti-LAG-3 antibody or antigen-binding fragment thereof. In some embodiments, one or more tertiary doses comprise (a) 350 mg of an anti-PD-1 antibody or antigen-binding fragment thereof, and (b) 400 mg, 800 mg, 1000 mg, 1400 mg, 1600 mg, or 2000 mg of an anti-LAG-3 antibody or antigen-binding fragment thereof.

[0034] The various aspects provided herein may be applied to any one or each of the other methods provided herein. Illustratively, in any one of the above-described methods, a single dose of the anti-PD-1 antibody or antigen-binding fragment thereof comprises 50 to 1500 mg, or 350 mg. In any one of the above-described methods, a single dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 50 to 8000 mg, or 400 mg, or 1600 mg. In any one of the above-described methods, the anti-LAG-3 antibody or antigen-binding fragment thereof is administered before, simultaneously with, or after the anti-PD-1 antibody or antigen-binding fragment thereof. In any one of the above-described methods, the anti-LAG-3 antibody or antigen-binding fragment thereof is administered before the anti-PD-1 antibody or antigen-binding fragment thereof. In any one of the above-described methods, the anti-LAG-3 antibody or antigen-binding fragment thereof is administered on the same day as the anti-PD-1 antibody or antigen-binding fragment thereof. In any one of the above methods, two or more doses of the anti-LAG-3 antibody or antigen-binding fragment thereof are administered in combination with two or more doses of the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof comprises 350 mg. In some embodiments, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 50-8000 mg. In some embodiments, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 1600 mg. In some embodiments, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 400 mg. In any one of the above methods, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof comprises 200 mg, 250 mg, or 350 mg, and each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 400 mg, 800 mg, 1000 mg, 1400 mg, or 1600 mg. In some embodiments, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof is administered 0.5 to 12 weeks after the immediately preceding dose. In some embodiments, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof is administered 0.5 to 12 weeks after the immediately preceding dose. 18. In some embodiments, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof is administered once every 6 weeks.In some embodiments, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof is administered once every six weeks. In some embodiments, each dose of the anti-PD-1 antibody or antigen-binding fragment thereof is administered once every three weeks. In some embodiments, each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof is administered once every three weeks. In any one of the above methods, the antibody is administered intravenously, subcutaneously, or intraperitoneally. In some embodiments, the melanoma is unresectable locally advanced melanoma. In some embodiments, the melanoma is unresectable metastatic melanoma. In some embodiments, the melanoma is completely resected high-risk melanoma.

[0035] In some embodiments, the patient meets the following criteria: (i) At least 12 years of age on the date of providing informed consent; (ii) have histologically confirmed melanoma that is either stage IIC, III, or stage IV according to the AJCC 8th edition (Amin, 2017) and has been completely surgically resected to qualify; (iii) Patients with stage IIIA disease must have at least one lymph node micrometastasis measuring greater than 1 mm in greatest diameter; (iv) stage IIC melanoma confirmed by a pathology-negative sentinel lymph node biopsy (SLNB) specimen and no evidence of regional or distant metastasis; (v) Complete surgical resection performed within 12 weeks prior to randomization, with treatment permitted only after satisfactory wound healing from surgery; (vi) disease-free status as demonstrated by a complete physical examination and imaging studies prior to treatment; (vii) the patient exhibits 1% or more LAG3 in tumor tissue as determined by IHC or iPET; and (viii) Patients must not have received systemic anti-cancer therapy or radiation therapy for melanoma within the past 5 years.

[0036] In some embodiments, the patient meets the following criteria: (1) 12 years of age or older; (2) Stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected within 12 weeks prior to randomization; (3) No prior systemic anticancer therapy or radiation therapy for melanoma within the past 5 years; (4) exhibiting ≥1% LAG3 in tumor tissue as determined by IHC or iPET; (5) no evidence of metastatic disease, and (6) Further selected as having one or more of the following: an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), a Karnofsky PS of greater than 70 (for patients over 16 years of age), or a Lansky PS of greater than 70 (for patients under 16 years of age).

[0037] According to any one of the methods provided herein, the treatment provides a therapeutic effect selected from the group consisting of delayed melanoma growth, reduced melanoma cell count, melanoma regression, increased survival, partial response, and complete response. In some embodiments, melanoma growth is delayed by at least 10 days compared to untreated subjects. In some embodiments, melanoma growth is inhibited by at least 50% compared to untreated subjects. In some embodiments, melanoma growth is inhibited by at least 20% compared to subjects administered either antibody as monotherapy.

[0038] In some embodiments, the methods provided herein further comprise administering to the subject an additional therapeutic agent or therapy, wherein the additional therapeutic agent or therapy is selected from the group consisting of radiation, surgery, a chemotherapeutic agent, a cancer vaccine, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a CD28 agonist, a CD38 inhibitor, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an angiopoietin-2 (Ang2) inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an antibody against a tumor-specific antigen, a Bacillus subtilisin, a steroid hormone receptor (SHR), ... The antibody is selected from the group consisting of Calmette-Guerin vaccine, granulocyte-macrophage colony-stimulating factor, oncolytic virus, cytotoxin, interleukin-6 receptor (IL-6R) inhibitor, interleukin-4 receptor (IL-4R) inhibitor, IL-10 inhibitor, IL-2, IL-7, IL-21, IL-12, IL-15, antibody-drug conjugate, GITR agonist, 4-1BB agonist, CD20xCD3 bispecific antibody, MUC16xCD3 bispecific antibody, and anti-inflammatory drug.

[0039] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody or antigen-binding fragment thereof, comprising heavy chain complementarity determining regions (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 comprises the amino acid sequence of SEQ ID NO: 3, HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 6, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8. In some aspects, the HCVR comprises the amino acid sequence of SEQ ID NO: 1, and the LCVR comprises the amino acid sequence of SEQ ID NO: 2. In some aspects, the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0040] In some embodiments, an anti-LAG-3 antibody or antigen-binding fragment thereof comprises the heavy chain CDRs of an HCVR (HCDR1, HCDR2, and HCDR3) and the three light chain CDRs of an LCVR (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18. In some aspects, the HCVR comprises the amino acid sequence of SEQ ID NO: 11, and the LCVR comprises the amino acid sequence of SEQ ID NO: 12. In some aspects, the anti-LAG-3 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0041] According to any one of the methods provided herein, the inhibition is more effective than administration of either antibody as a monotherapy.

[0042] Other embodiments will be apparent from consideration of the detailed description that follows. [Brief explanation of the drawings]

[0043] [Figure 1A] 1 shows a research flow diagram of Example 1. [Figure 1B] See legend to Figure 1A. [Figure 2] 1 shows a research flow diagram for Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0044] Detailed Description It is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. As used herein, the term "about," when used in reference to a specific recited numerical value, means that the value may vary by 1% or less from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0046] As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). In a typical antibody, each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region comprises three domains: C H1 , C H2 , and C H3 Each light chain comprises a light chain variable region (herein referred to as LCVR or V L The light chain constant region comprises one domain (C L1 ) included. V H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of an anti-IL-4R antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0047] The term "antibody," as used herein, also includes antigen-binding fragments of intact antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, 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 may be obtained from intact antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0048] 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) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0049] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V associated with domain H In an antigen-binding fragment having a domain, V H Domain and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H or V L It may also include a domain.

[0050] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -C H1 ;(ii)V H -C H2 ;(iii)V H -C H3 ;(iv)V H -C H1 -C H2 ;(v)V H -C H1 -C H2 -C H3 ;(vi)V H -C H2 -C H3 ;(vii)V H -C L ;(viii)V L -C H1 ;(ix)V L -C H2 ;(x)V L -C H3 ;(xi)V L -CH1 -C H2 ;(xii)V L -C H1 -C H2 -C H3 ;(xiii)V L -C H2 -C H3 and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association (e.g., via disulfide bonds) of the domains.

[0051] 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 different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format can be adapted for use in the context of the antibodies or antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art. For example, the present disclosure includes methods involving the use of bispecific antibodies in which one arm of the immunoglobulin is specific for PD-1 or LAG-3, and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety. Exemplary bispecific formats that may be used in the context of the present disclosure include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific antibodies include bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein for a review of the aforementioned formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0052] The antibody used in the methods of the present disclosure may be a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may nevertheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, particularly CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0053] The antibody used in the methods of the present disclosure may be a recombinant human antibody. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector (described in more detail below) transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library (described in more detail below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., 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 into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), thereby increasing the V H Area and V L The amino acid sequence of the region is human germline V H Sequence and V LWhile derived from and related to sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0054] Although any methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, exemplary methods and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety.

[0055] General method Standard methods in molecular biology are described by Sambrook, Fritsch and Maniatis (1982 & 1989 2 nd Edition, 2001 3 rd (1993) 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 can also be found 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), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0056] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, have been described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation are described (see, e.g., 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, 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 production, purification, and fragmentation of polyclonal and monoclonal antibodies have been 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, supra). Standard techniques for characterizing ligand / receptor interactions are available (e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0057] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford University 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, 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).

[0058] An alternative method for humanization is to use human antibody libraries displayed on phage or 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). Single-chain antibodies and diabodies have been described (see, e.g., Malecki 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. Pat. No. 4,946,778).Bifunctional antibodies have been provided (e.g., 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. 272:276-23; 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 the 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.

[0059] Purification of the antigen is typically not required for antibody production. Animals can be immunized with cells bearing the antigen of interest. Spleen cells can then be isolated from the immunized animal, and the spleen cells can be fused with a myeloma cell line to produce hybridomas (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithana et al. (1999) J. Immunol. 163:5157-5164).

[0060] Antibodies can be conjugated to, for example, small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapy, diagnosis, kits, or other purposes, including antibodies conjugated to, for example, dyes, radioisotopes, enzymes, or metals, such as 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).

[0061] Methods for flow cytometry, including fluorescence-activated cell sorting (FACS), are available (see, e.g., 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, polypeptides, and antibodies, for use as diagnostic reagents, are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

[0062] Standard methods for immune system histology have been described (see, e.g., 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).

[0063] For example, software packages and databases for determining antigen fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments are available (e.g., GenBank, Vector NTI® Suite (Informax, Inc., Bethesda, Md.), GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.), DeCypher® (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 See Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0064] 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. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, 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 approaches. See, e.g., 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 are also available for identifying CDR sequences within antibodies.

[0065] PD-1 inhibitors According to certain exemplary embodiments of the present disclosure, the method comprises administering a therapeutically effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof. The term "PD-1" refers to the programmed death-1 protein, also known as CD279, a T-cell co-inhibitor. The full-length PD-1 amino acid sequence is provided in GenBank under accession number NP_005009.2. PD-1 is a member of the CD28 / CTLA-4 / ICOS family of T-cell co-inhibitors. PD-1 is a 288-amino acid protein with an IgV-like extracellular N-terminal domain, a transmembrane domain, and an intracellular domain containing immunoreceptor tyrosine-based inhibition (ITIM) motifs and immunoreceptor tyrosine-based switch (ITSM) motifs (Chattopadhyay et al. 2009, Immunol. Rev.). The PD-1 receptor has two ligands, PD-ligand-1 (PD-L1) and PD-L2.

[0066] PD-L1 is a 290-amino acid protein with an extracellular IgV-like domain, a transmembrane domain, and a highly conserved intracellular domain of approximately 30 amino acids. PD-L1 is constitutively expressed on many cells, including antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells), as well as hematopoietic and non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islets, and immune-privileged sites). PD-L1 is also expressed on a wide variety of tumors, virus-infected cells, and autoimmune tissues, where it is a component of the immunosuppressive environment (Ribas 2012, NEJM 366:2517-2519).

[0067] PD-1 inhibitors include antibodies and antigen-binding fragments thereof, 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 the present disclosure, the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that binds to PD-L1 or PD-L2.

[0068] In an embodiment of the present disclosure, the PD-1 inhibitor is an antibody or antigen-binding fragment thereof described in US 9,987,500.

[0069] According to certain embodiments, the antibodies used in the methods of the present disclosure specifically bind to PD-1. The term "specifically binds" and the like means that the antibody or antigen-binding fragment thereof forms a complex with the antigen that is relatively stable 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, and the like. For example, as used in the context of the present disclosure, an antibody that "specifically binds" to PD-1 has a K 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, as measured by a surface plasmon resonance assay. D However, an isolated antibody that specifically binds human PD-1 may exhibit cross-reactivity to other antigens, such as PD-1 molecules from other (non-human) species.

[0070] According to certain exemplary embodiments of the present disclosure, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity-determining region (CDR) comprising any of the amino acid sequences of the anti-PD-1 antibodies described in U.S. Patent No. 9,987,500.

[0071] In certain exemplary embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof that may be used in the context of the methods of the present disclosure comprises a heavy chain complementarity-determining region (HCDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 1 and a light chain complementarity-determining region (LCDR) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 2. According to certain embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), where HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 6, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8. In yet other embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof comprises a HCVR comprising SEQ ID NO: 1 and a LCVR comprising SEQ ID NO: 2. In certain embodiments, the methods of the disclosure involve the use of an anti-PD-1 antibody, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10. An exemplary antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 is the fully human anti-PD-1 antibody known as REGN2810 (cemiplimab, LIBTAYO®).

[0072] According to certain exemplary embodiments, the methods of the present disclosure involve the use of REGN2810, or a biological equivalent thereof. As used herein, the term "bioequivalent" refers to an anti-PD-1 antibody or PD-1 binding protein, or fragment thereof, that is a pharmaceutical equivalent or pharmaceutical substitute that exhibits no significant difference in rate and / or extent of absorption from REGN2810 when administered in either single or multiple doses at the same molar dose under similar experimental conditions. In the context of the present disclosure, the term refers to an antigen binding protein that binds to PD-1 that has no clinically meaningful differences in safety, purity, and / or potency from REGN2810.

[0073] Other anti-PD-1 antibodies that may be used in the context of the methods of the present disclosure include antibodies referred to or known in the art as, for example, nivolumab (U.S. Pat. No. 8,008,449), pembrolizumab (U.S. Pat. No. 8,354,509), MEDI0608 (U.S. Pat. No. 8,609,089), pidilizumab (U.S. Pat. No. 8,686,119), or any of the anti-PD-1 antibodies described in U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757, 8,354,509, 8,779,105, or 8,900587. In one embodiment of the present disclosure, the PD-1 inhibitor is as described in any of US2011 / 0008369, US2013 / 0017199, US2013 / 0022595, WO2006 / 121168, WO2009 / 1154335, WO2012 / 145493, WO2013 / 014668, WO2009 / 101611, EP2262837, and EP2504028.

[0074] Anti-PD-1 antibodies used in the context of the methods of the present disclosure may have pH-dependent binding characteristics. For example, an anti-PD-1 antibody for use in the methods of the present disclosure may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, an anti-PD-1 antibody of the present disclosure may exhibit enhanced binding to its antigen at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​below about 6.2, e.g., 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 lower. As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0075] In certain instances, "reduced binding to PD-1 at acidic pH compared to neutral pH" refers to the K of an antibody that binds to PD-1 at acidic pH. DK values ​​of antibodies that bind to PD-1 at neutral pH D For example, an antibody or antigen-binding fragment thereof may be used where the antibody or antigen-binding fragment thereof has an acidic / neutral K of about 3.0 or greater. D When a ratio is presented, it may be considered to indicate "reduced binding to PD-1 at acidic pH compared to neutral pH" for purposes of this disclosure. In certain exemplary embodiments, the acidic / neutral K ratio for an antibody or antigen-binding fragment of the disclosure is D The ratio can be about 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 more.

[0076] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for decreased (or increased) binding to a specific antigen at acidic pH compared to neutral pH. In addition, modification of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent properties. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, an antibody with decreased antigen binding at acidic pH compared to neutral pH can be obtained. As used herein, the term "acidic pH" refers to a pH of 6.0 or less.

[0077] LAG-3 inhibitors The term "LAG-3" refers to the lymphocyte-activation gene-3 protein, also known as CD223, an immune checkpoint receptor or T cell co-inhibitor. The full-length LAG-3 amino acid sequence is provided 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 1 transmembrane protein with four extracellular Ig-like domains, D1-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).

[0078] As used herein, the term "T cell co-inhibitor" refers to ligands and / or receptors that regulate immune responses through T cell activation or suppression. The term "T cell co-inhibitor" is also known as T cell co-signaling molecules, including, but not limited to, programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), CD-28, 2B4, LY108, T cell immunoglobulin and mucin 3 (TIM3), T cell immunoglobulin and T cell immunoreceptor with 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), V-domain Ig inhibitor of T cell activation (VISTA), and CD160.

[0079] LAG-3 inhibitors include antibodies and antigen-binding fragments thereof, as well as other substances (e.g., peptides and small molecules) that specifically bind to LAG-3 and antagonize one or more biological activities of LAG-3. Molecules that specifically bind to LAG-3 may be referred to as "anti-LAG-3."

[0080] In an embodiment of the present disclosure, the LAG-3 inhibitor is an antibody or antigen-binding fragment thereof described in US2017 / 0101472.

[0081] According to certain embodiments, the antibody used in the methods of the present disclosure specifically binds to LAG-3. The term "specifically binds" or the like means that the antibody or antigen-binding fragment thereof forms a complex with the antigen that is relatively stable 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, and the like. For example, as used in the context of the present disclosure, an antibody that "specifically binds" to LAG-3 has a K 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 as measured by a surface plasmon resonance assay. D However, an isolated antibody that specifically binds human LAG-3 may have cross-reactivity to other antigens, such as LAG-3 molecules from other (non-human) species.

[0082] According to certain exemplary embodiments of the present disclosure, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity determining region (CDR) comprising any of the amino acid sequences of the anti-LAG-3 antibodies described in US2017 / 0101472.

[0083] In certain exemplary embodiments, an anti-LAG-3 antibody or antigen-binding fragment thereof that can be used in the context of the methods of the present disclosure comprises a heavy chain complementarity-determining region (HCDR) of the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 11 and a light chain complementarity-determining region (LCDR) of the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 12. According to certain embodiments, an anti-LAG-3 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18. In still other embodiments, the anti-LAG-3 antibody, or antigen-binding fragment thereof, comprises an HCVR comprising SEQ ID NO: 11 and an LCVR comprising SEQ ID NO: 12. In certain embodiments, the methods of the disclosure comprise the use of an anti-LAG-3 antibody, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-LAG-3 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. An exemplary antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12 is the fully human anti-LAG-3 antibody known as REGN3767 (fianlimab).

[0084] According to certain exemplary embodiments, the methods of the present disclosure include the use of REGN3767, or a biological equivalent thereof. As used herein, the term "bioequivalent" refers to an anti-LAG-3 antibody or LAG-3 binding protein, or fragment thereof, that is a pharmaceutical equivalent or pharmaceutical substitute that exhibits no significant difference in rate and / or extent of absorption from REGN3767 when administered in either single or multiple doses at the same molar dose under similar experimental conditions. In the context of the present disclosure, this term refers to an antigen-binding protein that binds to LAG-3 that has no clinically meaningful differences from REGN3767 in safety, purity, and / or efficacy.

[0085] Other anti-LAG-3 antibodies that may be used in the context of the methods of the present disclosure include, for example, antibodies referred to and known in the art as rialtimab (U.S. Publication No. 2011 / 0150892), LAG525 (WO2017 / 037203), GSK2831781 (US2016 / 0017037), Sym022 (WO2018 / 069500), INCAGN02385 (US2018 / 0127499), or antibodies described in U.S. Patent / Publication Nos. 5,976,877, 6,143,273, 6,197,524, 8,551,481, 2011 / 0070238, 2011 / 0150892. , Patent Publication Nos. 2013 / 0095114, 2014 / 0093511, 2014 / 0127226, 2014 / 0286935, as well as any of the anti-LAG-3 antibodies described in WO95 / 30750, WO97 / 03695, WO98 / 58059, WO2004 / 078928, WO2008 / 132601, WO2010 / 019570, WO2014 / 008218, EP0510079B1, EP0758383B1, EP0843557B1, EP0977856B1, EP1897548B2, EP2142210A1, and EP2320940B1.

[0086] Methods for treating melanoma or inhibiting melanoma tumor growth The present disclosure includes methods for treating, ameliorating, or reducing the severity of at least one symptom or sign, or inhibiting the growth of melanoma in a subject. The methods according to this embodiment include administering to a subject in need thereof an antibody or antigen-binding fragment thereof that specifically binds PD-1 in combination with an antibody or antigen-binding fragment thereof that specifically binds LAG-3. In some embodiments, the methods include administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds PD-1 in combination with a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds LAG-3. As used herein, the terms "treat," "treating," and the like refer to alleviating symptoms, eliminating the cause of symptoms, either temporarily or permanently, slowing or inhibiting the growth of melanoma, reducing melanoma cell mass or tumor burden, promoting melanoma regression, causing shrinkage, necrosis, and / or disappearance of melanoma, preventing recurrence of melanoma, and / or prolonging the survival of a subject.

[0087] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal exhibiting one or more symptoms or signs of melanoma and / or a human or non-human mammal diagnosed with melanoma and in need of treatment therefor. 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 signs including, but not limited to, enlarged lymph nodes, abdominal bloating, unexplained pain, chest pain / tightness, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, enlarged spleen, and itching. In certain embodiments, the manifestations include a human subject with unresectable locally advanced melanoma or metastatic melanoma and in need of treatment therefor. In some embodiments, the human subject has not received prior systemic treatment for advanced disease. In certain embodiments, the manifestations include a human subject with completely resected high-risk melanoma in the adjuvant setting and in need of treatment therefor. In certain embodiments, the phrase "subject in need thereof" includes patients with melanoma that is resistant, refractory, or inadequately controlled to prior therapy (e.g., treatment with conventional anti-cancer agents, or therapies such as radiation, chemotherapy, or surgery, or treatment with anti-cancer biologics). For example, the phrase includes subjects who have been treated with a PD-1 or PD-L1 inhibitor (e.g., an anti-PD-1 antibody). The phrase also includes subjects with melanoma for which conventional anti-cancer therapy is not advisable, e.g., due to toxic side effects. For example, the phrase includes patients who have received one or more cycles of chemotherapy with toxic side effects. In certain embodiments, the phrase "subject in need thereof" includes patients with melanoma that has been treated but has subsequently recurred or metastasized. For example, a patient with melanoma who has been treated with one or more anti-cancer agents and may have experienced regression of the melanoma, but has subsequently recurred with melanoma that is resistant to the one or more anti-cancer agents (e.g., chemotherapy-resistant melanoma), is treated with the methods provided herein.

[0088] In some cases, patients with unresectable locally advanced or metastatic melanoma meet the following criteria: (i) At least 12 years of age on the date of providing informed consent; (ii) have histologically confirmed melanoma that is either stage IIC, III, or stage IV according to the AJCC 8th edition (Amin, 2017) and has been completely surgically resected to qualify; (iii) Patients with stage IIIA disease must have at least one lymph node micrometastasis measuring greater than 1 mm in greatest diameter; (iv) stage IIC melanoma confirmed by a pathology-negative SLNB specimen and no evidence of regional or distant metastasis; (v) complete surgical resection performed within 12 weeks prior to treatment, and enrollment may occur only after satisfactory wound healing from surgery; (vi) disease-free status documented by a complete physical examination and imaging studies within 4 weeks prior to treatment; (vii) For patients with abnormal or suspicious findings on screening scans, residual malignancy and / or metastatic disease is: 18 Must be ruled out by F-FDG PET-CT and / or biopsy, and (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 had CNS RT after definitive resection. Adjuvant radiation therapy to the primary site and / or nodal base after definitive surgical resection is permitted if it is considered institutional standard of care.

[0089] In some cases, having a completely resected high-risk melanoma in the adjuvant setting meets the following criteria: (1) 12 years of age or older; (2) Stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected less than 12 weeks prior to treatment; (3) No prior systemic anticancer therapy or radiation therapy for melanoma within the past 5 years; (4) no evidence of metastatic disease on staging; and (5) Further selected as having one or more of the following: an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), a Karnofsky PS of greater than 70 (for patients over 16 years of age), or a Lansky PS of greater than 70 (for patients under 16 years of age).

[0090] The phrase "subject in need thereof" also includes subjects at risk of developing melanoma, such as those with a family history of melanoma, those with a previous occurrence of melanoma, or those with a compromised immune system. In some embodiments, the subject is resistant or inadequately responsive to a prior therapy, or has relapsed after a prior therapy.

[0091] In certain embodiments, the methods provided herein may be used to treat patients who exhibit elevated levels of one or more cancer-associated biomarkers (e.g., PD-L1 or LAG-3). For example, a method of the invention comprises administering a therapeutically effective amount of an anti-LAG-3 antibody in combination with an anti-PD-1 antibody to a patient with elevated levels of LAG-3 and / or PD-L1. In one embodiment, the method is used in a patient with melanoma who is selected based on LAG-3 expression in the cancer tissue, wherein the cancer tissue comprises melanoma cells and tumor-infiltrating immune cells. In certain embodiments, the method is used to treat a patient with melanoma who is selected based on LAG-3 expression in the cancer tissue and / or immune cells of 1% or more. In one embodiment, the method is used in a patient with melanoma who is selected based on LAG-3 expression in the cancer tissue, wherein the cancer tissue comprises melanoma cells and tumor-infiltrating immune cells. In certain embodiments, the method is used to treat a patient with melanoma who is selected based on LAG-3 expression in the cancer tissue and / or immune cells of 1% or more. 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 certain embodiments, LAG-3 expression in tumor tissues is determined by any assay known in the art, for example, by ELISA assay or by immunohistochemistry (IHC) assay (e.g., as described in He et al. 2017, J. Thoracic Oncol. 12:814-823, WO2016 / 124558, or WO2016 / 191751). In certain embodiments, LAG-3 or PD-L1 expression is determined by quantifying RNA expression, for example, by in situ hybridization or RT-PCR.In certain embodiments, LAG-3 expression is determined by imaging with a labeled anti-LAG-3 antibody, e.g., by immunopositron emission tomography or iPET (see, e.g., The Oncologist, 12:1379 (2007); Journal of Nuclear Medicine, 52(8):1171 (2011); U.S. Patent Application Publication No. 2018 / 0228926). In certain embodiments, PD-L1 expression is determined by imaging with a labeled anti-PD-L1 antibody, e.g., by immunopositron emission tomography or iPET (U.S. Patent Application Publication No. 2018 / 0161464).

[0092] In certain embodiments, the methods provided herein are used in a subject with cancer. The terms "tumor," "cancer," and "malignant tumor" are used interchangeably herein.

[0093] In certain embodiments, the cancer or tumor is melanoma. References throughout to "tumor" or "cancer" include melanoma, e.g., the tumor or cancer is melanoma. In some aspects, the melanoma is unresectable locally advanced melanoma. In some aspects, the melanoma is metastatic melanoma. In some aspects, the patient has not received prior systemic treatment for advanced disease. In other aspects, the melanoma is a completely resected high-risk melanoma. In some aspects, the melanoma is a completely resected high-risk melanoma in the adjuvant setting.

[0094] According to certain embodiments, the present disclosure includes methods for treating melanoma tumors or slowing or inhibiting the growth of melanoma tumors. In certain embodiments, this includes methods for promoting melanoma regression. In certain embodiments, this includes methods for reducing tumor cell burden or reducing tumor burden. In certain embodiments, the present disclosure includes methods for preventing melanoma recurrence. The method according to this aspect includes sequentially administering to a subject in need thereof a therapeutically effective amount of an anti-PD-1 antibody in combination with an anti-LAG-3 antibody, wherein each antibody is administered to the subject in multiple doses, e.g., as part of a particular therapeutic dosing regimen. For example, a therapeutic dosing regimen may involve administering one or more doses of an anti-PD-1 antibody about once daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every month, once every 6 weeks, once every 2 months, once every 3 months, once every 4 months, or less frequently. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered in combination with one or more therapeutically effective doses of an anti-LAG-3 antibody, and the one or more doses of the anti-LAG-3 antibody are administered to a subject about once per day, once every two days, once every three days, once every four days, once every five days, once every six days, once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every six weeks, once every two months, once every three months, once every four months, or less frequently.

[0095] In certain embodiments, the present disclosure includes a method for inhibiting, slowing, or halting melanoma metastasis or invasion into peripheral organs. The method according to this aspect includes administering a therapeutically effective amount of an anti-PD-1 antibody to a subject in need thereof. In certain embodiments, the anti-PD-1 antibody is administered in combination with an anti-LAG-3 antibody.

[0096] In certain embodiments, the present disclosure provides methods for increasing anti-tumor efficacy or increasing melanoma inhibition, hi certain embodiments, the methods provide an increase in melanoma inhibition of, e.g., 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 administered either antibody as monotherapy.

[0097] Methods provided herein, according to certain embodiments, include administering a therapeutically effective amount of an anti-PD-1 antibody to a subject with melanoma prior to, concurrently with, or after administering a therapeutically effective amount of an anti-LAG-3 antibody. In some aspects, the anti-PD-1 antibody may be administered about 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 before the anti-LAG-3 antibody. In some aspects, the anti-PD-1 antibody and the anti-LAG-3 antibody are administered simultaneously, or within 30 minutes, or within 60 minutes, or within 2 hours, or within 3 hours, or within 1 day of each other.

[0098] In certain embodiments, the methods provided herein comprise administering a therapeutically effective amount of an anti-PD-1 antibody to a subject with melanoma. In certain embodiments, the melanoma is indolent or aggressive. In certain embodiments, the subject has not responded to or has relapsed after a prior therapy. The prior therapy may include surgery, radiation, and / or chemotherapy, or treatment with a PD-1 inhibitor, a PD-L1 inhibitor, and / or any other anti-cancer biologic.

[0099] In certain embodiments, the methods of the disclosure comprise administering an anti-PD-1 antibody in combination with an anti-LAG-3 antibody to a subject in need thereof as a "first-line" treatment (e.g., initial treatment). In other embodiments, the anti-PD-1 antibody in combination with an anti-LAG-3 antibody is administered as a "second-line" treatment (e.g., after prior therapy). For example, the anti-PD-1 antibody in combination with an anti-LAG-3 antibody is administered as a "second-line" treatment to a subject who has relapsed after prior therapy, e.g., with chemotherapy or rituximab.

[0100] In certain embodiments, the methods of the present disclosure are used to treat patients with MRD-positive disease. Minimal residual disease (MRD) refers to the small number of cancer cells that remain in a patient during or after treatment, and the patient may or may not exhibit symptoms or signs of disease. If not removed, these residual cancer cells often lead to disease recurrence. The present disclosure includes methods for inhibiting and / or removing residual cancer cells in patients at the time of MRD testing. MRD can be assayed according to methods known in the art (e.g., MRD flow cytometry). The method according to this aspect of the disclosure includes administering an anti-PD-1 antibody in combination with an anti-LAG-3 antibody to a subject in need thereof.

[0101] Certain embodiments of the methods disclosed herein include 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, such as radiation, chemotherapy, surgery, a cancer vaccine, CART, a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody), a CD3 inhibitor, a CD20 inhibitor, a CTLA-4 inhibitor, a CD38 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an antibody against a tumor-specific antigen (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA)), or a Bacillus subtilis. The agent may be selected from the group consisting of Calmette-Guerin vaccine, granulocyte macrophage colony-stimulating factor, oncolytic virus, cytotoxin, CD28 agonist, GITR agonist, 4-1BB agonist, CD20xCD3 bispecific antibody (e.g., REGN1979), MUC16xCD3 bispecific antibody, vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), vaccine (e.g., Bacillus Calmette-Guerin), granulocyte macrophage colony-stimulating factor, cytotoxin, chemotherapeutic agent, cytokine such as IL-6R inhibitor, IL-4R inhibitor, IL-10 inhibitor, IL-2, IL-7, IL-12, IL-21, and IL-15, anti-inflammatory agent such as corticosteroid, and non-steroidal anti-inflammatory agent.

[0102] In certain embodiments, the antibody may be administered in combination with a therapy including a chemotherapy agent, radiation, or surgery. As used herein, the phrase "in combination with" means that the antibody is administered to the subject simultaneously with, immediately before, or immediately after the administration of a third therapeutic agent. In certain embodiments, the third therapeutic agent is administered as a co-formulation with the antibody. In related embodiments, the present disclosure includes methods comprising administering a therapeutically effective amount of an anti-PD-1 antibody in combination with an anti-LAG-3 antibody to a subject receiving a background anti-cancer treatment regimen. The background anti-cancer treatment regimen may include, for example, the administration of a series of chemotherapy agents or radiation. The anti-PD-1 antibody in combination with the anti-LAG-3 antibody may be added at the beginning of the background anti-cancer treatment regimen. In some embodiments, the antibody is added as part of a "background step-down" scheme, in which the background anti-cancer therapy is gradually withdrawn from the subject over time (e.g., in a stepwise manner) while the antibody is administered to the subject at a constant dose, or at increasing or decreasing doses over time.

[0103] In certain embodiments, the methods of the disclosure comprise administering to a subject in need thereof a therapeutically effective amount of an anti-PD-1 antibody in combination with a therapeutically effective amount of an anti-LAG-3 antibody, wherein administration of the antibodies leads to increased inhibition of melanoma growth. In certain 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 administered either antibody as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody and / or an anti-LAG-3 antibody to a subject leads to increased melanoma regression, tumor shrinkage, and / or elimination. In certain embodiments, administration of an anti-PD-1 antibody and / or an anti-LAG-3 antibody leads to a delay in melanoma growth and progression; e.g., melanoma growth may be delayed by about 3 days, more than 3 days, about 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 an untreated subject or a subject treated with either antibody as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody in combination with an anti-LAG-3 antibody prevents melanoma recurrence and / or extends subject survival; e.g., 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 compared to an untreated subject or a subject treated with either antibody as monotherapy. In certain embodiments, administration of the combined antibodies extends progression-free survival or overall survival. In certain embodiments, administration of an anti-PD-1 antibody in combination with an anti-LAG-3 antibody increases the response and duration of response in a subject by, for example, 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 untreated subjects or subjects receiving either antibody as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody and / or an anti-LAG-3 antibody to a subject with melanoma leads to the complete disappearance of all signs of melanoma cells (a "complete response"). In certain embodiments, administration of an anti-PD-1 antibody and / or an anti-LAG-3 antibody to a subject with melanoma leads to at least a 30% or greater reduction in melanoma cells or tumor size (a "partial response").In certain embodiments, administration of an anti-PD-1 antibody and / or an anti-LAG-3 antibody to a subject with melanoma leads to the complete or partial disappearance of melanoma cells / lesions, including new measurable lesions. Melanoma tumor reduction 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), cytology, histology, or molecular genetic analysis. In some aspects, administration of an anti-PD-1 antibody in combination with an anti-LAG-3 antibody to a patient population unexpectedly results in more patients responding to treatment, unexpectedly results in patients responding longer to treatment even if more patients do not respond, and / or patients who respond to therapy have a more robust response.

[0104] In certain embodiments, the administered antibody combination is safe or well tolerated by patients, with no increase or a tolerable increase in serious side effects compared to patients receiving either antibody as monotherapy.

[0105] Combination therapy The disclosed methods, according to certain embodiments, include administering an anti-LAG-3 antibody in combination with an anti-PD-1 antibody to a subject. In certain embodiments, the disclosed methods include administering the antibodies for additive or synergistic activity to treat melanoma. As used herein, the term "in combination with" means that the anti-LAG-3 antibody is administered before, after, or simultaneously with the anti-PD-1 antibody. The term "in combination with" also includes sequential or simultaneous administration of the anti-PD-1 antibody and the anti-LAG-3 antibody. For example, when administered "before" an 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 before administration of the anti-LAG-3 antibody. When administered "after" an anti-LAG-3 antibody, the anti-PD-1 antibody may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or more than 72 hours after administration of the anti-LAG-3 antibody. Administration "concurrently" with an anti-LAG-3 antibody means that the anti-PD-1 antibody is administered to the subject in a separate dosage form within less than 5 minutes (before, after, or simultaneously) of administration of the anti-LAG-3 antibody, e.g., within 5 minutes of completion of the anti-LAG-3 antibody infusion, or is administered to the subject as a single combined dosage formulation containing both the anti-PD-1 antibody and the anti-LAG-3 antibody. In some embodiments, the anti-PD-1 antibody is administered on the same day as the anti-LAG-3 antibody. In some embodiments, the anti-PD-1 antibody and the anti-LAG-3 antibody are in separate dosage forms but are administered within 8 hours of each other, e.g., within 6 hours, or within 5 hours, or within 4 hours, or within 3 hours, or within 2 hours, or within 60 minutes of each other.

[0106] In certain embodiments, the methods provided herein include administering a third therapeutic agent, wherein the third therapeutic agent is an anti-cancer agent. As used herein, "anti-cancer agent" refers to any agent useful in treating cancer, including, but not limited to, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotics, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radioactive agents. As used herein, "cytotoxin or cytotoxic agent" also refers to chemotherapeutic agents and refers to any agent that is harmful to cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinbiastine, coichicin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydroterotosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. In certain embodiments, the methods provided herein may be administered in combination with other therapeutic agents, including radiation, surgery, a cancer vaccine, a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody), a CD20 inhibitor, a CD3 inhibitor, a CTLA-4 inhibitor (e.g., ipilimumab), a CD38 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an antagonist of another T cell co-inhibitor or ligand (e.g., an antibody against CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., aflibercept or steroids described in U.S. Patent No. 7,087,513, or ...Other VEGF inhibitory fusion proteins, such as "VEGF-Trap" as described in US Pat. No. 411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib), Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), agonists for costimulatory receptors (e.g., Glycerin, Glycerol), and the like. agonists for glucocorticoid-induced TNFR-associated 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, CD20×CD3 bispecific antibodies (e.g., REGN1979), MUC16×CD3 bispecific antibodies, vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), vaccines (e.g., Bacillus Calmette-Guérin, cancer vaccines), adjuvants that increase antigen presentation (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., sarilumab), 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 ADC, and anti-DS6-DM4 ADC). ADC), chimeric antigen receptor T cells (e.g., CD19-targeted T cells) or other cell therapy, and administration of a third therapeutic agent selected from the group consisting of anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs).

[0107] In certain embodiments, the methods provided herein involve administering an anti-PD-1 antibody and an anti-LAG-3 antibody in combination with radiation therapy / chemotherapy to generate a long-term durable anti-tumor response and / or enhance survival in patients with melanoma.

[0108] In some embodiments, the methods of the disclosure include administering radiation therapy before, concurrently with, or after administering an anti-PD-1 antibody and an anti-LAG-3 antibody to a melanoma patient. For example, radiation therapy may be administered in one or more doses to melanoma lesions after administration of one or more doses of the antibodies. In some embodiments, radiation therapy may be administered locally to melanoma lesions before or after systemic administration of an anti-PD-1 antibody and / or an anti-LAG-3 antibody to enhance the local immunogenicity of the patient's melanoma (adjuvant radiation) and / or kill melanoma cells (ablative radiation). In certain embodiments, the antibodies may be administered in combination with radiation therapy and a chemotherapeutic agent (e.g., temozolomide or cyclophosphamide) or a VEGF antagonist (e.g., aflibercept).

[0109] Pharmaceutical Compositions and Administration Methods are provided herein that involve administering an anti-PD-1 antibody in combination with an anti-LAG-3 antibody to a subject, where the antibodies are contained in separate or combined (single) pharmaceutical compositions. The pharmaceutical compositions of the present disclosure may be formulated with suitable carriers, excipients, and other agents that provide for suitable transport, delivery, tolerability, etc. Numerous suitable formulations can be found in formularies known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols 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.

[0110] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure. For example, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (See, e.g., 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 by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents.

[0111] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In one embodiment, the syringe is a pre-filled syringe. Further, for subcutaneous delivery, pen delivery devices are readily adapted to deliver the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device comes pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0112] In certain circumstances, pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed in the vicinity of the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0113] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose, and other auxiliary agents, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injection solution prepared in this manner is preferably filled into an appropriate ampule.

[0114] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for the dosage of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0115] Dosing regimen The present disclosure includes methods comprising administering to a subject an anti-PD-1 antibody at a dosing frequency of about 4 times per week, twice per week, once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 8 weeks, once per 12 weeks, or less frequently, so long as a therapeutic response is achieved. In certain embodiments, the present disclosure includes methods comprising administering to a subject an anti-LAG-3 antibody at a dosing frequency of about 4 times per week, twice per week, once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 8 weeks, once per 12 weeks, or less frequently, so long as a therapeutic response is achieved. In certain embodiments, the methods involve administering an anti-PD-1 antibody in combination with an anti-LAG-3 antibody at a dosing frequency of about 4 times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 9 weeks, once every 12 weeks, or less frequently, so long as a therapeutic response is achieved.

[0116] According to certain embodiments of the present disclosure, multiple doses of an anti-PD-1 antibody in combination with an anti-LAG-3 antibody may be administered to a subject over a defined time course. Methods according to this aspect of the disclosure include sequentially administering one or more doses of an anti-PD-1 antibody in combination with one or more doses of an anti-LAG-3 antibody to a subject. As used herein, "sequentially administering" means that each dose of antibody is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering to a patient a single initial dose of an anti-PD-1 antibody, followed by one or more secondary doses of an anti-PD-1 antibody, and optionally followed by one or more tertiary doses of an anti-PD-1 antibody. In certain embodiments, the method further comprises sequentially administering to the patient a single initial dose of an anti-LAG-3 antibody, followed by one or more secondary doses of an anti-LAG-3 antibody, and optionally followed by one or more tertiary doses of an anti-LAG-3 antibody.

[0117] According to certain embodiments of the present disclosure, multiple doses of an anti-PD-1 antibody and an anti-LAG-3 antibody may be administered to a subject over a defined time course. The method according to this aspect of the disclosure includes sequentially administering multiple doses of an anti-PD-1 antibody and an anti-LAG-3 antibody to a subject. As used herein, "sequentially administering" means that each dose of an anti-PD-1 antibody in combination with an anti-LAG-3 antibody is administered to the subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months).

[0118] According to certain embodiments of the present disclosure, multiple doses of an anti-LAG-3 antibody may be administered to a subject once every 3 or 6 weeks for several months or years, and then the subject is administered an anti-PD-1 antibody in combination with the anti-LAG-3 antibody for several months or years. In some aspects, the anti-LAG-3 antibody dosing differs between monotherapy and combination therapy. In some aspects, the anti-LAG-3 antibody dosing is the same whether administered as monotherapy or in combination with an anti-PD-1 antibody.

[0119] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of antibody (anti-PD-1 antibody or anti-LAG-3 antibody). However, in certain embodiments, the amounts contained in the initial, secondary, and / or tertiary doses differ from one another (e.g., adjusted upward or downward) during the course of treatment. In certain embodiments, one or more doses (e.g., 1, 2, 3, 4, or 5) are administered at the beginning of a treatment regimen as a "loading dose," followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis. For example, an anti-PD-1 antibody may be administered to a patient with melanoma at a loading dose of about 1-20 mg / kg, followed by one or more maintenance doses of about 3 mg / kg of patient body weight.

[0120] In one exemplary embodiment of the present disclosure, each secondary dose and / or tertiary dose is administered ½ to 14 weeks after the immediately preceding dose (e.g., ½ week, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks, 8.5 weeks, 9 weeks, 9.5 weeks, 10 weeks, 10.5 weeks, 11 weeks, 11.5 weeks, 12 weeks, 12.5 weeks, 13 weeks, 13.5 weeks, 14 weeks, 14.5 weeks, or more). As used herein, the phrase "immediately preceding dose" refers to a dose of an anti-PD-1 antibody (and / or anti-LAG-3 antibody) administered to a patient prior to the administration of the immediately succeeding dose in a multiple-administration series, without any intervening doses.

[0121] Methods according to some aspects may include administering any number of secondary and / or tertiary doses of an anti-PD-1 antibody (and / or anti-LAG-3 antibody) to a patient. For example, in certain embodiments, only a single secondary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to a patient. Similarly, in certain embodiments, only a single tertiary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to a patient.

[0122] In embodiments comprising 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 1-2 weeks after the immediately preceding dose. Similarly, in embodiments comprising 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 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment, depending on the needs of the individual patient after clinical testing.

[0123] In certain embodiments, one or more doses of the anti-PD-1 antibody and / or anti-LAG-3 antibody are administered more frequently (e.g., twice weekly, once weekly, or once every two weeks) at the start of the treatment regimen as an "induction dose," followed by subsequent doses ("bolstering doses" or "maintenance doses") administered less frequently (e.g., once every 4-12 weeks).

[0124] Contemplated herein are several embodiments of co-administration of an anti-PD-1 antibody and an anti-LAG-3 antibody administered in separate doses at similar or different frequencies compared to the anti-PD-1 antibody. In some embodiments, the anti-LAG-3 antibody is administered before, after, or simultaneously with the anti-PD-1 antibody. In certain embodiments, the anti-LAG-3 antibody is administered in a single dosage formulation with the anti-PD-1 antibody.

[0125] The present disclosure includes methods comprising sequentially administering an anti-PD-1 antibody in combination with an anti-LAG-3 antibody to a patient to treat melanoma. In some embodiments, the method comprises administering one or more doses of an anti-PD-1 antibody, followed by one or more doses of an anti-LAG-3 antibody. In certain embodiments, the method comprises administering a single dose of an anti-PD-1 antibody, followed by one or more doses of an anti-LAG-3 antibody. In some embodiments, one or more doses of about 0.1 mg / kg to about 20 mg / kg of an anti-PD-1 antibody, followed by one or more doses of about 0.1 mg / kg to about 50 mg / kg of an anti-LAG-3 antibody may be administered to inhibit melanoma growth and / or prevent melanoma recurrence in a subject with melanoma. In some embodiments, an anti-PD-1 antibody is administered in one or more doses, followed by one or more doses of an anti-LAG-3 antibody, resulting in increased anti-tumor efficacy (e.g., greater inhibition of melanoma growth, increased prevention of melanoma recurrence compared to untreated subjects or subjects receiving either antibody as monotherapy).

[0126] The present disclosure also includes methods comprising sequentially administering an anti-LAG-3 antibody in combination with an anti-PD-1 antibody to a patient to treat melanoma. In some embodiments, the method comprises administering one or more doses of an anti-LAG-3 antibody, followed by one or more doses of an anti-PD-1 antibody. In certain embodiments, the method comprises administering a single dose of an anti-LAG-3 antibody, followed by one or more doses of an anti-PD-1 antibody. In some embodiments, one or more doses of about 0.1 mg / kg to about 50 mg / kg of an anti-LAG-3 antibody, followed by one or more doses of about 0.1 mg / kg to about 20 mg / kg of an anti-PD-1 antibody may be administered to inhibit tumor growth and / or prevent recurrence of melanoma in a subject with melanoma. In some embodiments, one or more doses of about 50 mg to about 8000 mg of an anti-LAG-3 antibody may be administered to inhibit melanoma growth and / or prevent melanoma recurrence in a subject with melanoma, followed by one or more doses of about 50 mg to about 1500 mg of an anti-PD-1 antibody. In some embodiments, administration of one or more doses of an anti-LAG-3 antibody, followed by one or more doses of an anti-PD-1 antibody, results in increased anti-tumor efficacy (e.g., greater inhibition of melanoma growth, increased prevention of melanoma recurrence compared to untreated subjects or subjects administered either antibody as monotherapy).

[0127] Dosage The amount of anti-PD-1 antibody and / or anti-LAG-3 antibody administered to a subject according to the methods of the present disclosure is generally a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to an amount of antibody (anti-PD-1 antibody or anti-LAG-3 antibody) that results in one or more of the following: (a) a reduction in melanoma severity or duration of symptoms; (b) inhibition of melanoma growth or increased melanoma cell necrosis, melanoma tumor shrinkage and / or melanoma tumor elimination; (c) delay in melanoma growth and progression; (d) inhibition, slowing, or halt of melanoma metastasis; (e) prevention of recurrence of melanoma growth; (f) increased survival of subjects with melanoma; and / or (g) a reduction in the use of or need for conventional anti-cancer therapy (e.g., reduction or elimination of the use of chemotherapeutic or cytotoxic agents), compared to untreated subjects or subjects administered either antibody as monotherapy.

[0128] In the case of an anti-PD-1 antibody, the therapeutically effective amount is 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, or about 320 mg. , about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg of the anti-PD-1 antibody. In certain embodiments, 350 mg of anti-PD-1 antibody is administered. In certain embodiments, 1050 mg of anti-PD-1 antibody is administered.

[0129] In the case of an anti-LAG-3 antibody, the therapeutically effective amount is about 10 mg to about 8000 mg, for example, about 10 mg, about 20 mg, about 50 mg, about 70 mg, about 100 mg, about 120 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about The amount of anti-LAG-3 antibody may be 1050 mg, about 1100 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 2000 mg, about 2050 mg, about 2100 mg, about 2200 mg, about 2500 mg, about 2700 mg, about 2800 mg, about 2900 mg, about 3000 mg, about 3200 mg, about 4000 mg, about 5000 mg, about 6000 mg, about 7000 mg, or about 8000 mg.

[0130] The amount of either anti-PD-1 antibody or anti-LAG-3 antibody contained within an individual dose may be expressed in milligrams of antibody per kilogram of subject body weight (i.e., mg / kg). In certain embodiments, either the anti-PD-1 antibody or anti-LAG-3 antibody used in the methods of the present disclosure may be administered to a subject at a dose of about 1 to about 50 mg / kg of subject body weight. For example, an anti-PD-1 antibody may be administered at a dose of about 0.1 mg / kg of subject body weight to about 20 mg / kg of subject body weight. An anti-LAG-3 antibody may be administered at a dose of about 0.1 mg / kg of subject body weight to about 50 mg / kg of subject body weight. [Example]

[0131] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. The compositions and methods described in the examples form part of the present disclosure.

[0132] Therapeutic monoclonal antibodies (mAbs) targeting immune inhibitory receptors (e.g., cytotoxic T-lymphocyte-associated protein 4 [CTLA-4] and programmed cell death 1 [PD-1]) have demonstrated excellent clinical activity with acceptable benefit-to-risk ratios in several tumor types (Topalian, 2014, Wolchok, 2013, Larkin, 2015a, Baksh, 2015). However, durable responses were achieved in only a minority of patients, suggesting that combination approaches may be required to overcome tumor immune inhibitory mechanisms (Topalian, 2012).

[0133] In the following examples, this antibody (REGN3767 (INN: fianlimab), an antibody against LAG-3 that blocks LAG-3 / MHC II-mediated T cell inhibition, was tested in combination with cemiplimab (REGN2810), an antibody against the PD-1 receptor that blocks PD-1 / programmed death-ligand 1 (PD-L1)-mediated T cell inhibition. LIBTAYO® (INN: cemiplimab, known in the U.S. as cemiplimab rwlc) has been approved by several health authorities for the treatment of patients with different tumor types.

[0134] Example 1: Phase 3 Clinical Trial of Anti-LAG3 (REGN3767, fianlimab) and Anti-PD-1 (REGN2810, cemiplimab) Compared to Pembrolizumab in the Adjuvant Setting in Patients with Completely Resected High-Risk Melanoma This phase 3 study evaluates the combination of adjuvant fianlimab and cemiplimab compared with pembrolizumab in patients with completely resected high-risk melanoma.

[0135] the purpose The primary objective of the study is to demonstrate superiority of fianlimab plus cemiplimab compared with pembrolizumab as measured by recurrence-free survival (RFS).

[0136] Secondary objectives include: To demonstrate superiority of fianlimab plus cemiplimab compared with pembrolizumab as measured by overall survival (OS). To demonstrate superiority of fianlimab plus cemiplimab compared with pembrolizumab as measured by melanoma-specific survival (MSS). To evaluate whether adjuvant postoperative therapy improves distant metastasis-free survival (DMFS) in patients with stage IIC or III disease receiving fianlimab plus cemiplimab compared with pembrolizumab. To assess the impact of fianlimab plus cemiplimab compared with pembrolizumab on quality of life in adults. To assess the safety and tolerability of fianlimab plus cemiplimab compared with pembrolizumab. To characterize the pharmacokinetics (PK) of fianlimab plus cemiplimab using sparse PK sampling in patients aged 12 years and older. To evaluate the immunogenicity of fianlimab and its immunogenicity relative to cemiplimab.

[0137] Study design This randomized, double-blind, phase 3 study in patients aged 12 years and older with completely resected high-risk melanoma in the adjuvant setting has three arms. Arm A: fianlimab (1600 mg) + cemiplimab (350 mg) IV every 3 weeks (Q3W) Group B: fianlimab (400 mg) + cemiplimab (350 mg) Q3W IV Arm C: Pembrolizumab (200 mg) + saline / dextrose placebo Q3W IV

[0138] Patients will be randomized 1:1:1 into Arm A, Arm B, and Arm C. Patients will have no prior systemic therapy for melanoma and will have completely resected stage IIC, III, or IV melanoma (American Joint Committee on Cancer [AJCC] v8).

[0139] The study population was patients with high-risk stage IIC, III, or IV melanoma within 12 weeks of complete surgical resection (8th edition of the American Joint Committee on Cancer melanoma classification, 2017) (Amin, 2017).

[0140] Inclusion criteria Patients must meet the following criteria to be eligible for inclusion in this study:

[0141] 1. At least 12 years of age on the day of providing informed consent. Note: Patients under the age of 18 will be included where accepted in accordance with local laws, regulations, and ethics committees.

[0142] 2. All patients have histologically confirmed melanoma that is either stage IIC, III, or stage IV according to the AJCC 8th edition (Amin, 2017) and has been completely surgically resected to be eligible. Patients with stage IIIA disease must have at least one lymph node micrometastasis measuring greater than 1 mm in greatest diameter. Patients with stages IIC, IIIA, and IV (M1c / d) will each represent up to 10% of the total population (i.e., 30% in total). · Patients with acral and mucosal melanoma are allowed, provided they have undergone complete resection and have a combined upper limit of 10% for both types. Patients with melanoma of unknown primary may be admitted, provided they have undergone complete resection. Stage IIC melanoma must be confirmed by a pathology-negative sentinel lymph node biopsy (SLNB) specimen and no evidence of regional or distant metastasis.

[0143] 3. Complete surgical excision must be performed within 12 weeks prior to treatment, and enrollment may only occur after satisfactory wound healing from surgery. Complete surgical resection is defined as negative microscopic margins on the resected primary tumor along with SLNB. Complete lymphadenectomy is not mandatory in cases of positive SLNB, provided the patient is clinically node-negative and the resection margins are microscopically clear. Surgical / pathology reports documenting complete resection must be reviewed and signed by the investigator prior to study enrollment for all patients. Solitary cutaneous melanoma lesions without epithelial components should be treated as primary tumors and undergo SLNB. For all resected stage IV M1d patients, neurosurgery and pathology reports documenting complete resection and clear margins must be reviewed and signed by the investigator prior to enrollment.

[0144] 4. All patients must be disease-free as documented by a complete physical examination and imaging studies within 4 weeks prior to treatment. Imaging must include scans of the chest, abdomen, and pelvis (preferably with contrast X-ray CT), as well as all relevant anatomical regions and sites of resected disease (for patients with stage III and IV disease). Contrast-enhanced MRI of the brain must be performed at the time of staging for all patients. For patients with abnormal or suspicious findings on screening scans, residual malignancy and / or metastatic disease 18 It must be ruled out by F-FDG PET-CT and / or biopsy. For patients with resected stage IV M1d disease, a repeat brain MRI with contrast showing no evidence of residual or recurrent disease must be performed at least 4 weeks after surgery / RT and prior to study enrollment.

[0145] 5. Patients must not have received any systemic anticancer therapy or radiation therapy for melanoma within the past 5 years, except for stage IV M1d patients who may have had CNS-directed RT after definitive resection. Adjuvant radiation therapy to the primary site and / or nodal base after definitive surgical resection is permitted if it is considered institutional standard of care.

[0146] 6. Performance Status: For adult patients: ECOG PS 0 or 1. For patients under 18 years of age: Karnofsky PS greater than 70 (for patients over 16 years of age) or Lansky PS greater than 70 (for patients under 16 years of age).

[0147] 7. Adequate bone marrow function as determined by hematological parameters: Absolute neutrophil count (ANC) 1.5 x 10^9 / L (1500 / mm 3 ) End Hemoglobin level 9.0g / dL (5.59mmol / L) or higher ·Platelet count 75,000 / mm 3 End

[0148] 8. Adequate liver function as determined by: AST / ALT for adults: aspartate aminotransferase (AST) less than 3 x upper limit of normal (ULN) and alanine aminotransferase (ALT) less than 3 x ULN AST / ALT for adolescents: AST and ALT less than 2.5 ULN Serum bilirubin ≤ 1.5 x ULN, except in patients with clinically documented Gilbert syndrome, in which ≤ 3 x ULN is acceptable

[0149] 9. Adequate renal function: For adult patients: ≥30 ml / min as determined by estimated glomerular filtration rate (eGFR) (using the creatine phosphokinase (CPK)-EPI equation) For pediatric patients: (i) Creatinine clearance or radioisotope glomerular filtration rate (GFR) 70 mL / min / 1.73 m 2 super or (ii) Serum creatinine based on age / gender as shown in Table 1: Table 1. Serum Creatinine by Age / Gender TIFF2026508608000001.tif47128

[0150] 10. For biomarker analysis, all patients must submit a minimum of 25 slides or 125 mm of tumor tissue sample (FFPE) excised from a tissue block. 3 Cut slides must be shipped to the testing laboratory within two weeks of slide preparation. Bone biopsies are not acceptable.

[0151] 11. Women of childbearing potential (WOCBP*) must have negative serum (beta-human chorionic gonadotropin [beta-hCG]) at screening and agree not to become pregnant during study treatment and for up to 6 months after receiving the last dose of study treatment.

[0152] *WOCBP are defined as women who remain fertile after menarche until postmenopause, unless permanently infertile. Permanent methods of contraception include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy.

[0153] Postmenopausal status is defined as 12 months of amenorrhea without any other medical cause. Elevated follicle-stimulating hormone (FSH) levels within the postmenopausal range can be used to confirm postmenopausal status in women who do not use hormonal contraception or hormone replacement therapy. However, in the absence of 12 months of amenorrhea, a single FSH measurement is insufficient to determine the occurrence of postmenopausal status. The above definition follows guidance from the Clinical Trial Facilitation Group (CTFG). Pregnancy testing and contraception are not required for women with documented evidence of hysterectomy.

[0154] Male study participants with WOCBP partners are required to use condoms for six months after receiving their last therapeutic dose unless they have undergone a vasectomy or practice sexual abstinence.

[0155] The vasectomized partner or vasectomized study participant must have undergone a medical evaluation of surgical success.

[0156] Periodic abstinence (calendar, symptom-temperature, postovulatory), withdrawal (withdrawal), spermicide only, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female and male condoms should not be used together.

[0157] 12. WOCBPs must agree not to donate eggs (eggs, oocytes) for assisted reproduction purposes during the entire study and until 6 months after their final treatment.

[0158] 13. All men must agree not to donate sperm during the study and for 6 months after receiving their last therapeutic dose.

[0159] 14. Study patient provides signed informed consent (patients aged 12-17 years provide approval supplemented by parental or legal guardian consent).

[0160] 15. Willing and able to comply with clinic visit and study-related procedures.

[0161] 16. Able to understand and complete study-related questionnaires.

[0162] Exclusion criteria Patients who meet any of the following criteria will be excluded from the study:

[0163] 1.Uveal melanoma.

[0164] 2. Any evidence of residual disease after surgery by imaging, pathology, or cytology (including clinical or radiological suspicion of leptomeningeal metastasis).

[0165] 3. Current or recent (within 2 years) evidence of clinically significant autoimmune disease requiring systemic treatment with immunosuppressants, including but not limited to: vitiligo, resolved childhood asthma, residual hypothyroidism requiring hormone replacement only, and psoriasis not requiring systemic treatment.

[0166] 4. Diagnosis of uncontrolled infection with HIV, HBV, or HCV infection, or immunodeficiency associated with or resulting in chronic infection.

[0167] Note: Patients with known HIV whose infection is controlled (undetectable viral load and CD4 count >350 either naturally or on a stable antiviral regimen) are permitted. Monitoring of patients with controlled HIV infection will be performed according to local standards. Patients with known hepatitis B whose infection is controlled (HepBsAg+) (serum hepatitis B virus DNA PCR below the limit of detection and receiving antiviral therapy for hepatitis B) are accepted. Patients with controlled infection must undergo regular monitoring of HBV DNA according to local standards and must continue antiviral therapy for at least 6 months past the last dose of study drug. Patients who are known HCV Ab+ and whose infection is controlled (HCV RNA undetectable by PCR, either naturally or in response to a prior successful course of anti-HCV therapy) are allowed. Patients with HIV or hepatitis must be reviewed by a specialist qualified to manage this disease (e.g., infectious disease or hepatologist) before study initiation and periodically throughout study participation.

[0168] 5. Another malignancy that is currently progressing or has required active treatment within the past 5 years, except for those with a negligible risk of metastasis or death (e.g., adequately treated cervical carcinoma in situ, basal or squamous cell skin cancer, localized early stage prostate cancer, or ductal carcinoma in situ of the breast). Note: Any uncertain cases must be discussed with the medical monitor before enrollment.

[0169] 6. Pregnant or breastfeeding women.

[0170] 7. WOCBP who are not willing to use highly effective contraception prior to the first dose / initiation of first treatment, during the study, and for at least 6 months after the last dose. Highly effective contraception methods include: Stable use of a combined (estrogen and progesterone-containing) hormonal contraceptive method (oral, vaginal, transdermal) or a progesterone-only hormonal contraceptive method (oral, injectable, implantable) associated with the inhibition of ovulation, initiated for at least two menstrual cycles prior to screening; Intrauterine devices (IUDs), intrauterine hormone-releasing systems (IUSs), Bilateral tubal ligation (occlusion), A vasectomized partner (provided that the male vasectomized partner is the WOCBP study participant's only sexual partner and the vasectomized partner has undergone a medical evaluation of the surgical success of the procedure), and / or ·Sexual abstinence†, ‡. (i)†Sexual abstinence is considered highly effective only when defined as abstinence from heterosexual intercourse for the entire period of risk associated with the investigational drug. The reliability of sexual abstinence needs to be assessed in relation to the duration of the clinical trial and the subject's preferred and usual lifestyle. (ii) Periodic abstinence (calendar, symptom-temperature, or postovulatory), withdrawal (withdrawal), spermicide alone, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female and male condoms should not be used together.

[0171] Prior / concomitant therapy: 8.Systemic immunosuppression: Use of immunosuppressive doses of corticosteroids (greater than 10 mg prednisolone or equivalent per day) within 14 days of the first dose of study drug. Physiologic replacement doses up to and including 10 mg prednisone / day or equivalent are permitted. Inhaled or topical steroids are permitted. ·Other clinically significant forms of systemic immunosuppression.

[0172] 9. Treatment with any anti-cancer therapy for malignancies other than melanoma, including immunotherapy, chemotherapy, radiation therapy, or biotherapy, within the 5 years prior to treatment. Adjuvant hormone therapy used for breast cancer or other hormone-sensitive cancers in long-term remission is permitted.

[0173] Other comorbidities: 10. History or current evidence of significant (CTCAE Grade ≥ 2) local or systemic infection (e.g., cellulitis, pneumonia, sepsis) requiring systemic antibiotic treatment within 2 weeks prior to the first dose of study drug.

[0174] Other exclusions: 11. Known hypersensitivity to the active substance or any of the excipients, or any contraindication to either cemiplimab or pembrolizumab according to local prescribing information.

[0175] 12. Presence of severe intercurrent illness or other conditions (e.g., psychological, familial, sociological, or geographical circumstances) that would not allow adequate follow-up and adherence to the protocol.

[0176] 13. Cardiovascular disease as defined below: New York Heart Association (NYHA) class II, III, or IV heart failure classification, or Myocardial infarction (MI) or acute coronary syndrome (ACS) within 6 months, or Transient ischemic attack (TIA) or stroke within one year.

[0177] 14. Received a live vaccine within 30 days of the planned start of the investigational product. Note: Receipt of a live or live attenuated vaccine with potential for replication. If the patient plans to receive a COVID-19 vaccine before starting the investigational product, study participation must be postponed at least 1 week after any COVID-19 vaccination. During treatment, it is recommended that COVID-19 vaccination be postponed until the patient has received and tolerated the investigational product. Booster vaccine doses should not occur more than 48 hours before or after administration of the investigational product.

[0178] 15. Major surgical procedure (i.e., requiring general anesthesia) or significant traumatic injury within 4 weeks prior to screening.

[0179] 16. Exception: Minor melanoma-related surgery / biopsy is permitted, provided there is satisfactory wound healing prior to receiving study treatment.

[0180] 17. History of allogeneic stem cell transplantation or solid organ transplantation

[0181] 18. Any medical condition where, in the opinion of the investigator, study participation is not in the patient's best interest.

[0182] 19. Members of the investigational site research team and / or their immediate families, unless previously approved by the sponsor.

[0183] 20. Patients who are institutionalized pursuant to an order issued by either a judicial or administrative authority will be excluded from this study.

[0184] Adolescent patients (ages 12 to 18) weighing less than 21.40 kg.

[0185] Dose / Route / Schedule: The combination of cemiplimab and fianlimab, and the combination of pembrolizumab and placebo will be prepared by unblinded pharmacists at the investigational site and administered in a blinded manner in the outpatient setting. Adult and adolescent patients will receive co-infusions of the study drugs via 30-minute (± 10-minute) IV infusion Q3W. Similarly, pembrolizumab and placebo will be infused via 30-minute (± 10-minute) IV infusion once every 3 weeks.

[0186] Fianlimab and cemiplimab (combined co-infusion) Fianlimab 1600 mg will be supplied as a liquid in a sterile, single-use vial. Instructions for dose preparation are provided in the Investigational Product Control Procedures.

[0187] Fianlimab 400 mg will be supplied as a liquid in a sterile, single-use vial. Instructions for dose preparation are provided in the Investigational Product Control Procedures.

[0188] Cemiplimab 350 mg will be supplied as a liquid in a sterile, single-use vial. Instructions for dose preparation will be provided in the Investigational Product Control Procedures.

[0189] Pembrolizumab Pembrolizumab 200 mg will be prepared at the investigational site as a liquid in a sterile, single-use vial for co-infusion (with saline / dextrose placebo). Adolescent patients will receive pembrolizumab at 2 mg / kg (maximum 200 mg total). Instructions for dose preparation will be provided in the Investigational Drug Administration Procedures.

[0190] Evaluation items The primary endpoint for all patients was investigator-assessed RFS, defined as the time from treatment to the first documented recurrence of disease at any site (excluding new primary melanoma) or death from any cause, whichever occurred first.

[0191] For efficacy, the key secondary endpoint is overall survival, defined as the time from treatment to the date of death. Additional efficacy endpoints include melanoma-specific survival, defined as death from melanoma (death from other causes or undefined causes is censored), and distant metastasis-free survival, defined as the time between the date of treatment and the date of the first distant metastasis.

[0192] Patient-reported outcomes Secondary endpoints of general health status included patient-reported outcomes (PROs) for adult patients as measured by the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire C30 (EORTC-QLQ-C30), European Quality of Life Dimension 5 (EQ-5D-5L), Functional Assessment of Cancer Therapy (FACT)-Melanoma (melanoma subscale only), Patient Global Impression Scale (PGIS), and Patient Global Impression of Change Scale (PGIC).

[0193] Methods and Evaluation The primary endpoint is RFS, which is an accepted primary endpoint in this clinical setting. Secondary efficacy endpoints include OS, MSS, DMFS, and PROs in adults only, as measured by EORTC QLQ-C30, EQ-5D-5L, FACT-Melanoma (melanoma subscale only), PROs for fatigue (CTCAE, PGIS, and PGIC). The Pediatric Quality of Life Inventory (PEDSQL) will be administered in adolescents. Analysis of RFS will be based on investigator assessment using imaging or pathology evaluation at the time of disease recurrence or patient death.

[0194] Potential disease recurrences initially detected clinically are evaluated by CT or MRI. Isolated suspicious lesions, or lesions of unknown etiology detected by imaging, require biopsy for histologic confirmation of recurrence, if medically appropriate. Skin lesions also require biopsy for histologic confirmation of disease recurrence, if medically appropriate. New cases of primary melanoma are distinguished from recurrences by a local pathologist by reviewing skin lesion biopsy and excision specimens to identify the presence of an intraepidermal component and whether they are consistent with a new primary melanoma, as opposed to a local or distant metastatic recurrence, according to established dermatology practice guidelines.

[0195] Blood samples for measuring fianlimab and cemiplimab in serum and immunogenicity (presence of ADA and NAb against fianlimab and cemiplimab in serum) will be taken from all patients.

[0196] Blood samples will be collected for analysis of additional biomarkers in plasma and serum. Potential pharmacodynamic, predictive, and prognostic biomarkers related to fianlimab and cemiplimab treatment exposure, clinical activity, and / or underlying disease will be investigated in plasma, serum, and tumor tissue.

[0197] The safety and tolerability of fianlimab in combination with cemiplimab will be monitored by clinical assessment of treatment-emergent adverse events (TEAEs) / immune-mediated adverse events (imAEs), serious adverse events (SAEs), adverse events of special interest (AESIs), and by repeated measurements and clinical assessment of vital signs (temperature, blood pressure, pulse, and respiration), physical examination, 12-lead electrocardiogram (ECG), and standard hematology, chemistry, urinalysis, and other laboratory tests (including blood cortisol and thyroid-stimulating hormone [TSH]).

[0198] result Surprisingly, the combination of fianlimab (REGN3767, anti-LAG-3) and cemiplimab Q3W improves recurrence-free survival (RFS) compared with pembrolizumab in the adjuvant setting in patients with completely resected high-risk melanoma.

[0199] Example 2: Phase 3 Clinical Trial of an Anti-LAG-3 Antibody (REGN3767, fianlimab) and an Anti-PD-1 Antibody (REGN2810, cemiplimab) in Patients with Previously Untreated Unresectable Locally Advanced or Metastatic Melanoma This phase 3 study evaluates the combination of fialimab and cemiplimab compared with pembrolizumab in patients with previously untreated unresectable locally advanced or metastatic melanoma.

[0200] the purpose The primary objective of this study is to demonstrate the superiority of fianlimab 1600 mg plus cemiplimab and / or fianlimab 400 mg plus cemiplimab compared to pembrolizumab as measured by progression-free survival (PFS).

[0201] Secondary objectives include: To demonstrate superiority of fianlimab 1600mg plus cemiplimab and / or fianlimab 400mg plus cemiplimab compared to pembrolizumab as measured by overall survival (OS). To demonstrate superior ORR with fianlimab 1600mg plus cemiplimab and / or fianlimab 400mg plus cemiplimab compared to pembrolizumab. To characterize ORR, PFS, and OS with fianlimab 1600mg + cemiplimab and / or fianlimab 400mg + cemiplimab compared to cemiplimab, and to provide information on the contribution of each component. To evaluate the immunogenicity of fianlimab 1600 mg and / or 400 mg and cemiplimab. To assess the effects of fianlimab 1600mg plus cemiplimab and / or fianlimab 400mg plus cemiplimab compared with pembrolizumab on physical and role function and global health status / quality of life in adults. To characterize the safety and tolerability of treatment in patients aged 12 to 18 years. To characterize ORR, PFS, and OS with treatment in patients aged 12 to 18 years. To assess the safety and tolerability of fianlimab plus cemiplimab compared with pembrolizumab and cemiplimab. To characterize the pharmacokinetics (PK) of the treatment using sparse PK sampling in patients aged 12 years and older.

[0202] Study design This is a phase 3 study in patients aged 12 years and older with unresectable locally advanced or metastatic melanoma who have not received prior systemic treatment for advanced disease.

[0203] There are four main arms to the study, which is conducted in a randomized, double-blind fashion in adult and adolescent patients. Group A: fianlimab (1600 mg intravenously every 3 weeks [Q3W]) + cemiplimab (350 mg Q3W IV) Group A1: fianlimab (400 mg every 3 weeks [Q3W], intravenously [IV]) + cemiplimab (350 mg Q3W IV) Arm B: Pembrolizumab (200 mg Q3W IV) + saline / dextrose placebo (placebo) Group C: Cemiplimab (350 mg Q3W IV) + saline / dextrose placebo (placebo)

[0204] The study population included patients with unresectable stage III or IV (metastatic) melanoma (8th edition of the American Joint Committee on Cancer melanoma classification) who had not received prior systemic anticancer therapy for progressive unresectable and metastatic disease. The patient population included male and female patients aged 12 years and older.

[0205] Inclusion criteria Patients must meet the following criteria to be eligible for inclusion in this study:

[0206] 1. 12 years of age or older on the day of providing informed consent. Note: Patients under the age of 18 will be included where accepted in accordance with local laws, regulations, and ethics committees.

[0207] 2. Patients with histologically confirmed unresectable stage III and stage IV (metastatic) melanoma according to AJCC, 8th revision (Amin, 2017) who have not received prior systemic therapy for progressive unresectable disease. Patients who have received adjuvant and / or neoadjuvant systemic therapy are eligible if they have had no evidence of disease progression or recurrence and / or have discontinued such therapy due to the occurrence of a Grade 3 or higher immune-related adverse event (irAE) (excluding endocrinopathy fully controlled with hormone replacement). Patients must also have a treatment-free and disease-free interval of greater than 6 months. Patients with acral and mucosal melanoma are eligible. The increase is limited to approximately 10% of the total population.

[0208] 3. Measurable disease according to RECIST v1.1 Previously radiotherapy-treated lesions may only be considered target lesions if progression is demonstrated and no other target lesions are available Skin lesions should be evaluated as non-target lesions.

[0209] 4. Performance Status: For adult patients: Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0 or 1 For pediatric patients: Karnofsky performance status of 70 or higher (patients 16 years of age or older) or Lansky performance status of 70 or higher (patients under 16 years of age)

[0210] 5. An expected life expectancy of at least 3 months.

[0211] 6. Adequate bone marrow function as determined by hematological parameters: Absolute neutrophil count (ANC) 1.5 x 10^9 / L (1500 / mm 3 ) End Hemoglobin 9.0g / dL (5.59mmol / L) or higher. ·Platelet count 75,000 / mm 3 That's all.

[0212] 7. Adequate liver function as determined by: AST / ALT for adults: aspartate aminotransferase (AST) ≤ 3 x ULN, alanine aminotransferase (ALT) ≤ 3 x ULN, and alkaline phosphatase < 2.5 x ULN (or < 5 x ULN if liver or bone metastases are present) AST / ALT for adolescents: AST, ALT, and ALP ≤2.5 ULN (and no liver involvement) (or <5 x ULN if liver or bone metastases are present) Serum bilirubin ≤ 1.5 x ULN, except in patients with clinically documented Gilbert syndrome, in which ≤ 3 x ULN is acceptable

[0213] 8. Adequate renal function: For adult patients: ≥30 mL / min as determined by estimated glomerular filtration rate (eGFR) (using the CPK-EPI equation) For pediatric patients: (i) Creatinine clearance or radioisotope glomerular filtration rate (GFR) 70 mL / min / 1.73 m 2 End or (ii) Serum creatinine based on age / gender as shown in Table 2: Table 2. Serum creatinine by age and gender TIFF2026508608000002.tif44128

[0214] 9. Biopsy LAG-3 IHC Result Requirements: Patients must have a valid, centrally tested LAG-3 IHC result to be enrolled in the study. Any LAG-3 level (0-100% expression) is acceptable.

[0215] Sample requirements: Patients must submit tumor tissue samples (formalin-fixed, paraffin-embedded [FFPE]) from tissue blocks ≤1 year without interim therapy prior to enrollment. Cut slides must be shipped to the testing laboratory within 2 weeks of slide preparation.

[0216] If archived tissue is unavailable or insufficient for testing, a new biopsy may be requested prior to enrollment, provided that it is deemed by the investigator not to represent a significant potential risk to the subject's health, safety, or welfare. If archived or new biopsy tissue cannot be obtained, the patient will not be eligible for enrollment.

[0217] Lesions eligible for biopsy must be non-target lesions and must either have not received prior radiation therapy or have histologic evidence of viable tumor after the final course of radiation therapy. Bone biopsies are not permitted.

[0218] 10. Women of childbearing potential (WOCBP*) must have negative serum (beta-human chorionic gonadotropin [beta-hCG]) at screening.

[0219] *WOCBP are defined as women who remain fertile after menarche until postmenopause, unless permanently infertile. Permanent methods of contraception include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy.

[0220] Postmenopausal status is defined as 12 months of amenorrhea without any other medical cause. Elevated follicle-stimulating hormone (FSH) levels within the postmenopausal range can be used to confirm postmenopausal status in women who do not use hormonal contraception or hormone replacement therapy. However, in the absence of 12 months of amenorrhea, a single FSH measurement is insufficient to determine the occurrence of postmenopausal status. The above definition follows guidance from the Clinical Trial Facilitation Group (CTFG). Pregnancy testing and contraception are not required for women with documented evidence of hysterectomy.

[0221] Male study participants with WOCBP partners are required to use condoms for six months unless they have a vasectomy or practice sexual abstinence. · Vasectomized partners or vasectomized study participants must have undergone a medical evaluation of surgical success. Periodic abstinence (calendar, symptom-temperature, or postovulatory methods), withdrawal (withdrawal), spermicide alone, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female and male condoms should not be used together.

[0222] 11. WOCBPs must agree not to donate eggs (eggs, oocytes) for assisted reproduction purposes during the entire study and until 6 months after their final treatment.

[0223] 12. All men must agree not to donate sperm during the study and for 6 months after receiving their last therapeutic dose.

[0224] 13. Study patients provide signed informed consent (patients aged 12-17 years provide consent supplemented by parental or legal guardian consent, unless they are incapacitated due to a mental disability).

[0225] 14. Willing and able to comply with clinic visit and study-related procedures.

[0226] 15. I am able to understand and complete all study-related questionnaires.

[0227] Exclusion criteria Patients who meet any of the following criteria will be excluded from the study:

[0228] medical conditions 1. Uveal melanoma

[0229] 2. Current or recent (within 2 years) evidence of an autoimmune disease requiring systemic treatment with immunosuppressants, including but not limited to: vitiligo, resolved childhood asthma, residual hypothyroidism requiring hormone replacement only, and psoriasis not requiring systemic treatment.

[0230] 3. Diagnosis of immunodeficiency associated with or resulting in uncontrolled infection or chronic infection with human immunodeficiency virus (HIV), hepatitis B (HBV), or hepatitis C virus (HCV) infection.

[0231] Note: Patients with known HIV whose infection is controlled (undetectable viral load and CD4 count >350 either naturally or on a stable antiviral regimen) will be admitted. Monitoring will be performed according to local standards for patients with controlled HIV infection. Patients with known hepatitis B whose infection is controlled (HepBsAg+) (serum hepatitis B virus DNA PCR below the limit of detection and receiving antiviral therapy for hepatitis B) are accepted. Patients with controlled infection must undergo regular monitoring of HBV DNA according to local standards and must continue antiviral therapy for at least 6 months past the last dose of study drug. Patients with known hepatitis C virus antibody positivity (HCV Ab+) and controlled infection (HCV RNA undetectable by PCR, either spontaneously or in response to a prior successful course of anti-HCV therapy) are accepted. Patients with HIV or hepatitis must be reviewed by a specialist qualified to manage this disease (e.g., infectious disease or hepatologist) before study initiation and periodically throughout study participation.

[0232] 4. Unknown BRAF V600 mutation status. Patients with BRAF-mutated melanoma who present with rapidly progressive disease and who are deemed, by the investigator's assessment, likely to benefit from prior treatment with a BRAF / MEK inhibitor will not be enrolled in the study.

[0233] 5. Another malignancy that is progressing or has required active treatment within the past 2 years, except for those with a negligible risk of metastasis or death (e.g., adequately treated cervical carcinoma in situ, basal or squamous cell skin cancer, localized prostate cancer, or ductal carcinoma in situ of the breast).

[0234] Note: In case of uncertainty, consultation with the medical monitor should be sought.

[0235] 6. Pregnant or breastfeeding women.

[0236] 7. WOCBP who are not willing to use highly effective contraception prior to the first dose / initiation of first treatment, during the study, and for at least 6 months after the last dose. Highly effective contraception methods include: Stable use of a combined (estrogen and progesterone-containing) hormonal contraceptive method (oral, vaginal, transdermal) or a progesterone-only hormonal contraceptive method (oral, injectable, implantable) associated with the inhibition of ovulation, initiated for at least two menstrual cycles prior to screening; Intrauterine devices (IUDs), intrauterine hormone-releasing systems (IUSs), Tubal ligation, A vasectomized partner (provided that the male vasectomized partner is the WOCBP study participant's only sexual partner and the vasectomized partner has undergone a medical evaluation of the surgical success of the procedure), and / or ·Sexual abstinence†, ‡. (i)†Sexual abstinence is considered highly effective only when defined as abstinence from heterosexual intercourse for the entire period of risk associated with the investigational drug. The reliability of sexual abstinence needs to be evaluated in relation to the duration of the clinical trial and the patient's preferred usual lifestyle. (ii) Periodic abstinence (calendar, symptom-temperature, or postovulatory), withdrawal (withdrawal), spermicide alone, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female and male condoms should not be used together.

[0237] Prior / concomitant therapy 8.Systemic immunosuppression: Use of immunosuppressive doses of corticosteroids (up to 10 mg prednisolone or equivalent per day) within 14 days of the first dose of investigational drug. Physiologic replacement doses up to 10 mg prednisone / day or equivalent, including but not limited to, are permitted. Inhaled or topical steroids are permitted if not intended to treat an autoimmune disorder. ·Other clinically significant forms of systemic immunosuppression.

[0238] 9. Treatment with other anti-cancer therapies, including immunotherapy, chemotherapy, major surgery, or biologic therapy, within 21 days prior to the first dose of study treatment. Adjuvant hormone therapy used for breast cancer or other hormone-sensitive cancers in long-term remission is permitted.

[0239] 10. Patients with a history of Grade 3 or higher immune-mediated adverse events from prior checkpoint inhibitor therapy (excluding endocrinopathy fully controlled with hormone replacement) will be excluded.

[0240] Other comorbidities 11. History or current evidence of significant (CTCAE grade ≥ 2) local or systemic infection (e.g., cellulitis, pneumonia, sepsis) requiring systemic antibiotic treatment within 14 days prior to the first dose of study drug.

[0241] 12. Active or untreated brain tumor or spinal cord compression. Patients with leptomeningeal disease are excluded. Patients with known brain metastases are eligible if: received radiation therapy or another appropriate standard therapy for brain metastases; -Return to neurological baseline (excluding residual signs and symptoms related to CNS treatment) for at least 14 days prior to enrollment. - Not requiring immunosuppressive doses of corticosteroid therapy (greater than 10 mg prednisone per day or equivalent) in the 14 days prior to enrollment.

[0242] Note: Asymptomatic patients with a single untreated brain metastasis less than 10 mm in size are eligible.

[0243] Other Exclusions 13. Known hypersensitivity to any of the active ingredients or excipients

[0244] 14. Presence of severe intercurrent illness or other conditions (e.g., psychological, familial, sociological, or geographical circumstances) that would not allow adequate follow-up and adherence to the protocol.

[0245] 15. Received a live vaccine, i.e., a replicative live vaccine or a live attenuated vaccine, within 30 days of the planned start of the investigational product. If the patient plans to receive a COVID-19 vaccine before starting the investigational product, participation in the study must be postponed at least 1 week after any COVID-19 vaccination. It is recommended that COVID-19 vaccination be postponed until the patient has received and tolerated a consistent dose of the investigational product during the treatment period. The vaccine dose must not precede or follow the investigational product administration more than 48 hours later.

[0246] 16. Major surgery, open biopsy, or significant traumatic injury within 4 weeks prior to screening.

[0247] 17. History of allogeneic stem cell transplant or solid organ transplant.

[0248] 18. Any medical condition where, in the opinion of the investigator, study participation is not in the patient's best interest.

[0249] 19. Members of the investigational site research team and / or their immediate families, unless previously approved by the sponsor.

[0250] 20. Patients who are institutionalized pursuant to an order issued by either a judicial or administrative authority will be excluded from this study.

[0251] Adolescent patients (ages 12 to 18) weighing less than 21.40 kg.

[0252] Dose / Route / Schedule The cemiplimab and fianlimab combination, the pembrolizumab and placebo combination, and the cemiplimab and placebo combination will be prepared by unblinded pharmacists at the investigational sites and administered in a blinded manner to all patients.

[0253] All infusions for adults and adolescents will be administered as a 30-minute (±10-minute) IV infusion in an outpatient setting every 3 weeks.

[0254] Cemiplimab Cemiplimab 350 mg IV once every 3 weeks is an approved regimen (LIBTAYO®, cemiplimab) for the treatment of cutaneous squamous cell carcinoma (CSCC), basal cell carcinoma (BCC), and non-small cell lung cancer (NSCLC).

[0255] Cemiplimab will be supplied as a liquid in sterile, single-use vials. Instructions for dose preparation will be provided in the Investigational Product Control Procedures.

[0256] Fianlimab and cemiplimab (for combination / co-infusion administration) Based on efficacy and safety data from the ongoing Phase 1 study, a dose of fianlimab 1600 mg Q3W IV, along with cemiplimab 350 mg Q3W IV, was recommended for progression to Phase 3. The efficacy of fianlimab 400 mg Q3W IV with cemiplimab 350 mg Q3W IV has not been studied but has been added because it is in line with expectations for considering lower doses in the oncology setting and is consistent with doses used for other anti-LAG-3 antibodies utilized in Phase 2 and 3 clinical trials in melanoma and other solid tumors.

[0257] Fianlimab 1600 mg and 400 mg Q3W IV will be supplied as a liquid in sterile, single-use vials. Instructions for dose preparation are provided in the Investigational Product Control Procedures.

[0258] Fianlimab and cemiplimab are administered simultaneously as a combined co-infusion.

[0259] Pembrolizumab Pembrolizumab 200 mg Q3W IV is the approved dose for the treatment of advanced and metastatic melanoma.

[0260] For adolescent patients randomized to Arm B, pembrolizumab will be administered at 2.0 mg / kg Q3W IV (maximum 200 mg) based on weight.

[0261] Pembrolizumab will be prepared at the investigational site as a sterile liquid for co-infusion (with saline placebo). Instructions for dose preparation will be provided in the Investigational Product Administration Procedures.

[0262] Evaluation items The primary endpoint is progression-free survival (PFS) (according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 by a blinded independent central review committee [BICR]).

[0263] The primary efficacy endpoints are overall survival and objective response rate (ORR), defined as the proportion of patients achieving a best overall response of CR (complete response) or PR (partial response) (per RECIST 1.1 per BICR). Additional efficacy endpoints include disease control rate (DCR), defined as the proportion of patients achieving a best overall response of CR or PR, or stable disease (SD) (per RECIST 1.1 per BICR; SD is assessed at least 6 months after the first dose).

[0264] DoR (duration of response), defined as the time from initial response (CR or PR according to RECIST 1.1) to the first occurrence of PD (progressive disease) (according to RECIST 1.1 via BICR), or death from any cause, whichever occurs first.

[0265] PFS, ORR, DCR, and DoR based on investigator assessment according to RECIST 1.1 and iRECIST (immune RECIST).

[0266] Patient-reported outcomes: Secondary endpoints of general health status were: Patient-reported outcomes as measured by EORTC QLQ-C30, EQ-5D-5L, FACT-Melanoma (melanoma subscale only), PGIS, and PGIC. Change from baseline in physical function according to EORTC QLQ-C30 at week 25 Change from baseline in role functioning at week 25 according to EORTC QLQ-C30 Change from baseline in GHS / QoL according to EORTC QLQ-C30 at week 25 Change from baseline in physical function during the study according to EORTC QLQ-C30 Change from baseline in role function during the study according to EORTC QLQ-C30 · Change from baseline in GHS / QoL during the study according to EORTC QLQ-C30.

[0267] Methods and Evaluation Efficacy endpoints include antitumor activity. For all patients in the study, this will be assessed by computed tomography (CT) or magnetic resonance imaging (MRI). Digital photography may be used for skin lesions.

[0268] The safety and tolerability of fianlimab in combination with cemiplimab will be monitored by clinical assessment of TEAEs / immune-related adverse events (irAEs), SAEs, AESIs, and by repeated measurements and clinical assessment of vital signs (temperature, blood pressure, pulse, and respiration), physical examination, 12-lead electrocardiogram (ECG), and standard hematology, chemistry, urinalysis, and other laboratory tests (including blood cortisol and TSH).

[0269] Blood samples will be taken from all patients to measure functional fianlimab and cemiplimab in serum and to measure immunogenicity (presence of ADA and NAb to fianlimab and cemiplimab in serum).

[0270] A centrally determined LAG-3 immunohistochemistry (IHC) result is required for patient eligibility. Patients must have a valid, centrally tested LAG-3 IHC result to be enrolled in the study. Any LAG-3 level (0-100% expression) is acceptable. Patient samples will be collected for analysis of additional biomarkers. Putative pharmacodynamic and / or predictive and prognostic biomarkers related to fianlimab and cemiplimab treatment exposure, clinical activity, and / or underlying disease may be investigated. Serum, plasma, peripheral blood mononuclear cells (PBMCs), and tumor tissue will be collected.

[0271] result Surprisingly, the combination of fianlimab (REGN3767, anti-LAG-3) and cemiplimab improves progression-free survival (PFS) compared with pembrolizumab in patients with previously untreated, unresectable locally advanced or metastatic melanoma. Furthermore, the combination of fianlimab and cemiplimab is expected to demonstrate a PK, safety, and efficacy profile in adolescent patients with previously untreated, unresectable locally advanced or metastatic melanoma similar to that observed in adults.

[0272] Example 3: Phase 3 Clinical Trial Comparing Anti-LAG-3 (Fianlimab) and Anti-PD-1 (Cemiplimab) with Pembrolizumab in Patients with Completely Resected High-Risk Melanoma Background: Melanoma accounts for the majority of skin cancer-related deaths. Most patients with newly diagnosed melanoma have resectable disease and are potentially cured by surgery. However, local nodular and / or distant recurrence can occur after curative-intent resection. Postoperative adjuvant therapy with immune checkpoint inhibitors improves recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) in high-risk patients with melanoma. Fianlimab (anti-LAG-3) and cemiplimab (anti-PD-1) are both high-affinity, fully human, IgG4 monoclonal antibodies (MAbs), which, in combination, have shown high clinical activity in patients with advanced melanoma in phase 1 studies.

[0273] Methods: This is a three-way, double-blind, phase 3 trial comparing fianlimab plus cemiplimab with pembrolizumab in the adjuvant treatment of high-risk resected melanoma. The primary objective is recurrence-free survival, and secondary objectives are overall survival, safety, pharmacology, and immunogenicity.

[0274] Patient eligibility: (1) 12 years of age or older; (2) stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected less than 12 weeks prior to treatment; (3) no prior systemic anticancer therapy or radiation therapy for melanoma within the past 5 years; (4) no evidence of metastatic disease on staging studies; and (5) an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), a Karnofsky PS of greater than 70 (for patients older than 16 years), or a Lansky PS of greater than 70 (for patients younger than 16 years).

[0275] Study arms (all treatments administered intravenously every 3 weeks for 1 year): A. fianlimab (1600 mg) + cemiplimab (350 mg), B. fianlimab (400 mg) + cemiplimab (350 mg), C. pembrolizumab (200 mg) + saline / dextrose placebo. The placebo-controlled trial will enroll patients randomized 1:1:1 to arms A:B:C, treated for up to 1 year. The trial will be stratified by disease stage (stage IIIA vs. IIC-IIIB-IIIC vs. IIID-IV [M1a / b] vs. IV [M1c / d]) and geography (North America vs. Europe vs. rest of the world).

[0276] The primary endpoint is investigator-assessed recurrence-free survival. Secondary endpoints include efficacy (overall survival, distant metastasis-free survival, and melanoma-specific survival), safety (treatment-emergent adverse events (TEAEs), drug interruptions or discontinuation due to TEAEs), pharmacokinetics (serum fianlimab and cemiplimab concentrations over time), immunogenicity (serum anti-drug and neutralizing antibodies to fianlimab or cemiplimab), and patient-reported outcomes. Analysis will be performed when 242 recurrence-free survival events are observed.

[0277] Results: Enrolled in the trial. Surprisingly, administration of fianlimab in combination with cemiplimab results in enhanced tumor regression and improved disease control in patients with completely resected high-risk melanoma.

[0278] The present disclosure is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present disclosure, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0279] (Table 3) Unofficial sequence listing TIFF2026508608000003.tif219142TIFF2026508608000004.tif171142

Claims

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

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

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

4. The method of any one of claims 1 to 3, wherein a single dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 50 to 8000 mg.

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

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

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

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

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

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

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

12. The method of claim 10 or 11, wherein each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises 50 to 8000 mg.

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

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

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

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

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

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

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

21. 21. The method of any one of claims 10 to 18 or 20, wherein each dose of the anti-LAG-3 antibody or antigen-binding fragment thereof 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 unresectable locally advanced melanoma.

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

25. Patients meet the following criteria: (i) at least 12 years of age on the date of providing informed consent; (ii) have histologically confirmed melanoma that is either stage IIC, III, or IV according to the AJCC 8th edition (Amin, 2017) and has been completely surgically resected; (iii) Patients with stage IIIA disease must have at least one lymph node micrometastasis measuring greater than 1 mm in greatest dimension; (iv) Stage IIC melanoma confirmed by a pathology-negative sentinel lymph node biopsy (SLNB) specimen and no evidence of regional or distant metastasis; (v) Complete surgical excision performed within 12 weeks prior to treatment, and treatment can occur only after satisfactory wound healing from surgery; (vi) disease-free status as demonstrated by a complete physical examination and imaging studies prior to treatment; (vii) the patient exhibits 1% or more LAG3 in tumor tissue as determined by IHC or iPET; and (viii) Patients must not have received systemic anticancer therapy or radiation therapy for melanoma within the past 5 years.

25. The method of claim 23 or 24, further selected as having one or more of:

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

27. Patients meet the following criteria: (1) 12 years of age or older; (2) Stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected less than 12 weeks prior to treatment; (3) No prior systemic anticancer therapy or radiation therapy for melanoma in the past 5 years; (4) exhibiting 1% or more LAG3 in tumor tissue as determined by IHC or iPET; (5) no evidence of metastatic disease; and (6) Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), Karnofsky PS of greater than 70 (for patients over 16 years of age), or Lansky PS of greater than 70 (for patients under 16 years of age).

27. The method of claim 26, further selected as having one or more of:

28. 28. The method of any one of claims 1-27, wherein the treatment provides a therapeutic benefit selected from the group consisting of delayed melanoma growth, reduction in melanoma cell count, melanoma regression, increased survival, partial response, and complete response.

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

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

31. 29. The method of claim 28, wherein the melanoma growth is inhibited by at least 20% compared to subjects administered either antibody as monotherapy.

32. The method further comprises administering to the subject an additional therapeutic agent or therapy, wherein the additional therapeutic agent or therapy is selected from the group consisting of radiation, surgery, a chemotherapy agent, a cancer vaccine, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a CD28 agonist, a CD38 inhibitor, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an angiopoietin-2 (Ang2) inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, an antibody against a tumor-specific antigen, and a Bacillus subtilis.

32. The method of any one of claims 1 to 31, wherein the therapeutic agent is selected from the group consisting of Calmette-Guerin vaccine, granulocyte-macrophage colony-stimulating factor, oncolytic virus, cytotoxin, interleukin-6 receptor (IL-6R) inhibitor, interleukin-4 receptor (IL-4R) inhibitor, IL-10 inhibitor, IL-2, IL-7, IL-21, IL-12, IL-15, antibody-drug conjugate, GITR agonist, 4-1BB agonist, CD20xCD3 bispecific antibody, MUC16xCD3 bispecific antibody, and anti-inflammatory agent.

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

8.

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

2.

35. 35. The method of any one of claims 1 to 34, wherein the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising 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 antigen-binding fragment thereof comprises the heavy chain CDRs of HCVR (HCDR1, HCDR2, and HCDR3) and the three light chain CDRs of LCVR (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

18.

37. 37. The method of claim 36, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 11 and the LCVR comprises 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 comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO:

20.

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

40. (1) selecting a patient with melanoma, wherein the patient has unresectable locally advanced melanoma, unresectable metastatic melanoma, or completely resected high-risk melanoma; (2) administering to the patient (a) 350 mg of an anti-PD-1 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 / 2, and (b) 400 mg or 1600 mg of an anti-LAG-3 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 11 / 12; 10. A method of treating or inhibiting the growth of melanoma, comprising:

41. 41. The method of claim 40, wherein the administration of step (2) occurs once every three weeks.

42. 41. The method of claim 40, wherein the administration of step (2) occurs once every six weeks.

43. The patient meets the following criteria: (1) 12 years of age or older; (2) Stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected less than 12 weeks prior to treatment; (3) No prior systemic anticancer therapy or radiation therapy for melanoma within the past 5 years; (4) no evidence of metastatic disease; and (5) Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), Karnofsky PS of greater than 70 (for patients over 16 years of age), or Lansky PS of greater than 70 (for patients under 16 years of age).

41. The method of claim 40, further selected as having one or more of:

44. (1) selecting a patient with unresectable locally advanced melanoma, unresectable metastatic melanoma, or completely resected high-risk melanoma, wherein the selected patient has not received prior systemic treatment for progressive disease; (2) administering to the patient (a) 350 mg of an anti-PD-1 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 / 2, and (b) 400 mg or 1600 mg of an anti-LAG-3 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 11 / 12; 10. A method of treating or inhibiting the growth of melanoma, comprising:

45. 45. The method of claim 44, wherein the administration of step (2) occurs once every three weeks.

46. 45. The method of claim 44, wherein the administration of step (2) is once every six weeks.

47. The patient meets the following criteria: (1) 12 years of age or older; (2) Stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected less than 12 weeks prior to treatment; (3) No prior systemic anticancer therapy or radiation therapy for melanoma within the past 5 years; (4) no evidence of metastatic disease on staging; and (5) Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), Karnofsky PS of greater than 70 (for patients over 16 years of age), or Lansky PS of greater than 70 (for patients under 16 years of age).

45. The method of claim 44, further selected as having one or more of:

48. (1) selecting a patient with melanoma; (2) administering to the patient (a) 400 mg or 1600 mg of an anti-LAG-3 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 11 / 12, and in combination with (a), (b) 350 mg of an anti-PD-1 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 / 2; 10. A method of treating or inhibiting the growth of melanoma, comprising:

49. 49. The method of claim 48, wherein said administering step occurs once every three weeks.

50. 49. The method of claim 48, wherein the step of administering occurs once every six weeks.

51. (a) selecting a patient having melanoma, the patient having completed surgery to treat the melanoma; (b) administering to the patient: (1) an initial loading dose comprising an anti-PD-1 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 1 / 2, and an anti-LAG-3 antibody or antigen-binding fragment thereof comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 11 / 12; and (2) one or more secondary doses, the one or more secondary doses being administered 1 to 4 weeks after the immediately preceding administration; and administering 10. A method of treating or inhibiting the growth of melanoma, comprising:

52. 52. The method of claim 51, wherein the patient exhibits greater than 1% or greater LAG3 in the melanoma tissue.

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

54. The patient meets the following criteria: (1) 12 years of age or older; (2) Stage IIc, III, or IV (all M stage) histologically confirmed melanoma completely resected less than 12 weeks prior to treatment; (3) No prior systemic anticancer therapy or radiation therapy for melanoma within the past 5 years; (4) exhibiting 1% or more LAG3 in tumor tissue as determined by IHC or iPET; (5) no evidence of metastatic disease; and (6) The method of claim 51, further selected as having one or more of the following: an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1 (for adult patients), a Karnofsky PS of greater than 70 (for patients over 16 years of age), or a Lansky PS of greater than 70 (for patients under 16 years of age).

55. For patients who need it, (3) One or more tertiary doses, wherein the one or more tertiary doses are administered 3 to 12 weeks after the immediately preceding administration.

52. The method of claim 51, further comprising administering:

56. 52. The method of claim 51, wherein the one or more secondary doses are administered three weeks after the immediately preceding administration.

54. 56. The method of claim 55, wherein the one or more tertiary doses are administered 3 weeks or 6 weeks after the immediately preceding administration.

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

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

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

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