Methods and Compositions for Treating Cancer

Abstract

The present invention relates to methods and compositions for use in treating cancer in a subject by administering to the subject a bispecific antibody that targets programmed cell death protein 1 (PD-1) and lymphocyte activation gene-3 (LAG3).

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. Said XML copy, created on July 27, 2022, is titled 50474-304WO2_Sequence_Listing_7_27_22 and is 68,756 bytes in size.

[0002] FIELD OF THE INVENTION The present invention relates to methods and compositions for use in treating cancer in a subject by administering to the subject a bispecific antibody targeting programmed cell death protein 1 (PD-1) and lymphocyte activation gene-3 (LAG3) (PD1-LAG3), optionally together with an anti-TIGIT antagonist antibody (e.g., tiragolumab) or a VEGF antagonist (e.g., bevacizumab). [Background technology]

[0003] Cancer is characterized by the uncontrolled proliferation of a cell subpopulation. It is the leading cause of death in developed countries and the second leading cause of death in developing countries, with over 14 million new cancer cases diagnosed and over 8 million cancer deaths occurring each year. Thus, cancer treatment represents a significant and growing societal burden.

[0004] In particular, there is an urgent need for treatment methods for common, difficult-to-treat cancers.

[0005] Melanoma is a malignant tumor of melanocytes. This potentially fatal skin cancer is one of the fastest growing malignancies. Currently, more than 300,000 people worldwide are diagnosed with melanoma each year, and 57,000 die from the disease. Most advanced melanomas have a poor prognosis. Patients with lymph node metastasis (stage III) are at high risk for local and distant recurrence after surgery, and the 5-year survival rate in this patient population ranges from 32% to 93%. While few patients present with metastatic disease (stage IV) at presentation, some develop metastasis after initial definitive treatment. Immunotherapy and targeted therapies have improved the prognosis for these patients, with a 5-year survival rate of approximately 50%. Melanoma remains a serious health problem with a high medical need and incidence rates steadily increasing over the past 30 years. Therefore, there remains a great need for novel therapeutic approaches in this population.

[0006] Liver cancer is the fifth most common cancer worldwide and the second leading cause of cancer-related deaths, with 854,000 new cases and 810,000 deaths annually. Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, accounting for approximately 90% of all primary liver malignancies. Less common primary liver cancers include intrahepatic cholangiocarcinoma (iCCA), angiosarcoma, and hepatoblastoma. At the time of diagnosis, most patients with primary liver cancer have advanced disease, a stage at which treatment with curative therapies is not recommended. The WHO estimates that more than one million people will die from liver cancer in 2030, highlighting a significant global public health problem.

[0007] Thus, there is an unmet need in the art for the development of effective immunotherapies and methods of administration thereof for the treatment of cancer, including melanoma and liver cancer. Summary of the Invention

[0008] In one aspect, the disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject one or more dosage cycles of a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks.

[0009] In some embodiments, the cancer is a solid tumor.

[0010] In some aspects, the cancer is locally advanced or metastatic.

[0011] In some embodiments, the cancer is skin cancer, liver cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, or esophageal cancer. In some embodiments, the skin cancer is melanoma. In some embodiments, the skin cancer is previously untreated unresectable or metastatic melanoma. In some embodiments, the melanoma is (a) Stage III melanoma with measurable lymph node metastasis; (b) unresectable Stage III melanoma; or (c) Stage IV melanoma, optionally, the melanoma is not mucosal or uveal melanoma. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the kidney cancer is renal cell carcinoma (RCC). In some embodiments, the bladder cancer is metastatic urothelial carcinoma (mUC). In some embodiments, the breast cancer is triple-negative breast cancer (TNBC). In some embodiments, the esophageal cancer is esophageal squamous cell carcinoma (ESCC).

[0012] In another aspect, the disclosure provides methods for treating a subject having melanoma, comprising administering to the subject one or more dosage cycles of a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks, and the melanoma is (a) unresectable Stage III melanoma; or (b) Stage IV melanoma. In some aspects, the subject does not have intraocular melanoma.

[0013] In another aspect, the disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject one or more dosage cycles of a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks.

[0014] In some embodiments, the liver cancer is hepatocellular carcinoma (HCC). In some embodiments, the HCC is locally advanced, metastatic, and / or unresectable.

[0015] In some embodiments, the subject has not previously received systemic anti-cancer therapy.

[0016] In some embodiments, each of the one or more dosing cycles is 21 days in length. In some embodiments, the method comprises administering a bispecific antibody to the subject on day 1 of each of the one or more dosing cycles.

[0017] In some embodiments, the methods comprise administering the bispecific antibody intravenously to the subject.

[0018] In some embodiments, the method further comprises administering bevacizumab to the subject at a dose of about 15 mg / kg every three weeks. In some embodiments, each of the one or more dosing cycles is 21 days long, and the method comprises administering bevacizumab to the subject on day 1 of each of the one or more dosing cycles. In some embodiments, the bevacizumab is administered intravenously.

[0019] In some aspects, the subject has not been previously treated for metastatic or unresectable disease.

[0020] In some embodiments, the subject has not been previously treated with an anti-cancer therapy that includes an immunomodulatory agent.

[0021] In some embodiments, the subject has not been previously treated with an anti-LAG3 therapy.

[0022] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds PD-1, comprising a VH domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 25; (ii) an HVR-H2 sequence comprising the amino acid sequence GGR; and (iii) an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 26; and a VL domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 27; (ii) an HVR-L2 sequence comprising the amino acid sequence RSS; and (iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 28.

[0023] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises a VH domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 31; (ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 32; and (iii) an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 33; and a second antigen-binding domain that specifically binds LAG3, comprising a VL domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 34; (ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 35; and (iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 36.

[0024] In some embodiments, the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.

[0025] In some embodiments, the bispecific antibody is a full-length antibody.

[0026] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG, and optionally, the IgG Fc domain is an IgG1 Fc domain or an IgG4 Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, and optionally, the Fc receptor is an Fcγ receptor.

[0027] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises (a) an Fc domain of the human IgG1 subclass having the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index); and / or (b) an Fc domain comprising a modification that promotes association of a first subunit with a second subunit of the Fc domain.

[0028] In some aspects, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0029] In some embodiments, a bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain, a first Fab fragment comprising a first antigen-binding domain, and a second Fab fragment comprising a second antigen-binding domain. In some embodiments, in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3, the variable domains VL and VH are swapped for each other such that the VH domain is part of a light chain and the VL domain is part of a heavy chain, and optionally, the variable domains VL and VH are swapped for each other in the first Fab fragment. In some aspects, in the constant domain CL of one of the Fab fragments the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU index); and in the constant domain CHI the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index); and optionally in the constant domain CL of a second Fab fragment the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU index); and in the constant domain CHI the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0030] In some embodiments, the bispecific antibody comprises a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 39, a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 41, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 42. In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a first light chain comprising the amino acid sequence of SEQ ID NO: 40, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 41, and a second light chain comprising the amino acid sequence of SEQ ID NO: 42.

[0031] In some embodiments, the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in the tumor.

[0032] In some embodiments, the subject is a human.

[0033] In another aspect, the present disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject with cancer, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, the method comprising administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks.

[0034] In some embodiments, the cancer is a solid tumor.

[0035] In some aspects, the cancer is locally advanced or metastatic.

[0036] In some embodiments, the cancer is skin cancer, liver cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, or esophageal cancer. In some embodiments, the skin cancer is melanoma. In some embodiments, the skin cancer is previously untreated unresectable or metastatic melanoma. In some embodiments, the melanoma is (a) Stage III melanoma with measurable lymph node metastasis; (b) unresectable Stage III melanoma; or (c) Stage IV melanoma, optionally, the melanoma is not mucosal or uveal melanoma. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the kidney cancer is renal cell carcinoma (RCC). In some embodiments, the bladder cancer is metastatic urothelial carcinoma (mUC). In some embodiments, the breast cancer is triple-negative breast cancer (TNBC). In some embodiments, the esophageal cancer is esophageal squamous cell carcinoma (ESCC).

[0037] In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject with melanoma, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, the method comprising administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks, wherein the melanoma is (a) unresectable Stage III melanoma; or (b) Stage IV melanoma. In some aspects, the patient does not have intraocular melanoma.

[0038] In another aspect, the present disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method for treating a subject with liver cancer, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, the method comprising administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks. In some aspects, the liver cancer is HCC. In some aspects, the HCC is locally advanced, metastatic, and / or unresectable.

[0039] In some embodiments, the subject has not previously received systemic anti-cancer therapy.

[0040] In some embodiments, each of the one or more dosing cycles is 21 days in length. In some embodiments, the method comprises administering a bispecific antibody to the subject on day 1 of each of the one or more dosing cycles.

[0041] In some embodiments, the methods comprise administering the bispecific antibody intravenously to the subject.

[0042] In some embodiments, the method further comprises administering bevacizumab to the subject at a dose of about 15 mg / kg every three weeks. In some embodiments, each of the one or more dosing cycles is 21 days long, and the method comprises administering bevacizumab to the subject on day 1 of each of the one or more dosing cycles. In some embodiments, the bevacizumab is administered intravenously.

[0043] In some aspects, the subject has not been previously treated for metastatic or unresectable disease.

[0044] In some embodiments, the subject has not been previously treated with an anti-cancer therapy that includes an immunomodulatory agent.

[0045] In some embodiments, the subject has not been previously treated with an anti-LAG3 therapy.

[0046] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds PD-1, comprising a VH domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 25; (ii) an HVR-H2 sequence comprising the amino acid sequence GGR; and (iii) an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 26; and a VL domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 27; (ii) an HVR-L2 sequence comprising the amino acid sequence RSS; and (iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 28.

[0047] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises a VH domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 31; (ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 32; and (iii) an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 33; and a second antigen-binding domain that specifically binds LAG3, comprising a VL domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 34; (ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 35; and (iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 36.

[0048] In some embodiments, the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.

[0049] In some embodiments, the bispecific antibody is a full-length antibody.

[0050] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG, and optionally, the IgG Fc domain is an IgG1 Fc domain or an IgG4 Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, and optionally, the Fc receptor is an Fcγ receptor.

[0051] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises (a) an Fc domain of the human IgG1 subclass having the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index); and / or (b) an Fc domain comprising a modification that promotes association of a first subunit with a second subunit of the Fc domain.

[0052] In some aspects, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0053] In some embodiments, a bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain, a first Fab fragment comprising a first antigen-binding domain, and a second Fab fragment comprising a second antigen-binding domain. In some embodiments, in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3, the variable domains VL and VH are swapped for each other such that the VH domain is part of a light chain and the VL domain is part of a heavy chain, and optionally, the variable domains VL and VH are swapped for each other in the first Fab fragment. In some aspects, in the constant domain CL of one of the Fab fragments the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU index); and in the constant domain CHI the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index); and optionally in the constant domain CL of a second Fab fragment the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU index); and in the constant domain CHI the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0054] In some embodiments, the bispecific antibody comprises a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 39, a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 41, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 42. In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a first light chain comprising the amino acid sequence of SEQ ID NO: 40, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 41, and a second light chain comprising the amino acid sequence of SEQ ID NO: 42.

[0055] In some embodiments, the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in the tumor.

[0056] In some embodiments, the subject is a human.

[0057] In another aspect, the disclosure provides use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a medicament for treating a subject with cancer, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, and wherein the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks.

[0058] In some embodiments, the cancer is a solid tumor.

[0059] In some aspects, the cancer is locally advanced or metastatic.

[0060] In some embodiments, the cancer is skin cancer, liver cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, or esophageal cancer. In some embodiments, the skin cancer is melanoma. In some embodiments, the skin cancer is previously untreated unresectable or metastatic melanoma. In some embodiments, the melanoma is (a) Stage III melanoma with measurable lymph node metastasis; (b) unresectable Stage III melanoma; or (c) Stage IV melanoma, optionally, the melanoma is not mucosal or uveal melanoma. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the kidney cancer is renal cell carcinoma (RCC). In some embodiments, the bladder cancer is metastatic urothelial carcinoma (mUC). In some embodiments, the breast cancer is triple-negative breast cancer (TNBC). In some embodiments, the esophageal cancer is esophageal squamous cell carcinoma (ESCC).

[0061] In another aspect, the disclosure provides use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a medicament for treating a subject having melanoma, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, and the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks, and the melanoma is (a) unresectable Stage III melanoma; or (b) Stage IV melanoma. In some aspects, the subject does not have intraocular melanoma.

[0062] In another aspect, the present disclosure provides use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a medicament for treating a subject with liver cancer, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks. In some aspects, the liver cancer is HCC. In some aspects, the HCC is locally advanced, metastatic, and / or unresectable.

[0063] In some embodiments, the subject has not previously received systemic anti-cancer therapy.

[0064] In some embodiments, each of the one or more dosing cycles is 21 days in length, hi some embodiments, the bispecific antibody is administered to the subject on day 1 of each of the one or more dosing cycles.

[0065] In some embodiments, the bispecific antibody is administered intravenously to a subject.

[0066] In some embodiments, bevacizumab is administered to the subject at a dose of about 15 mg / kg every 3 weeks. In some embodiments, each of the one or more dosing cycles is 21 days long, and bevacizumab is administered to the subject on day 1 of each of the one or more dosing cycles. In some embodiments, bevacizumab is administered intravenously.

[0067] In some aspects, the subject has not been previously treated for metastatic or unresectable disease.

[0068] In some embodiments, the subject has not been previously treated with an anti-cancer therapy that includes an immunomodulatory agent.

[0069] In some embodiments, the subject has not been previously treated with an anti-LAG3 therapy.

[0070] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds PD-1, comprising a VH domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 25; (ii) an HVR-H2 sequence comprising the amino acid sequence GGR; and (iii) an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 26; and a VL domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 27; (ii) an HVR-L2 sequence comprising the amino acid sequence RSS; and (iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 28.

[0071] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises a VH domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 31; (ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 32; and (iii) an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 33; and a second antigen-binding domain that specifically binds LAG3, comprising a VL domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 34; (ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 35; and (iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 36.

[0072] In some embodiments, the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.

[0073] In some embodiments, the bispecific antibody is a full-length antibody.

[0074] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG, and optionally, the IgG Fc domain is an IgG1 Fc domain or an IgG4 Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, and optionally, the Fc receptor is an Fcγ receptor.

[0075] In some embodiments, the bispecific antibody targeting PD-1 and LAG3 comprises (a) an Fc domain of the human IgG1 subclass having the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index); and / or (b) an Fc domain comprising a modification that promotes association of a first subunit with a second subunit of the Fc domain.

[0076] In some aspects, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0077] In some embodiments, a bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain, a first Fab fragment comprising a first antigen-binding domain, and a second Fab fragment comprising a second antigen-binding domain. In some embodiments, in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3, the variable domains VL and VH are swapped for each other such that the VH domain is part of a light chain and the VL domain is part of a heavy chain, and optionally, the variable domains VL and VH are swapped for each other in the first Fab fragment. In some aspects, in the constant domain CL of one of the Fab fragments the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU index); and in the constant domain CHI the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index); and optionally in the constant domain CL of a second Fab fragment the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU index); and in the constant domain CHI the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0078] In some embodiments, the bispecific antibody comprises a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 39, a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 41, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 42. In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a first light chain comprising the amino acid sequence of SEQ ID NO: 40, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 41, and a second light chain comprising the amino acid sequence of SEQ ID NO: 42.

[0079] In some embodiments, the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in the tumor.

[0080] In some embodiments, the subject is a human. [Brief explanation of the drawings]

[0081] [Figure 1] Figure 1 is a flowchart showing the study design of a phase Ib / II clinical trial in patients with melanoma. Atezo = atezolizumab; CIT = cancer immunotherapy; CLND = complete lymph node dissection; Ipi = ipilimumab; Nivo = nivolumab; R = randomized; Tira = tiragolumab. [Figure 2] Figure 2 is a schematic diagram of the study scheme outlining the study timeline and activities in Cohort 1 of the Phase Ib / II clinical trial in patients with melanoma. CLND = complete lymph node dissection; Comp. = completion; CT = computed tomography; Discon. = discontinuation; M = month; R = randomization; Q3M = every 3 months; SFU = survival follow-up; Tx = treatment; W = week. [Figure 3]Figure 3 is a schematic diagram showing the minimal physiologically based pharmacokinetic (mPBPK) model of pembrolizumab. Jt = influx of pembrolizumab into the tumor compartment; kint = pembrolizumab-PD-1 complex internalization rate constant; koff = dissociation rate constant; kon = binding rate constant; kdeg = PD-1 degradation rate; Ksyn = PD-1 synthesis rate; Lt = lymph flow from the tumor compartment; L1 = lymph flow from the close tissue compartment; L2 = lymph flow from the leaking tissue compartment; mAb = pembrolizumab concentration; Qt = tumor plasma flow; RC = pembrolizumab-PD-1 complex concentration; Rmax = total PD-1 concentration; Vleaky = volume of tissue compartment with leaky vasculature; Vlymph = volume of lymphatic compartment; Vp = volume of plasma compartment; Vtight = volume of tissue compartment with tight vasculature; Vtumor = volume of tumor compartment; σtight = vascular reflectance coefficient of tight tissue; σleaky = vascular reflectance coefficient of leaky tissue; σLy = lymphatic reflectance coefficient. [Figure 4] Figure 4 is a schematic diagram showing the additional LAG3 receptor added to the PD1-LAG3 mPBPK model. [Figure 5] Figure 5 is a table showing a summary of adverse events in safety-evaluable patients in the dose escalation (Part A1, Q2W) portion of the NP41300 study. [Figure 6] Figure 6 is a table showing a summary of adverse events in safety-evaluable patients in the dose escalation (Part A1, Q2W) portion of the NP41300 study. [Figure 7]Figure 7 shows a series of boxplots showing predicted Ctrough after the first and third doses of PD1-LAG3 at doses of 600 mg or 1200 mg in a Q2W (every 2 weeks) or Q3W (every 3 weeks) dosing regimen. The lower and upper hinges correspond to the first and third quartiles (25th and 75th percentiles). The upper whiskers extend from the hinge to a maximum value no more than 1.5*IQR from the hinge (where IQR is the interquartile range, i.e., the distance between the first and third quartiles). The lower whiskers extend from the hinge to a minimum value up to 1.5*IQR of the hinge. Data beyond the ends of the whiskers are referred to as "outside" points and are plotted separately. Simulations were performed using a population pharmacokinetic model (a nonlinear mixed-effects modeling approach). For each dosing regimen, 500 individuals were simulated using a set of covariates bootstrapped (with replacement) from the original analysis dataset. [Figure 8] FIG. 8 is a graph showing simulated PD1 and LAG3 binding across a dose range for RO7247669 administered Q3W after 3 cycles. [Figure 9] Figure 9 is a flow chart showing the study design for the BP43963 trial in melanoma patients. [Figure 10] Figure 10 is a flowchart showing the study design of the GO42216 clinical trial. CIT = cancer immunotherapy; HCC = hepatocellular carcinoma; R = randomized. [Figure 11] Figure 11 is a flowchart showing the detailed study design of the GO42216 clinical trial. Bev = bevacizumab; HCC = hepatocellular carcinoma; Q2W = every 2 weeks; Q3W = every 3 weeks; R = randomized. DETAILED DESCRIPTION OF THE INVENTION

[0082] The present invention provides therapeutic methods and compositions for the treatment of cancer. Compositions, uses and kits comprising such combinations and / or dosing regimens are also provided herein.

[0083] I. Definition The following abbreviations are used herein: TIFF2024529451000001.tif100170

[0084] The term "about" as used herein refers to a normal error range for each value, which is readily understood by those skilled in the art. Reference to "about" a value or parameter herein includes (and describes) aspects directed to the value or parameter itself. For example, the description "about X" includes the description of "X."

[0085] As used herein, "TIGIT" or "T-cell immunoreceptor with Ig and ITIM domains" refers to any naturally occurring TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIG9, VSTM3, and WUCAM. This term encompasses "full-length," unprocessed TIGIT (e.g., full-length human TIGIT having the amino acid sequence of SEQ ID NO: 20) as well as any form of TIGIT resulting from processing within a cell (e.g., processed human TIGIT without a signal sequence and having the amino acid sequence of SEQ ID NO: 21). The term also encompasses naturally occurring variants of TIGIT, such as splice variants or allelic variants. An exemplary amino acid sequence of human TIGIT can be found, for example, at UniProt Accession No. Q495A1.

[0086] As used herein, "tiragolumab" is a fully human IgG1 / kappa MAb derived from Open Monoclonal Technology (OMT) rats that binds to TIGIT and comprises the heavy chain sequence of SEQ ID NO: 23 and the light chain sequence of SEQ ID NO: 24. Tiragolumab contains two N-linked glycosylation sites (N306) in the Fc domain. Tiragolumab is also listed in the WHO National Information on Drugs (International Nonproprietary Names of Medicinal Substances), proposed INN: List 117, Vol. 31, No. 2, published July 7, 2017 (see page 343).

[0087] The term "anti-TIGIT antagonist antibody" refers to an antibody or its antigen-binding fragment or variant that can bind to TIGIT with sufficient affinity to substantially or completely inhibit the biological activity of TIGIT. For example, an anti-TIGIT antagonist antibody can block signal transduction via PVR, PVRL2, and / or PVRL3 to restore functional responses to antigen stimulation by T cells from a dysfunctional state (e.g., proliferation, cytokine production, target cell killing). For example, an anti-TIGIT antagonist antibody can block signal transduction via PVR without affecting PVR-CD226 interaction. It will be understood by those skilled in the art that in some instances, an anti-TIGIT antagonist antibody can antagonize one TIGIT activity without affecting another TIGIT activity. For example, an anti-TIGIT antagonist antibody for use in certain methods or uses described herein is an anti-TIGIT antagonist antibody that antagonizes TIGIT activity in response to, for example, one of PVR interaction, PVRL3 interaction, or PVRL2 interaction, without affecting or minimally affecting any of the other TIGIT interactions. In one embodiment, the extent of binding of the anti-TIGIT antagonist antibody to an unrelated, non-TIGIT protein is less than about 10% of the binding of the antibody to TIGIT, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, TIGIT binding to the anti-TIGIT antagonist antibody is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10- -13 Dissociation constant (K D). In certain embodiments, the anti-TIGIT antagonist antibody binds to an epitope on TIGIT that is conserved among TIGITs from different species, or an epitope on TIGIT that allows cross-species reactivity. In some embodiments, the anti-TIGIT binding antibody has intact Fc-mediated effector function (e.g., tiragolumab, vibostolimab, etiglimab, EOS084448, or TJ-T6). In some embodiments, the anti-TIGIT binding antibody has enhanced Fc-mediated effector function (e.g., SGN-TGT). In other embodiments, the anti-TIGIT binding antibody lacks Fc-mediated effector function (e.g., domvanalimab, BMS-986207, ASP8374, or COM902). In some embodiments, the anti-TIGIT binding antibody is an IgG1 class antibody (e.g., tiragolumab, vibostolimab, domvanalimab, BMS-986207, etigilimab, BGB-A1217, SGN-TGT, EOS084448 (EOS-448), TJ-T6, or AB308). In some embodiments, the anti-TIGIT antagonist antibody is an IgG4 class antibody (e.g., ASP8374 or COM902). In one embodiment, the anti-TIGIT antagonist antibody is tiragolumab.

[0088] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with any one or more of its binding partners to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis, thereby restoring or enhancing T cell function (e.g., proliferation, cytokine production, and / or target cell killing). As used herein, PD-1 axis binding antagonists include PD-L1 binding antagonists, PD-1 binding antagonists, and PD-L2 binding antagonists. In some instances, the PD-1 axis binding antagonist is a PD-L1 binding antagonist or a PD-1 binding antagonist. In preferred embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[0089] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L1 with any one or more of its binding partners, such as PD-1 and / or B7-1. In some instances, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In particular aspects, PD-L1 binding antagonists inhibit the binding of PD-L1 to PD-1 and / or B7-1. In some instances, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In one example, the PD-L1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes via signaling through PD-L1, such that dysfunctional T cells become non-dysfunctional (e.g., enhance effector responses to antigen recognition). In some instances, the PD-L1 binding antagonist binds to PD-L1. In some instances, the PD-L1 binding antagonist is an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). Exemplary anti-PD-L1 antagonist antibodies include atezolizumab, MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), SHR-1316, CS1001, embafolimab, TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, cosibelimab, lodapolimab, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, RC98, PDL-GEX, KD036, KY1003, YBL-007, and HS-636. In some embodiments, the anti-PD-L1 antibody is atezolizumab, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab).In one particular embodiment, the PD-L1 binding antagonist is MDX-1105. In another particular embodiment, the PD-L1 binding antagonist is MEDI4736 (durvalumab). In another particular embodiment, the PD-L1 binding antagonist is MSB0010718C (avelumab). In other embodiments, the PD-L1 binding antagonist can be a small molecule, such as GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK041, which in some instances can be administered orally. Other exemplary PD-L1 binding antagonists include AVA-004, MT-6035, VXM10, LYN192, GB7003, and JS-003. In a preferred embodiment, the PD-L1 binding antagonist is atezolizumab.

[0090] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-1 and / or B7-1. PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." An exemplary human PD-1 is set forth in UniProtKB / Swiss-Prot Accession No. Q15116. In some instances, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In certain aspects, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signaling resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one example, the PD-1 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes via signaling through PD-1, so as to prevent dysfunctional T cells from becoming dysfunctional (e.g., enhancing effector responses to antigen recognition). In some instances, the PD-1 binding antagonist binds to PD-1. In some instances, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody).Exemplary anti-PD-1 antagonist antibodies include nivolumab, pembrolizumab, MEDI-0680, PDR001 (spartalizumab), REGN2810 (cemiplimab), BGB-108, prorugolimab, canrelizumab, sintilimab, tislelizumab, toripalimab, dostarimab, retifanlimab, sasanlimab, penprimab, CS1003, HLX10, SCT-I10A, zimberelimab, balstilimab, genolimuzumab, BI Examples of PD-1 binding antagonists include 754091, cetrelimab, YBL-006, BAT1306, HX008, budicalimab, AMG404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103, and hAb21. In a specific embodiment, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific embodiment, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific embodiment, the PD-1 binding antagonist is a PD-L2 Fc fusion protein, e.g., AMP-224. In another specific embodiment, the PD-1 binding antagonist is MED1-0680. In another specific embodiment, the PD-1 binding antagonist is PDR001 (spartalizumab). In another specific embodiment, the PD-1 binding antagonist is REGN2810 (cemiplimab). In another specific embodiment, the PD-1 binding antagonist is BGB-108. In another specific embodiment, the PD-1 binding antagonist is prorugolimab. In another specific embodiment, the PD-1 binding antagonist is canrelizumab. In another specific embodiment, the PD-1 binding antagonist is sintilimab. In another specific embodiment, the PD-1 binding antagonist is tislelizumab. In another specific embodiment, the PD-1 binding antagonist is toripalimab. Other additional exemplary PD-1 binding antagonists include BION-004, CB201, AUNP-012, ADG104, and LBL-006.

[0091] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, prevents, or interferes with signal transduction resulting from the interaction of PD-L2 with any one or more of its binding partners, such as PD-1. PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1LG2," "CD273," "B7-DC," "Btdc," and "PDL2." An exemplary human PD-L2 is set forth in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some instances, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In certain aspects, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. Exemplary PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signaling resulting from the interaction of PD-L2 with any one or more of its binding partners, such as PD-1. In one aspect, the PD-L2 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that mediate signaling through PD-L2, such that dysfunctional T cells become less dysfunctional (e.g., so as to enhance effector responses to antigen recognition). In some aspects, the PD-L2 binding antagonist binds to PD-L2. In some aspects, the PD-L2 binding antagonist is an immunoadhesin. In other aspects, the PD-L2 binding antagonist is an anti-PD-L2 antagonist antibody.

[0092] The terms "programmed death-ligand 1" and "PD-L1," as used herein, refer to native-sequence human PD-L1 polypeptide. Native-sequence PD-L1 polypeptide is provided by Uniprot Accession No. Q9NZQ7. For example, native-sequence PD-L1 may have the amino acid sequence set forth in Uniprot Accession No. Q9NZQ7-1 (isoform 1) (SEQ ID NO: 22). In another example, native-sequence PD-L1 may have the amino acid sequence set forth in Uniprot Accession No. Q9NZQ7-2 (isoform 2). In yet another example, native-sequence PD-L1 may have the amino acid sequence set forth in Uniprot Accession No. Q9NZQ7-3 (isoform 3). PD-L1 is also referred to in the art as "programmed cell death 1 ligand 1," "PDCD1LG1," "CD274," "B7-H," and "PDL1."

[0093] The Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (see, e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in immunoglobulin heavy chain constant regions (e.g., the EU index reported in Kabat et al., supra). The "Kabat EU index" refers to the residue numbering of the human IgG1 EU antibody.

[0094] As used herein, "atezolizumab" refers to an Fc-engineered, humanized, aglycosylated IgG1 kappa immunoglobulin that binds PD-L1 and comprises the heavy chain sequence of SEQ ID NO: 62 and the light chain sequence of SEQ ID NO: 63. Atezolizumab contains a single amino acid substitution (asparagine to alanine) (N297A) at position 297 on the heavy chain using EU numbering of Fc region amino acid residues, resulting in an aglycosylated antibody with minimal binding to Fc receptors. Atezolizumab is also listed in the WHO National Pharmaceutical Information (International Nonproprietary Names for Medicinal Substances), proposed INN: List 112, Vol. 28, No. 4, published January 16, 2015 (see page 485).

[0095] The term "cancer" refers to a disease caused by the uncontrolled division of abnormal cells in a part of the body. In one example, the cancer is skin cancer (e.g., melanoma, basal cell carcinoma (BCC), squamous cell carcinoma, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans (DFSP), Merkel cell carcinoma, or sebaceous carcinoma). In another example, the cancer is liver cancer (e.g., hepatocellular carcinoma (HCC), e.g., locally advanced or metastatic HCC and / or unresectable HCC). Cancer includes solid tumor cancer and non-solid tumor cancer, as well as locally advanced or metastatic cancer (e.g., locally advanced or metastatic tumor). Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, locally advanced and metastatic UC (mUC), bladder cancer (e.g., muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC), e.g., BCG-refractory NMIBC), urothelial carcinoma (UC), including MIBC urothelial bladder cancer (UBC); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); urinary tract cancer; lung cancer, such as small cell lung cancer (SCLC), including extensive-stage SCLC (ES-SCLC); non-small cell lung cancer (NSCLC), including locally advanced unresectable NSCLC (e.g., IIIB NSCLC), or recurrent or metastatic NSCLC (e.g., stage IV NSCLC), adenocarcinoma of the lung, or squamous cell carcinoma (e.g., squamous cell carcinoma (e.g., squamous cell carcinoma of the lung)); pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC), e.g., metastatic PDAC); head and neck cancer (e.g., SCCHN, e.g., recurrent / metastatic PD-L1 positive SCCHN, head and neck squamous cell carcinoma (HNSCC); ovarian cancer (OC); esophageal cancer; peritoneal cancer; hepatocellular carcinoma; gastric cancer (GC) (e.g., gastroesophageal junction (GEJ) or gastric cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; glioblastoma; urinary tract cancer; liver cancer; breast cancer (e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC (e.g., early TNBC (eTNBC)), estrogen receptor (ER-), progesterone receptor (PgR-), and HER2 (HER2-) negative); prostate cancer such as castration-resistant prostate cancer (CRPC), peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC));Glioblastoma; cervical cancer (e.g., stage IVB, metastatic, recurrent, or persistent cervical cancer, e.g., metastatic and / or recurrent PD-L1-positive cervical cancer); ovarian cancer; liver cancer; colon cancer; rectal cancer; colorectal cancer (CRC; e.g., CRC with microsatellite stable (MSS) and microsatellite instability (MSI) low (MSI-Low)); endometrial or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma (including superficial metastatic melanoma, melanoma-associated melanoma, acral melanoma, and nodular melanoma); multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL ; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade non-cleaving cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myoblastic leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal blood vessel growth associated with phacomatosis, edema (such as that associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and related metastases.

[0096] In some instances, the cancer (eg, a skin cancer (eg, melanoma) or a liver cancer (eg, HCC)) is a tumor that has a tumor microenvironment that includes LAG3-expressing CD8+ T cells.

[0097] In some instances, the cancer may be unresectable (eg, unresectable locally advanced or metastatic cancer).

[0098] Further examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of cancer include, but are not limited to, esophageal cancer (e.g., squamous cell carcinoma (e.g., esophageal squamous cell carcinoma (ESCC)), adenocarcinoma (e.g., esophageal adenocarcinoma (EAC)), or esophageal cancer with neuroendocrine histopathology (e.g., esophageal neuroendocrine carcinoma (ENEC)). Further examples include metastatic esophageal cancer (e.g., metastatic ESCC, metastatic EAC, or metastatic ENEC). In one example, the cancer is colorectal cancer (CRC). As used herein, When used herein, the terms "colorectal cancer," "CRC," "colon cancer," or "intestinal cancer" refer to cancer that originates in the large intestine, e.g., the colon or rectum (e.g., colorectal adenocarcinoma). Examples of cancer include, but are not limited to, hematological cancers such as mature B-cell cancers, e.g., excluding Hodgkin's lymphoma, but non-Hodgkin's lymphoma (NHL), e.g., diffuse large B-cell lymphoma (DLBCL), which may be relapsed or refractory DLBCL or Richter's transformation.Other specific examples of cancer include germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), transformed FL, mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), transformed MZL, high-grade B-cell lymphoma, and primary mediastinal (thymic) large B-cell lymphoma (PMLBCL). , small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), transformed LL, Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassifiable, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, heavy chain leukemia, alpha heavy chain disease, gamma heavy chain disease, μ heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodular marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL, leg type, EBV-positive DLBCL of the elderly, DLBCL associated with chronic inflammation, lymphoma Examples of cancer include lymphomatoid granulomatosis, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma due to HV8-associated multicentric Castleman disease, primary effusion lymphoma, unclassifiable B-cell lymphoma with features intermediate between DLBCL and Burkitt's lymphoma, and unclassifiable B-cell lymphoma with features intermediate between DLBCL and classical Hodgkin's lymphoma. Further examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and lymphoid malignancies, including leukemia or B-cell lymphoma.More specific examples of such cancers include, but are not limited to, multiple myeloma (MM); low-grade / follicular NHL; small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphocytic NHL; high-grade small non-dividing cell NHL; bulky mass disease NHL; AIDS-related lymphoma; and acute lymphocytic leukemia (ALL); chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD).

[0099] The terms "B-cell proliferative disorder" or "B-cell malignancy" refer to diseases associated with some degree of abnormal B-cell proliferation and include, for example, lymphoma, leukemia, myeloma, and myelodysplastic syndrome. In some instances, the B-cell proliferative disorder is a lymphoma, such as non-Hodgkin's lymphoma (NHL), including, for example, follicular lymphoma (FL) (e.g., relapsed and / or refractory FL or transformed FL), diffuse large B-cell lymphoma (DLBCL) (e.g., relapsed or refractory DLBCL or Richter's transformed), MCL, high-grade B-cell lymphoma, or PMLBCL. In another embodiment, the B-cell proliferative disorder is a leukemia, such as chronic lymphocytic leukemia (CLL). In one embodiment, the B-cell proliferative disorder is relapsed and / or refractory.

[0100] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0101] As used herein, "tumor cell" refers to any tumor cell present in a tumor or a sample thereof. Tumor cells can be distinguished from other cells, e.g., stromal cells and tumor-infiltrating immune cells, that may be present in a tumor sample using methods known in the art and / or described herein.

[0102] "Tumor immunity" refers to the process by which tumors evade immune recognition and elimination. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor regression, and tumor clearance.

[0103] As used herein, "metastasis" refers to the spread of cancer from its primary site to other locations in the body. Cancer cells may break away from the primary tumor, infiltrate lymphatic and blood vessels, circulate through the bloodstream, and grow (metastasize) at distant foci within normal tissue elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process in which tumor cells break away from the primary tumor, travel through the bloodstream, and arrest at a distant site. At the new site, the cells establish a blood supply and may grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within tumor cells control this behavior, and interactions between tumor cells and host cells at distant sites are also important.

[0104] As used herein, "treating" includes effective cancer treatment with an effective amount of a therapeutic agent (e.g., a bispecific antibody targeting PD-1 and LAG3). Treatment herein includes, inter alia, adjuvant therapy, neoadjuvant therapy, non-metastatic cancer therapy (e.g., locally advanced cancer therapy), and metastatic cancer therapy. Treatment can be first-line therapy (e.g., the patient may not have been previously treated or has not received prior systemic therapy) or second-line or subsequent therapy.

[0105] As used herein, an "effective amount" refers to the amount of a therapeutic agent (e.g., a bispecific antibody targeting PD-1 and LAG3 or a combination of therapeutic agents (e.g., a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, tiragolumab, or a VEGF antagonist, e.g., an anti-VEGF antibody (e.g., bevacizumab))) that achieves a therapeutic result. In some examples, an effective amount of a therapeutic agent or combination of therapeutic agents is the amount of a therapeutic agent or combination of therapeutic agents that achieves the clinical endpoints of improved pathological response rate (PRR), improved overall response rate (ORR), improved disease control rate (DCR), complete response (CR), pathological complete response (pCR), partial response (PR), improved survival (e.g., disease-free survival (DFS), and / or progression-free survival (PFS), and / or overall survival (OS)), and / or improved duration of response (DOR).

[0106] As used herein, "complete response" and "CR" refer to the disappearance of all target lesions.

[0107] As used herein, "partial response" or "PR" refers to at least a 30% reduction in the sum of the longest diameters (SLD) of target lesions relative to the pre-treatment baseline SLD.

[0108] As used herein, "progressive disease" and "PD" refer to at least a 20% increase in the SLD of target lesions relative to the smallest total (nadir) during the study, including baseline. The appearance of one or more new lesions may also be considered PD.

[0109] As used herein, "stable disease" and "SD" do not mean a sufficient reduction to qualify for PR or an increase to qualify for PD, based on a minimum sum.

[0110] As used herein, "disease control rate" and "DCR" refer to the percentage of patients with advanced or metastatic cancer who achieve CR, PR, and stable disease (SD). For example, DCR is defined as the proportion of patients with 12 weeks or more of SD or CR or PR as determined by the investigator according to RECIST v1.1.

[0111] As used herein, "overall response rate," "objective response rate," and "ORR" refer interchangeably to the sum of the CR rate and PR rate. For example, an objective response may be defined as a CR or PR according to Response Evaluation Criteria in Solid Tumors (RECIST) v.1.1, as determined by investigator assessment and confirmed by repeat evaluation at least four weeks after initial recording. In another example, ORR may be defined as the proportion of patients with CR or PR on two consecutive occasions at least 4 weeks apart, as determined by the investigator according to RECIST v1.1.

[0112] As used herein, "pathological response rate" and "pRR" refer interchangeably to the proportion of patients who have a pathological complete response (pCR, e.g., the complete absence of viable tumor in the treated tumor bed), a pathological near complete response (pnCR, e.g., <10% of the treated tumor bed is occupied by viable tumor cells), and a pathological partial response (pPR, e.g., <50% of the treated tumor bed is occupied by viable tumor cells), e.g., at the time of surgery.

[0113] As used herein, "progression-free survival" and "PFS" refer to the length of time during and after treatment during which cancer does not worsen. PFS can include the amount of time a patient experiences CR or PR, and the amount of time a patient experiences stable disease. For example, PFS can be defined as the time from initial study treatment to the first occurrence of progression or death from any cause, as determined by RECIST v.1.1 as determined by the investigator, whichever occurs first. In another example, PFS can be defined as the time from initial study enrollment to the first occurrence of progression or death from any cause, as determined by RECIST v.1.1 as determined by the investigator, whichever occurs first.

[0114] As used herein, "overall survival" and "OS" refer to the length of time from either the date of diagnosis or the start of treatment for a disease (e.g., cancer) that a patient is still alive. For example, OS can be defined as the time from first study treatment to death from any cause.

[0115] As used herein, the terms "duration of response" and DOR refer to the length of time from documented tumor response to disease progression or death from any cause, whichever occurs first. For example, DOR can be defined as the time from the first occurrence of a documented objective response according to RECIST v1.1 as determined by the investigator to the first documented disease progression or death from any cause, whichever occurs first.

[0116] As used herein, the term "chemotherapeutic agent" refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include EGFR inhibitors (including small molecule inhibitors such as erlotinib (TARCEVA®, Genentech / OSI Pharm.); PD183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca; ZM105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim; PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine; CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)- 2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer); and dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016, or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[(2-methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine); tyrosine kinase inhibitors (e.g., EGFR inhibitors; small molecule HER2 tyrosine kinase inhibitors such as TAK165 (Takeda);CP-724,714, an oral selective inhibitor of ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2- and EGFR-overexpressing cells; PKI-166 (Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as ISIS-5132 (ISIS Pharmaceuticals), an antisense drug that inhibits Raf-1 signaling; non-HER-targeted tyrosine kinase inhibitors such as imatinib mesylate (GLEEVEC®, GlaxoSmithKline); multi-target tyrosine kinase inhibitors such as sunitinib (SUTENT®, Pfizer); vatalanib (PTK787 / ZK222584, Novartis / Schering) VEGF receptor tyrosine kinase inhibitors such as VEGF receptor tyrosine kinase inhibitors (VEGF receptor tyrosine kinase inhibitors (AG)); the MAPK extracellular regulated kinase I inhibitor CI-1040 (Pharmacia); quinazolines such as PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP59326, CGP60261, and CGP62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to nucleic acids encoding HER); quinoxalines (U.S. Patent No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); ZD6474 (Astra Zeneca; PTK-787 (Novartis / Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Afinitac (ISIS3521; Isis / Lilly); PKI166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Pfizer); ZD6474 (AstraZeneca);PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); and rapamycin (sirolimus, RAPAMUNE®); bortezomib (VELCADE®, Millennium Proteasome inhibitors such as fluticasone (fluticasone-3), fluticasone-4, fluticasone-5, fluticasone-6, fluticasone-7, fluticasone-8, fluticasone-9, fluticasone-10, fluticasone-11, fluticasone-12, fluticasone-13, fluticasone-14, fluticasone-15, fluticasone-16, fluticasone-17, fluticasone-18, fluticasone-19, fluticasone-20, fluticasone-21, fluticasone-22, fluticasone-23, fluticasone-24, fluticasone-25, fluticasone-26, fluticasone-30, fluticasone-31, fluticasone-32, fluticasone-33, fluticasone-44, fluticasone-45, fluticasone-46, fluticasone-47, fluticasone-48, fluticasone-49, fluticasone-50, fluticasone-51, fluticasone-52, fluticasone-53, fluticasone-54, fluticasone-55, fluticasone-55, fluticasone-55, fluticasone-56, fluticasone-57, fluticasone-58, fluticasone-59 ...9, fluticasone-59, flu Labs); alkylating agents such as AG1478, thiotepa, and Cytoxan® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylameramines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; kallistatin; C C-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductases (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat dolastatins; aldesleukin, talc; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatins;Nitrogen mustards such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1 and calicheamicin omega 1); dynemicin antibiotics, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoproteins (enediine antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin ), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; fludarabine, 6 Purine analogs such as mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calucelone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergic agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid;Eniluracil; Amsacrine; Bestravsil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diazicon; Elfomitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidamol; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK® Polysaccharide Complex (JHS Natural Products); Razoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacetocin; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Chlorambucil; GEMZAR® (Gemcitabine) ; 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP-16); ifosfamide; mitoxantrone; novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, prodrugs, and derivatives of any of the above.

[0117] Chemotherapeutic agents also include (i) antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators, including tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate). (ii) aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestani, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and A RIMIDEX® (anastrozole; AstraZeneca), etc.; (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid protein kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Ralf, and H-Ras; (vii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®;(ix) proliferation inhibitors, including vincas (e.g., vincristine and vinblastine), NAVELBINE® (vinorelbine), taxanes (e.g., paclitaxel, nab-paclitaxel, and docetaxel), topoisomerase II inhibitors (e.g., doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin), and DNA alkylating agents (e.g., tamoxigen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C); and (x) pharmaceutically acceptable salts, acids, prodrugs, and derivatives of any of the above;

[0118] As used herein, the term "cytotoxic agent" refers to any agent that is detrimental to cells (e.g., causing cell death, inhibiting growth, or interfering with cell function). Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212, and radioactive isotopes of Lu); chemotherapeutic agents; enzymes, such as nucleases and fragments thereof; and toxins, such as small molecule or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Exemplary cytotoxic agents may be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid biosynthesis, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism. In one example, the cytotoxic agent is a platinum-based chemotherapeutic agent (e.g., carboplatin or cisplatin). In one example, the cytotoxic agent is an EGFR antagonist, such as N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g., erlotinib). In one example, the cytotoxic agent is a RAF inhibitor, such as a BRAF and / or CRAF inhibitor. In one example, the RAF inhibitor is vemurafenib. In one example, the cytotoxic agent is a PI3K inhibitor.

[0119] The term "patient" or "subject" refers to a human patient or subject. For example, the patient or subject can be an adult.

[0120] The term "antibody" specifically encompasses monoclonal antibodies (such as full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. In one example, the antibody is a full-length monoclonal antibody.

[0121] The term IgG "isotype" or "subclass" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions.

[0122] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and are generally described, for example, in Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). An antibody can be part of a larger fusion molecule formed by covalent or noncovalent association of the antibody with one or more other proteins or peptides.

[0123] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment as described below. This term refers to an antibody that includes an Fc region.

[0124] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or a truncated variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447), may or may not be present. The amino acid sequence of a heavy chain comprising an Fc region is shown herein without the C-terminal lysine (Lys447) unless otherwise indicated. In one aspect, a heavy chain comprising an Fc region as specified herein contained in an antibody disclosed herein comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447). In one aspect, a heavy chain comprising an Fc region as specified herein contained in an antibody disclosed herein comprises an additional C-terminal glycine residue (G446). In one aspect, a heavy chain comprising an Fc region as specified herein contained in an antibody disclosed herein comprises an additional C-terminal lysine residue (K447). In one embodiment, the Fc region comprises a single amino acid substitution, N297A, in the heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to EU numbering, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0125] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0126] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (which may contain, for example, naturally occurring mutations or arise during the production of the monoclonal antibody preparation, and such variants are typically present in minor amounts). Each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen, in contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes). Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0127] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain, e.g., the "complementarity-determining regions" (CDRs), which are hypervariable in sequence and determine antigen-binding specificity.

[0128] Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact sites present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0129] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0130] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in the VH (or VL) in the following order: FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0131] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al. (see above). Using this numbering system, the actual linear amino acid sequence may contain fewer amino acids corresponding to a shortening of the FR or HVR of the variable domain or additional amino acids corresponding to an insertion into the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and may contain inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment with the "standard" Kabat numbering sequence in the homologous regions of the antibody sequence.

[0132] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each binding to the same epitope of the same antigen. As used herein, the term "bispecific" antibody refers to an antibody that can specifically bind to at least two different antigens, e.g., two binding sites formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), each binding to a different antigen or a different epitope on the same antigen. Such bispecific antibodies are in a 1+1 format. Other bispecific antibody formats are the 2+1 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or the 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, bispecific antibodies contain two antigen-binding sites, each specific for a different antigen.

[0133] As used herein, "PD-L1-positive tumor cell fraction" is the percentage of viable tumor cells that exhibit partial or complete membrane staining (excluding cytoplasmic staining) of any intensity relative to all viable tumor cells present in a sample following staining of the sample in association with an immunohistochemistry (IHC) assay, such as an IHC assay to stain PD-L1 using antibodies SP263, SP142, 22C3, or 28-8. Thus, the PD-L1-positive tumor cell fraction can be calculated using the PD-L1 IHC SP142 (Ventana) assay, for example, by the formula: PD-L1-positive tumor cell fraction = (number of PD-L1-positive tumor cells) / (total number of PD-L1-positive and PD-L1-negative tumor cells), where PD-L1 cytoplasmic staining of tumor cells and all non-tumor cells (e.g., tumor-infiltrating immune cells, normal cells, necrotic cells, and debris) is excluded from evaluation and scoring. It will be understood that any given diagnostic PD-L1 antibody may correspond to a particular IHC assay protocol and / or scoring terminology that can be used to obtain the PD-L1 positive tumor cell fraction. For example, the PD-L1 positive tumor cell fraction can be obtained from tumor cell samples stained with SP263, 22C3, SP142, or 28-8 using OPTIVIEW® detection on a Benchmark ULTRA, EnVision Flex on an AutostainerLink 48, OPTIVIEW® detection and amplification on a Benchmark ULTRA, or EnVision Flex on an AutostainerLink 48, respectively.

[0134] As used herein, the "Ventana SP142 IHC assay" is described in the Ventana PD-L1 (SP142) Assay Package Insert (Tucson, AZ: Ventana Medical Systems, Inc.), which is incorporated herein by reference in its entirety.

[0135] As used herein, the "Ventana SP263 IHC assay" is described in the Ventana PD-L1 (SP263) Assay Package Insert (Tucson, AZ: Ventana Medical Systems, Inc.), which is incorporated herein by reference in its entirety.

[0136] As used herein, a "pharmDx 22C3 IHC assay" is performed in accordance with the PD-L1 IHC 22C3 pharmDx package insert (Dako, Agilent Pathology Solutions, Carpinteria, CA), which is incorporated herein by reference in its entirety.

[0137] As used herein, a "pharmDx 28-8 IHC assay" is performed in accordance with the PD-L1 IHC 28-8 pharmDx package insert (Dako, Agilent Pathology Solutions, Carpinteria, CA), which is incorporated herein by reference in its entirety.

[0138] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings for use of such therapeutic product.

[0139] As used herein, "in combination with" refers to the administration of one therapeutic modality in addition to another, e.g., a therapeutic regimen that includes the administration of a bispecific antibody targeting programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG3) with an anti-TIGIT antagonist antibody (e.g., tiragolumab) or a VEGF antagonist, e.g., an anti-VEGF antibody (e.g., bevacizumab). Thus, "in combination with" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to a patient.

[0140] A drug that is administered "concurrently" with one or more other drugs is administered on the same treatment day as the other drug(s), and optionally at the same time as the other drug(s) during the same treatment cycle. For example, in the case of cancer therapy administered every three weeks, the concurrently administered drugs would be administered on day 1 of each three-week cycle.

[0141] As used herein, the term "adverse event" or "AE" refers to any untoward and unintended sign (including abnormal laboratory findings), symptom, or disease temporally associated with the use of a medical treatment or procedure, which may or may not be considered related to the medical treatment or procedure. Adverse events can be classified by "grade" as defined by the National Cancer Institute Common Terminology Criteria for Adverse Events v4.0 or v5.0 (NIH CTCAE). In some embodiments, the AE is a low-grade AE, e.g., a grade 1 or grade 2 AE. Grade 1 includes AEs that are asymptomatic or have mild symptoms. Grade 2 includes AEs that are moderate, limit age-appropriate instrumental activities of daily living (e.g., meal preparation, grocery or clothing shopping), and indicate the need for local or non-invasive intervention. In other instances, the AE is a high-grade AE, e.g., a grade 3, grade 4, or grade 5 AE. In some instances, the AE is a grade 3 or grade 4 AE. Grade 3 includes AEs that are serious or medically significant but not immediately life-threatening and indicate the need for hospitalization or prolonged hospitalization, Grade 4 includes AEs that have life-threatening consequences and indicate the need for urgent interventional treatment, and Grade 5 includes AEs that result in or are associated with death.

[0142] As used herein, the term "treatment-related AE" refers to an AE determined by the investigator to have occurred as a result of a treatment, e.g., a PD-1 axis binding antagonist therapy (e.g., atezolizumab therapy) and / or an anti-TIGIT antagonist antibody therapy (e.g., tiragolumab therapy).

[0143] The term "valent" as used within this application refers to the presence of a specific number of binding domains in an antigen-binding molecule. In this context, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding domains, four binding domains, and six binding domains, respectively, in an antigen-binding molecule. Bispecific antibodies of the present invention are at least "bivalent," and may also be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). In certain embodiments, antibodies of the present invention have two or more binding sites and are bispecific. That is, antibodies can be bispecific even when more than two binding sites are present (i.e., the antibody is trivalent or multivalent).

[0144] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibodies made from antibody fragments, and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have extended in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments that contain two antigen-binding domains that are bivalent or bispecific, and are described, e.g., in EP 404097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003), and Hollinger et al., Proc. See Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1).Additionally, antibody fragments comprise a single polypeptide chain characterized by a VH domain (i.e., capable of assembling with a VL domain) or characterized by a VL domain (i.e., capable of assembling with a VH domain into a functional antigen-binding site), thereby conferring antigen-binding properties of a full-length antibody. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0145] Papain digestion of an intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, as well as the light chain constant domain and the first heavy chain constant domain (CH1). Thus, as used herein, the term "Fab fragment" refers to an antibody fragment containing the light chain VL domain and constant domain (CL) and the heavy chain VH domain and first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment containing two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0146] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. Two possible chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the present invention. On the other hand, the variable regions of the Fab heavy and light chains have been exchanged, i.e., the crossover Fab molecule contains a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). This crossover Fab molecule is called CrossFab. (VLVH)On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule contains a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL), and a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1). This crossover Fab molecule is called CrossFab. (CLCH1) It is also called.

[0147] A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, where the antibody domains and linker are arranged in the following order from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; the linker is a polypeptide of at least 30 amino acids, preferably 32-50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules may be further stabilized by the creation of interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0148] A "crossover single-chain Fab fragment" or "x-scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have, from N- to C-terminus, one of the following orders: a) VH-CL-linker-VL-CH1; and b) VL-CH1-linker-VH-CL; together, VH and VL form an antigen-binding domain that specifically binds to a certain antigen, and the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules may be further stabilized by the creation of an interchain disulfide bond by the insertion of cysteine ​​residues (e.g., position 44 of the variable heavy chain and position 100 of the variable light chain according to the Kabat numbering system).

[0149] A "single-chain variable fragment (scFv)" is a fragment of an antibody heavy chain (V) linked using a short linker peptide of 10 to about 25 amino acids. H ) and light chain (V L ) variable region fusion proteins. The linker is usually rich in glycine for flexibility, as well as rich in serine or threonine for solubility, and V H N-terminus of V L The scFv antibody can be linked to the C-terminus of a full-length antibody, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 4696. In addition, antibody fragments contain a single polypeptide chain characterized by a VH domain (i.e., capable of assembling with a VL domain) or a VL domain (i.e., capable of assembling with a VH domain into a functional antigen-binding site), thereby conferring the antigen-binding properties of a full-length antibody.

[0150] Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domain was derived from the variable domain of an antibody heavy chain from a camel (nanobody or V). HFurthermore, the term single domain antibody refers to an antibody that contains an autonomous human heavy chain variable domain (aVH) or a shark-derived VH. NAR These include fragments. Fibronectin is a scaffold that can be engineered to bind to antigens. Adnectins consist of a backbone with the native amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the β-sandwich can be engineered to enable Adnectins to specifically recognize therapeutic targets of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent Application Publication No. 20080139791, WO 2005056764, and U.S. Patent No. 6,818,418. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted into the active site. For further details, see Expert Opin. Biol. Ther. 5, 783797 (2005). Microbodies are derived from naturally occurring microproteins that are 25-50 amino acids long and contain three to four cysteine ​​bridges; examples of microproteins include KalataBI, conotoxins, and knottins. The microproteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

[0151] The terms "a bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3," "a bispecific antibody that specifically binds PD-1 and LAG3," "a bispecific antigen-binding molecule specific for PD-1 and LAG3," or "anti-PD-1 / anti-LAG3 antibody" are used interchangeably herein and refer to a bispecific antibody that is capable of binding to PD-1 and LAG3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting PD-1 and LAG3.

[0152] The term "PD-1," also known as programmed cell death protein 1, is a 288-amino acid type I membrane protein first described in 1992 (Ishida et al., EMBO J., 11 (1992), 3887-3895). PD-1 is a member of the extended CD28 / CTLA-4 family of T cell regulatory factors and possesses two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The protein structure comprises an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located within an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively regulates TCR signaling. This is consistent with the binding of SHP-1 and SHP-2 phosphatases to the cytoplasmic tail of PD-1 upon ligand binding. PD-1 is not expressed on naive T cells but is upregulated following T cell receptor (TCR)-mediated activation and is observed on both activated and exhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772). These exhausted T cells have a dysfunctional phenotype and are unable to respond appropriately. PD-1 has a relatively broad expression pattern, but its most important role may be as a co-inhibitory receptor for T cells (Chinai et al., Trends in Pharmacological Sciences 36 (2015), 587-595). Therefore, current therapeutic approaches focus on blocking the interaction of PD-1 with its ligand to enhance T cell responses. The terms "programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PD1," "PDCD1," "hPD-1," and "hPD-I" can be used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one epitope in common with PD-1. The amino acid sequence of human PD-1 is set forth in UniProt (www.uniprot.org) Accession No. Q15116 (SEQ ID NO: 55).

[0153] The term "LAG3" or "Lag-3" or "lymphocyte activation gene-3" or "CD223," as used herein, unless otherwise specified, refers to any naturally occurring LAG3 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed LAG3, as well as any form of LAG3 that results from processing within a cell. The term also encompasses naturally occurring variants of LAG3, such as splice variants or allelic variants. In a preferred embodiment, the term "LAG3" refers to human LAG3. The amino acid sequence of an exemplary processed (signal sequence-less) LAG3 is set forth in SEQ ID NO:56. The amino acid sequence of an exemplary extracellular domain (ECD) LAG3 is set forth in SEQ ID NO:57.

[0154] The terms "anti-LAG3 antibody" and "antibody that binds to LAG3" refer to an antibody that can bind to LAG3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting LAG3. In one aspect, the binding of an anti-LAG3 antibody to an unrelated, non-LAG3 protein is less than about 10% of the binding of the antibody to LAG3 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to LAG3 has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 ~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D In certain aspects, the anti-LAG3 antibody binds to an epitope of LAG3 that is conserved among LAG3s from different species. In a preferred embodiment, "anti-LAG3 antibody," "antibody that specifically binds to human LAG3," and "antibody that binds to human LAG3" refer to an antibody that binds to the human LAG3 antigen or its extracellular domain (ECD) by a K D The value is 1.0x10 -8mol / l or less, in one embodiment K D The value is 1.0x10 -9 mol / l or less, in one embodiment K D The value is 1.0x10 -9 mol / l to 1.0x10 -13 "Anti-LAG3 antibody" refers to an antibody that specifically binds with a binding affinity of mol / l. In this respect, binding affinity is determined using standard binding assays, such as surface plasmon resonance technology (BIAcore®, GE-Healthcare Uppsala, Sweden), for example, using the LAG3 extracellular domain. The term "anti-LAG3 antibody" also encompasses bispecific antibodies capable of binding to LAG3 and a second antigen.

[0155] This knob-into-hole technique is described, for example, in U.S. Pat. No. 5,731,168 and U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, positioning the protrusion within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In a more specific embodiment, the Fc domain subunit containing the knob modification further comprises the amino acid substitution S354C, and the Fc domain subunit containing the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues forms a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0156] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); cytokine secretion; immune complex-mediated antigen uptake by antigen-presenting cells; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0157] An "activating Fc receptor" is an Fc receptor that, following binding by the Fc region of an antibody, triggers signaling events that stimulate the receptor-bearing cell to exert effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0158] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n , (SG4) n or G4 (SG4) na peptide linker, where "n" is usually a number from 1 to 10, typically 2 to 4, in particular 2, i.e. a peptide selected from the group consisting of GGGGS (SEQ ID NO:46), GGGGSGGGGS (SEQ ID NO:47), SGGGGSGGGG (SEQ ID NO:48) and GGGGSGGGGSGGGG (SEQ ID NO:49), but also including the sequences GSPGSSSSGS (SEQ ID NO:50), (GS) (SEQ ID NO:51), (GS) (SEQ ID NO:52), GSGSGSGS (SEQ ID NO:53), GSGSGNGS (SEQ ID NO:54), GGSGSGSG (SEQ ID NO:55), GGSGSG (SEQ ID NO:56), GGSG (SEQ ID NO:57), GGSGNGSG (SEQ ID NO:58), GGNGSGSG (SEQ ID NO:59) and GGNGSG (SEQ ID NO:60). Peptide linkers of particular interest are (G4S) (SEQ ID NO: 46), (G4S) or GGGGSGGGGS (SEQ ID NO: 47), (G4S) (SEQ ID NO: 51), and (G4S) (SEQ ID NO: 53), more specifically (G4S) (SEQ ID NO: 47) or GGGGSGGGGS (SEQ ID NO: 47).

[0159] By "fused to" or "linked to" is meant that the components (e.g., an antigen binding domain and an Fc domain) are linked by a peptide bond either directly or via one or more peptide linkers.

[0160] The term "VEGF antagonist" or "VEGF-specific antagonist" refers to a molecule capable of binding to VEGF, reducing VEGF expression levels, or neutralizing, blocking, inhibiting, abrogating, reducing, or interfering with the biological activities of VEGF (including, but not limited to, VEGF binding to one or more VEGF receptors, VEGF signaling, and VEGF-mediated angiogenesis, and endothelial cell survival or proliferation). For example, a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing, or interfering with the biological activities of VEGF may exert its effect by binding to one or more VEGF receptors (VEGFRs) (e.g., VEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF receptors (mbVEGFRs), or soluble VEGF receptors (sVEGFRs)). Such antagonists are also referred to herein as "VEGFR inhibitors." VEGF-specific antagonists useful in the methods of the present invention include polypeptides that specifically bind to VEGF, anti-VEGF antibodies and antigen-binding fragments thereof, receptor molecules and derivatives that specifically bind to VEGF and thereby block binding to one or more receptors, fusion proteins (e.g., VEGF-Trap (Regeneron)), and VEGF receptor molecules and derivatives that specifically bind to VEGF and thereby block binding to one or more receptors. 121-Gelonin (Peregrine). VEGF-specific antagonists also include antagonist variants of VEGF polypeptides, antisense nucleobase oligomers complementary to at least a fragment of a nucleic acid molecule encoding a VEGF polypeptide, small RNAs complementary to at least a fragment of a nucleic acid molecule encoding a VEGF polypeptide, ribozymes targeting VEGF, peptibodies against VEGF, and VEGF aptamers. VEGF antagonists also include polypeptides that bind to VEGFR, anti-VEGFR antibodies (e.g., bevacizumab) and antigen-binding fragments thereof, as well as derivatives or fusion proteins that bind to VEGFR and thereby block, inhibit, abrogate, reduce, or interfere with VEGF biological activity (e.g., VEGF signal transduction). VEGF-specific antagonists also include non-peptide small molecules that can bind to VEGF or VEGFR and block, inhibit, abrogate, reduce, or interfere with VEGF biological activity. Thus, the term "VEGF biological activity" specifically includes VEGF-mediated biological activity of VEGF. In certain embodiments, the VEGF antagonist reduces or inhibits the expression level or biological activity of VEGF by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the VEGF inhibited by the VEGF-specific antagonist is VEGF(8-109), VEGF(1-109), or VEGF 165 is.

[0161] As used herein, VEGF antagonists include anti-VEGFR2 antibodies and related molecules (e.g., ramucirumab, tanibirumab, aflibercept), anti-VEGFR1 antibodies and related molecules (e.g., icrucumab, aflibercept (VEGF Trap-Eye, EYLEA®), and dib-aflibercept (VEGF Trap, ZALTRAP®), bispecific VEGF antibodies (e.g., MP-0250, vanucizumab (VEGF-ANG2), and the bispecific antibodies disclosed in US 2001 / 0236388), bispecific antibodies comprising a combination of two of an anti-VEGF arm, an anti-VEGFR1 arm, and an anti-VEGFR2 arm, anti-VEGFA antibodies (e.g., bevacizumab, sevacizumab), anti-VEGFB antibodies, anti-VEGFC antibodies (e.g., VGX-100), anti-VEGFD antibodies, and non-peptide small molecule VEGF antagonists. In some examples, the VEGF antagonist can be a tyrosine kinase inhibitor, including a receptor tyrosine kinase inhibitor (e.g., a multi-targeted receptor tyrosine kinase inhibitor such as sunitinib or axitinib).

[0162] An "anti-VEGF antibody" is an antibody that binds to VEGF with sufficient affinity and specificity. In certain embodiments, the antibody has a sufficiently high binding affinity for VEGF, e.g., the antibody has a K of 100 nM to 1 pM. D Antibody affinity can be determined, for example, by surface plasmon resonance-based assays (such as the BIAcore® assay described in PCT Application Publication No. WO 2005 / 012359), enzyme-linked immunosorbent assays (ELISA), and competitive assays (e.g., radioimmunoassays (RIAs)).

[0163] In certain embodiments, anti-VEGF antibodies can be used as therapeutic agents in targeting and interfering with diseases or conditions involving VEGF activity. Antibodies can also be subjected to other biological activity assays, e.g., to assess their effectiveness as therapeutic agents. Such assays are known in the art and depend on the target antigen for the antibody and its intended use. Examples include HUVEC inhibition assays; tumor cell growth inhibition assays (e.g., those described in WO 89 / 06692); antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonist activity or hematopoietic assays (see WO 95 / 27062). Anti-VEGF antibodies typically do not bind to other VEGF homologs, such as VEGF-B or VEGF-C, or other growth factors, such as PIGF, PDGF, or bFGF. In one embodiment, the anti-VEGF antibody is a monoclonal antibody that binds to the same epitope as monoclonal anti-VEGF antibody A4.6.1, produced by hybridoma ATCC HB 10709. In another embodiment, the anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al. (Cancer Res. 57:4593-4599, 1997), including but not limited to the antibody known as bevacizumab (BV; AVASTIN®).

[0164] The anti-VEGF antibody "bevacizumab (BV)," also known as "rhuMAb VEGF" or "AVASTIN®," is a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (Cancer Res. 57:4593-4599, 1997). It contains mutated human IgG1 framework regions and antigen-binding complementarity-determining regions (ADRs) derived from the murine anti-hVEGF monoclonal antibody A.4.6.1, which blocks the binding of human VEGF to its receptor. Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgG1, and approximately 7% of the sequence is derived from the murine antibody A4.6.1. Bevacizumab has a molecular weight of approximately 149,000 daltons and is glycosylated. Bevacizumab and other humanized anti-VEGF antibodies are further described in US Pat. No. 6,884,879, issued Feb. 26, 2005, the entire disclosure of which is incorporated herein by reference.

[0165] II. Cancer Treatment Methods and Compositions A. Methods Involving Bispecific Antibodies Targeting PD-1 and LAG3 In one aspect, the disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject one or more dosage cycles of a bispecific antibody targeting programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG3), the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the bispecific antibody is administered to the subject every three weeks at a fixed dose of about 600 mg (e.g., a 600 mg fixed dose).

[0166] Exemplary bispecific antibodies that target PD-1 and LAG3 are provided below in Section VIII. A particular example of a bispecific antibody that targets PD-1 and LAG3 is PD1-LAG3, as defined herein.

[0167] Recently, clinical proof-of-concept was demonstrated for dual PD-1 and LAG3 blockade using the combination of leratolimab and nivolumab in patients with previously untreated unresectable or metastatic melanoma (Tawbi et al., N Engl J Med, 386(1):24-34, 2022). By targeting both PD-1 and LAG-3 on dysfunctional tumor-specific T lymphocytes, this bispecific antibody aims to restore effective antitumor immune responses and provide cancer patients with even greater survival benefits than currently available checkpoint inhibitors. By preferentially targeting PD-1 / LAG-3 co-expressing dysfunctional T cells and potentially reducing targeting of LAG-3-expressing Tregs in the tumor microenvironment, this bispecific antibody may avoid reactivating Treg-mediated immunosuppression while restoring antitumor immune responses.

[0168] In some embodiments, each of the one or more dosing cycles is 21 days in length. In some embodiments, the method comprises administering (e.g., intravenously) the bispecific antibody to the subject on day 1 of each of the one or more dosing cycles.

[0169] The cancer can be a solid tumor (e.g., a solid tumor having a tumor microenvironment comprising LAG3-expressing CD8+ T cells). For example, in some embodiments, the cancer can be skin cancer (e.g., melanoma), liver cancer (e.g., hepatocellular carcinoma (HCC)), lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer, renal cancer (e.g., renal cell carcinoma (RCC)), bladder cancer (e.g., bladder cancer (e.g., metastatic urothelial carcinoma (mUC)), breast cancer (e.g., triple-negative breast cancer (TNBC)), esophageal cancer (e.g., esophageal squamous cell carcinoma (ESCC)), pancreatic cancer, cervical cancer, head and neck cancer, gastric cancer, colorectal cancer, or ovarian cancer. In some embodiments, the cancer can be skin cancer (e.g., melanoma), liver cancer (e.g., HCC), lung cancer (e.g., NSCLC), kidney cancer, renal cancer, renal cancer. The cancer may be locally advanced or metastatic.

[0170] In embodiments where the skin cancer is melanoma, the melanoma can be, for example, a previously untreated unresectable or metastatic melanoma (e.g., a histologically confirmed unresectable or metastatic melanoma according to the American Joint Committee on Cancer (AJCC) staging system (unresectable stage III or stage IV)). In some embodiments, the melanoma is (a) stage III melanoma with measurable lymph node metastasis; (b) unresectable stage III melanoma; or (c) stage IV melanoma. In some embodiments, the melanoma is not mucosal or uveal melanoma.

[0171] In some embodiments, the subject has not previously received systemic anti-cancer therapy.

[0172] In some embodiments, the subject has not been previously treated with an anti-cancer therapy comprising an immunomodulatory agent, e.g., has not been treated with an anti-cancer agent comprising a checkpoint inhibitor (CPI), e.g., has not been treated with an anti-programmed death-ligand 1 (PD-L1) / PD-1 agent, or has not been treated with an anti-cytotoxic T-lymphocyte-associated antigen (CTLA-4) agent. In other embodiments, the subject has been previously treated with an immunomodulatory agent (e.g., a CPI) as adjuvant or neoadjuvant therapy.

[0173] In some aspects, the subject has not been previously treated for metastatic or unresectable disease.

[0174] In some embodiments, the subject has not been previously treated with an anti-LAG3 therapy.

[0175] In some embodiments, the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in the tumor, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RO in the tumor.

[0176] In some embodiments, the subject is a human.

[0177] B. Methods Comprising Bispecific Antibodies Targeting PD-1 and LAG3 and Bevacizumab In some embodiments, the method further includes administering (e.g., intravenously) to the subject a VEGF antagonist, e.g., an anti-VEGF antibody (e.g., bevacizumab). Exemplary VEGF antagonists are provided in Section IX, below.

[0178] In some embodiments, the VEGF antagonist is administered before the bispecific antibody targeting PD-1 and LAG3. In other embodiments, the VEGF antagonist is administered after the bispecific antibody targeting PD-1 and LAG3. In yet further embodiments, the VEGF antagonist and the bispecific antibody targeting PD-1 and LAG3 are administered simultaneously.

[0179] In some embodiments, the methods include administering a VEGF antagonist (eg, bevacizumab) to the subject at a dose of about 15 mg / kg (eg, a dose of 15 mg / kg) every three weeks.

[0180] In some embodiments, each of the one or more dosing cycles is 21 days in length, and the method comprises administering a VEGF antagonist (e.g., bevacizumab) to the subject on day 1 of each of the one or more dosing cycles.

[0181] Accordingly, in one aspect, the disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject one or more dosage cycles of (1) a bispecific antibody targeting PD-1 and LAG3 (e.g., the bispecific antibody targeting PD-1 and LAG3 provided in Section VIII below), wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, and (2) a VEGF antagonist (e.g., bevacizumab), wherein the method comprises administering to the subject the bispecific antibody at a fixed dose of 600 mg every three weeks, and the VEGF antagonist at a dose of 15 mg / kg every three weeks.

[0182] In other embodiments, the method further comprises administering a VEGF antagonist (e.g., bevacizumab) to the subject every two weeks at a dose of about 10 mg / kg (e.g., a dose of 10 mg / kg). In some embodiments, each of the one or more dosing cycles is 28 days in length, and the method comprises administering a VEGF antagonist (e.g., bevacizumab) to the subject on days 1 and 15 of each of the one or more dosing cycles.

[0183] In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject with cancer (e.g., a bispecific antibody targeting PD-1 and LAG3 for use in any of the methods described above), wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, and the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks. A particular example of a bispecific antibody targeting PD-1 and LAG3 is PD1-LAG3, as defined herein.

[0184] In another aspect, the disclosure provides use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a medicament for treating a subject with cancer, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, and wherein the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks.

[0185] C. Methods Comprising a Bispecific Antibody Targeting PD-1 and LAG3 and an Anti-TIGIT Antagonist Antibody In some embodiments, the method further includes administering (e.g., intravenously) an anti-TIGIT antagonist antibody (e.g., tiragolumab) to the subject.

[0186] Exemplary anti-TIGIT antagonist antibodies are provided below in Section VII.

[0187] In some embodiments, the anti-TIGIT antagonist antibody is administered before the bispecific antibody targeting PD-1 and LAG3. In other embodiments, the anti-TIGIT antagonist antibody is administered after the bispecific antibody targeting PD-1 and LAG3. In yet further embodiments, the anti-TIGIT antagonist antibody and the bispecific antibody targeting PD-1 and LAG3 are administered simultaneously.

[0188] In some embodiments, the method includes administering to the subject an anti-TIGIT antagonist antibody (e.g., tiragolumab) at a fixed dose of about 600 mg (e.g., a 600 mg fixed dose) every three weeks.

[0189] In some embodiments, each of the one or more dosing cycles is 21 days in length, and the method comprises administering an anti-TIGIT antagonist antibody to the subject on about day 1 (e.g., day 1) of each of the one or more dosing cycles.

[0190] Accordingly, in one aspect, the disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject one or more dosage cycles of (1) a bispecific antibody targeting PD-1 and LAG3 (e.g., a bispecific antibody targeting PD-1 and LAG3 provided in Section VIII below; particularly PD1-LAG3 as defined herein), wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, and (2) an anti-TIGIT antagonist antibody (e.g., tiragolumab), wherein the method comprises administering to the subject the bispecific antibody at a fixed dose of 600 mg every three weeks, and the anti-TIGIT antagonist antibody at a fixed dose of 600 mg every three weeks.

[0191] III. Methods and compositions for treating liver cancer A. Methods Involving Bispecific Antibodies Targeting PD-1 and LAG3 In one aspect, the present disclosure provides a method for treating a subject having liver cancer, the method comprising administering to the subject one or more dosage cycles of a bispecific antibody targeting programmed cell death protein 1 (PD-1) and lymphocyte-activation gene 3 (LAG3), the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the method comprises administering the bispecific antibody to the subject every three weeks at a fixed dose of about 600 mg (e.g., a 600 mg fixed dose).

[0192] Exemplary bispecific antibodies that target PD-1 and LAG3 are provided below in Section VIII. A particular example of a bispecific antibody that targets PD-1 and LAG3 is PD1-LAG3, as defined herein.

[0193] In another aspect, the disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject one or more dosage cycles of a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 1200 mg every three weeks.

[0194] In some embodiments, each of the one or more dosing cycles is 21 days in length. In some embodiments, the method comprises administering (e.g., intravenously) the bispecific antibody to the subject on day 1 of each of the one or more dosing cycles.

[0195] In another aspect, the disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject one or more dosage cycles of a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 2100 mg every two weeks.

[0196] In some embodiments, each of the one or more dosing cycles is 28 days in length. In some embodiments, the method comprises administering (e.g., intravenously) a bispecific antibody to a subject on days 1 and 15 of the one or more dosing cycles.

[0197] In some embodiments, the liver cancer is hepatocellular carcinoma (HCC). In some embodiments, the liver cancer (e.g., HCC) has a tumor microenvironment comprising LAG3-expressing CD8+ T cells. HCC can be, for example, locally advanced, metastatic, and / or unresectable.

[0198] In some embodiments, the subject has not previously received systemic anti-cancer therapy.

[0199] In some embodiments, the subject has not been previously treated with an anti-cancer therapy comprising an immunomodulatory agent, e.g., has not been treated with an anti-cancer agent comprising a checkpoint inhibitor (CPI), e.g., has not been treated with an anti-programmed death-ligand 1 (PD-L1) / PD-1 agent, or has not been treated with an anti-cytotoxic T-lymphocyte-associated antigen (CTLA-4) agent. In other embodiments, the subject has been previously treated with an immunomodulatory agent (e.g., a CPI) as adjuvant or neoadjuvant therapy.

[0200] In some aspects, the subject has not been previously treated for metastatic or unresectable disease.

[0201] In some embodiments, the subject has not been previously treated with an anti-LAG3 therapy.

[0202] In some embodiments, the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in the tumor, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RO in the tumor.

[0203] In some embodiments, the subject is a human.

[0204] B. Methods Comprising Bispecific Antibodies Targeting PD-1 and LAG3 and Bevacizumab In some embodiments, the method further includes administering (e.g., intravenously) to the subject a VEGF antagonist, e.g., an anti-VEGF antibody (e.g., bevacizumab). Exemplary VEGF antagonists are provided in Section IX, below.

[0205] In some embodiments, the anti-TIGIT antagonist antibody is administered before the bispecific antibody targeting PD-1 and LAG3. In other embodiments, the anti-TIGIT antagonist antibody is administered after the bispecific antibody targeting PD-1 and LAG3. In yet further embodiments, the anti-TIGIT antagonist antibody and the bispecific antibody targeting PD-1 and LAG3 are administered simultaneously.

[0206] In some embodiments, the methods include administering a VEGF antagonist (eg, bevacizumab) to the subject at a dose of about 15 mg / kg (eg, a dose of 15 mg / kg) every three weeks.

[0207] In some embodiments, each of the one or more dosing cycles is 21 days in length, and the method comprises administering a VEGF antagonist (e.g., bevacizumab) to the subject on day 1 of each of the one or more dosing cycles.

[0208] Accordingly, in one aspect, the disclosure provides a method for treating a subject having cancer, the method comprising administering to the subject one or more dosage cycles of (1) a bispecific antibody targeting PD-1 and LAG3 (e.g., the bispecific antibody targeting PD-1 and LAG3, particularly PD1-LAG3, provided in Section VIII below), wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3; and (2) a VEGF antagonist, wherein the method comprises administering to the subject the bispecific antibody at a fixed dose of 600 mg every three weeks, and the VEGF antagonist at a dose of 15 mg / kg every three weeks.

[0209] In another aspect, the disclosure provides a method for treating a subject having liver cancer, the method comprising administering to the subject one or more dosage cycles of (1) a bispecific antibody targeting PD-1 and LAG3 (e.g., a bispecific antibody targeting PD-1 and LAG3 provided in Section VIII below), the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, and (2) a VEGF antagonist, wherein the method comprises administering to the subject the bispecific antibody at a fixed dose of 1200 mg every three weeks, and the VEGF antagonist at a dose of 15 mg / kg every three weeks.

[0210] In other embodiments, the method further comprises administering a VEGF antagonist (e.g., bevacizumab) to the subject every two weeks at a dose of about 10 mg / kg (e.g., a dose of 10 mg / kg). In some embodiments, each of the one or more dosing cycles is 28 days in length, and the method comprises administering a VEGF antagonist (e.g., bevacizumab) to the subject on days 1 and 15 of each of the one or more dosing cycles.

[0211] Accordingly, in another aspect, the present disclosure provides a method for treating a subject having liver cancer, the method comprising administering to the subject one or more dosage cycles of (1) a bispecific antibody targeting PD-1 and LAG3 (e.g., the bispecific antibody targeting PD-1 and LAG3 provided in Section VIII below), wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, and (2) a VEGF antagonist (e.g., bevacizumab), wherein the method comprises administering to the subject the bispecific antibody at a fixed dose of 2100 mg every two weeks, and the VEGF antagonist at a dose of 10 mg / kg every two weeks.

[0212] IV. Methods and Compositions for Treating Melanoma A. Methods Comprising an Anti-TIGIT Antagonist Antibody and a Bispecific Antibody Targeting PD-1 and LAG3 In one aspect, the disclosure provides a method for treating a subject with melanoma, the method comprising administering to the subject an anti-TIGIT antagonist antibody and a bispecific antibody targeting programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG3), the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3. In some embodiments, the anti-TIGIT antagonist antibody and the bispecific antibody are administered to the subject in a dosing regimen comprising one or more dosing cycles.

[0213] In some embodiments, the method comprises administering to the subject: (a) an anti-TIGIT antagonist antibody at a fixed dose of about 600 mg (e.g., a 600 mg fixed dose) every three weeks; and (b) a bispecific antibody at a fixed dose of about 2100 mg (e.g., a 2100 mg fixed dose) every three weeks.

[0214] In some embodiments, the method comprises administering to the subject: (a) an anti-TIGIT antagonist antibody at a fixed dose of about 600 mg (e.g., a 600 mg fixed dose) every three weeks; and (b) a bispecific antibody at a fixed dose of about 600 mg (e.g., a 600 mg fixed dose) every three weeks.

[0215] In another aspect, the disclosure provides a method for treating a subject having melanoma, the method comprising administering to the subject one or more dosage cycles of a bispecific antibody targeting PD-1 and LAG3 (e.g., the bispecific antibody targeting PD-1 and LAG3, particularly PD1-LAG3, provided in Section VIII below), wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks, and wherein the melanoma is (a) unresectable, Stage III melanoma; or (b) Stage IV melanoma (e.g., histologically confirmed unresectable or metastatic melanoma according to the American Joint Committee on Cancer (AJCC) staging system (unresectable, Stage III or Stage IV)).

[0216] In some embodiments, each of the one or more dosing cycles is 21 days in length. In some embodiments, the method comprises administering to the subject an anti-TIGIT antagonist antibody and a bispecific antibody on about day 1 (e.g., day 1) of each of the one or more dosing cycles.

[0217] In some embodiments, the method comprises administering the bispecific antibody to the subject before the anti-TIGIT antagonist antibody, hi other embodiments, the method comprises administering the anti-TIGIT antagonist antibody to the subject before the bispecific antibody.

[0218] In some embodiments, the methods comprise intravenously administering the bispecific antibody and the anti-TIGIT antagonist antibody to the subject.

[0219] i. Neoadjuvant therapy In some embodiments, one or more dosing cycles are administered as neoadjuvant therapy.

[0220] In some embodiments, the anti-TIGIT antagonist antibody and the bispecific antibody targeting PD-1 and LAG3 are administered as neoadjuvant therapy.

[0221] In some embodiments, the melanoma is stage III melanoma with measurable lymph node metastasis.

[0222] In some embodiments, the subject does not have in-transit metastases within six months prior to initiating treatment.

[0223] In some embodiments, the subject has not been previously treated with a cancer immunotherapy.

[0224] In some embodiments, the melanoma is not mucosal or uveal melanoma.

[0225] In some embodiments, the first dosing cycle is initiated prior to surgery.

[0226] In some embodiments, at least one dosing cycle (e.g., 1, 2, 3, 4, or more than 4 dosing cycles) or at least two dosing cycles (e.g., 2, 3, 4, or more than 4 dosing cycles) are completed prior to surgery. In some embodiments, two dosing cycles are completed prior to surgery.

[0227] In some embodiments, the surgery occurs within about one week after the last dosing cycle.

[0228] In some embodiments, the surgery is a complete lymph node dissection (CLND).

[0229] In some embodiments, the treatment results in an increased pathological response rate (pRR) compared to a reference pRR. In some embodiments, the reference pRR is the pRR of a population of subjects who have received a control therapy. In some embodiments, the control therapy is a therapy that includes an anti-TIGIT antagonist antibody and does not include a bispecific antibody targeting PD-1 and LAG3; a therapy that includes a bispecific antibody targeting PD-1 and LAG3 and does not include an anti-TIGIT antagonist antibody; or a therapy that includes ipilimumab and nivolumab.

[0230] In some embodiments, the treatment results in an increase in event-free survival (EFS) compared to a baseline EFS; an increase in recurrence-free survival (RFS) compared to a baseline RFS; an increase in overall survival (OS) compared to a baseline OS; and / or an increase in overall response rate (ORR) compared to a baseline ORR. In some embodiments, the baseline EFS, RFS, OS, or ORR is one of a population of subjects who have received a control therapy. In some embodiments, the control therapy is a therapy that includes an anti-TIGIT antagonist antibody and does not include a bispecific antibody targeting PD-1 and LAG3; a therapy that includes a bispecific antibody targeting PD-1 and LAG3 and does not include an anti-TIGIT antagonist antibody; or a therapy that includes ipilimumab and nivolumab.

[0231] ii. Treatment of Stage IV Melanoma In some embodiments, the melanoma is stage IV melanoma.

[0232] In some embodiments, (a) the subject has received no more than two lines of prior systemic therapy; or (b) the melanoma is a BRAF-mutated melanoma and the subject has received no more than three lines of prior systemic therapy.

[0233] In some embodiments, the treatment results in an increased overall response rate (ORR) compared to a reference ORR. In some embodiments, the reference ORR is the ORR of a population of subjects who received (a) a treatment comprising a bispecific antibody targeting PD-1 and LAG3, and not an anti-TIGIT antagonist antibody; and / or (b) a treatment comprising an anti-TIGIT antagonist antibody, and not a bispecific antibody targeting PD-1 and LAG3.

[0234] In some embodiments, the treatment results in an increase in progression-free survival (PFS) compared to baseline PFS; an increase in duration of response (DOR) compared to baseline DOR; an increase in OS compared to baseline OS; or an increase in disease control rate (DCR, e.g., stable disease for 12 weeks or more, complete response (CR), or partial response (PR)) compared to baseline DCR. In some embodiments, the baseline PFS, OS, DOR, or DCR is one of a population of subjects who have received a control therapy. In some embodiments, the control therapy is a therapy that includes an anti-TIGIT antagonist antibody and does not include a bispecific antibody targeting PD-1 and LAG3; a therapy that includes a bispecific antibody targeting PD-1 and LAG3 and does not include an anti-TIGIT antagonist antibody; or a therapy that includes ipilimumab and nivolumab.

[0235] In some embodiments, the subject is a human.

[0236] B. Methods Involving Bispecific Antibodies Targeting PD-1 and LAG3 In another aspect, the disclosure features a method for treating a subject with melanoma, the method including administering to the subject a bispecific antibody that targets PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3. In some embodiments, the bispecific antibody is administered to the subject in a dosing regimen that includes one or more dosing cycles. In some embodiments, the one or more dosing cycles are administered as neoadjuvant therapy.

[0237] In some embodiments, the methods comprise administering the bispecific antibody to the subject at a fixed dose of about 2100 mg (e.g., a fixed dose of 2100 mg) every three weeks.

[0238] In certain embodiments, the methods comprise administering the bispecific antibody to the subject at a fixed dose of about 600 mg (e.g., a 600 mg fixed dose) every three weeks.

[0239] In some embodiments, each of the one or more dosing cycles is 21 days in length. In some embodiments, the method comprises administering the bispecific antibody to the subject on about day 1 (e.g., day 1) of each of the one or more dosing cycles.

[0240] In some embodiments, the methods comprise administering the bispecific antibody intravenously to the subject.

[0241] In some embodiments, the melanoma is stage III melanoma with measurable lymph node metastasis.

[0242] In some embodiments, the subject does not have in-transit metastases within six months prior to initiating treatment.

[0243] In some embodiments, the subject has not been previously treated with a cancer immunotherapy.

[0244] In some embodiments, the melanoma is not mucosal or uveal melanoma.

[0245] In some embodiments, the first dosing cycle is initiated prior to surgery.

[0246] In some embodiments, at least one dosing cycle (e.g., 1, 2, 3, 4, or more than 4 dosing cycles) or at least two dosing cycles (e.g., 2, 3, 4, or more than 4 dosing cycles) are completed prior to surgery. In some embodiments, two dosing cycles are completed prior to surgery.

[0247] In some embodiments, the surgery occurs within about one week after the last dosing cycle.

[0248] In some embodiments, the surgery is a complete lymph node dissection (CLND).

[0249] In some embodiments, the treatment results in an increased pathological response rate (pRR) compared to a reference pRR. In some embodiments, the reference pRR is the pRR of a population of subjects who received a control therapy. In some embodiments, the control therapy is a therapy comprising ipilimumab and nivolumab.

[0250] In some embodiments, the treatment results in an increase in event-free survival (EFS) compared to a baseline EFS; an increase in recurrence-free survival (RFS) compared to a baseline RFS; an increase in overall survival (OS) compared to a baseline OS; and / or an increase in overall response rate (ORR) compared to a baseline ORR. In some embodiments, the baseline EFS, RFS, OS, or ORR is one of a population of subjects who have received a control therapy. In some embodiments, the control therapy is a therapy comprising ipilimumab and nivolumab.

[0251] In some embodiments, the subject is a human.

[0252] C. Methods Involving Anti-TIGIT Antagonist Antibodies and PD-1 Axis Binding Antagonists In another aspect, the disclosure features a method for treating a subject having melanoma, the method including administering to the subject one or more dosage cycles of an anti-TIGIT antagonist antibody and a PD-1 axis-binding antagonist, where the one or more dosage cycles are administered as neoadjuvant therapy.In another aspect, the disclosure features a method for treating a subject having melanoma, the method including administering to the subject an anti-TIGIT antagonist antibody and a PD-1 axis-binding antagonist, where the anti-TIGIT antagonist antibody and the PD-1 axis-binding antagonist are administered as neoadjuvant therapy.

[0253] In some embodiments, the method includes administering to the subject: (a) an anti-TIGIT antagonist antibody at a fixed dose of about 600 mg (e.g., a 600 mg fixed dose) every three weeks; and (b) a PD-1 axis binding antagonist at a fixed dose of about 600 mg (e.g., a 600 mg fixed dose) every three weeks.

[0254] In some embodiments, each of the one or more dosing cycles is 21 days in length.

[0255] In some embodiments, the method includes administering to the subject an anti-TIGIT antagonist antibody and a PD-1 axis binding antagonist on about day 1 (e.g., day 1) of each of one or more dosing cycles.

[0256] In some embodiments, the method comprises administering to the subject a PD-1 axis-binding antagonist before the anti-TIGIT antagonist antibody. In some embodiments, the method comprises administering to the subject an anti-TIGIT antagonist antibody before the PD-1 axis-binding antagonist.

[0257] In some embodiments, the methods comprise intravenously administering to a subject a PD-1 axis binding antagonist and an anti-TIGIT antagonist antibody.

[0258] In some embodiments, the melanoma is stage III melanoma with measurable lymph node metastasis.

[0259] In some embodiments, the subject does not have in-transit metastases within six months prior to initiating treatment.

[0260] In some embodiments, the subject has not been previously treated with a cancer immunotherapy.

[0261] In some embodiments, the melanoma is not mucosal or uveal melanoma.

[0262] In some embodiments, the first dosing cycle is initiated prior to surgery.

[0263] In some embodiments, at least one dosing cycle (e.g., 1, 2, 3, 4, or more than 4 dosing cycles) or at least two dosing cycles (e.g., 2, 3, 4, or more than 4 dosing cycles) are completed prior to surgery. In some embodiments, two dosing cycles are completed prior to surgery.

[0264] In some embodiments, the surgery occurs within about one week after the last dosing cycle.

[0265] In some embodiments, the surgery is a complete lymph node dissection (CLND).

[0266] In some embodiments, the treatment results in an increased pathological response rate (pRR) compared to a reference pRR. In some embodiments, the reference pRR is the pRR of a population of subjects receiving a control therapy. In some embodiments, the control therapy is a therapy comprising an anti-TIGIT antagonist antibody and not a PD-1 axis-binding antagonist; a therapy comprising a PD-1 axis-binding antagonist and not an anti-TIGIT antagonist antibody; or a therapy comprising ipilimumab and nivolumab.

[0267] In some embodiments, the treatment results in an increase in event-free survival (EFS) compared to a reference EFS; an increase in recurrence-free survival (RFS) compared to a reference RFS; an increase in overall survival (OS) compared to a reference OS; and / or an increase in overall response rate (ORR) compared to a reference ORR. In some embodiments, the reference EFS, RFS, OS, or ORR is one of a population of subjects who have received a control therapy. In some embodiments, the control therapy is a therapy that includes an anti-TIGIT antagonist antibody and does not include a PD-1 axis-binding antagonist; a therapy that includes a PD-1 axis-binding antagonist and does not include an anti-TIGIT antagonist antibody; or a therapy that includes ipilimumab and nivolumab.

[0268] In some embodiments, the subject is a human.

[0269] D. Agents for Use in Methods of Treating Melanoma i. Bispecific antibodies targeting PD-1 and LAG3 Further examples of bispecific antibodies targeting PD-1 and LAG3, and dosing regimens thereof, are provided below in Section VIII. A particular example of a bispecific antibody targeting PD-1 and LAG3 is PD1-LAG3, as defined herein.

[0270] ii. Anti-TIGIT antagonist antibody Exemplary anti-TIGIT antagonist antibodies and their dosing regimens are provided below in Section VII.

[0271] iii.PD-1 axis binding antagonists Exemplary anti-TIGIT antagonist antibodies and their dosing regimens are provided below in Section VII.

[0272] V. Evaluation of PD-L1 Expression PD-L1 expression can be assessed in a subject treated according to any of the methods and compositions for use described herein. The methods and compositions for use can include determining the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from a subject with cancer (e.g., esophageal cancer (e.g., metastatic esophageal cancer)). In other examples, the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the subject has been determined before or after treatment has begun. PD-L1 expression can be determined using any suitable approach. For example, PD-L1 expression can be determined as described in U.S. Patent Application Publication Nos. 15 / 787,988 and 15 / 790,680. Any suitable tumor sample can be used, such as a formalin-fixed, paraffin-embedded (FFPE) tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0273] For example, PD-L1 expression may be determined in relation to the percentage of a tumor sample comprised by tumor-infiltrating immune cells that express a detectable expression level of PD-L1, as the percentage of tumor-infiltrating immune cells in a tumor sample that express a detectable expression level of PD-L1, and / or as the percentage of tumor cells in a tumor sample that express a detectable expression level of PD-L1. In any of the foregoing examples, it will be understood that the percentage of a tumor sample comprised by tumor-infiltrating immune cells may be the percentage of tumor area covered by tumor-infiltrating immune cells in a section of a tumor sample obtained from a subject, as assessed, for example, by IHC using an anti-PD-L1 antibody (e.g., SP142 antibody). Any suitable anti-PD-L1 antibody can be used, including, for example, SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28-8 (Dako), E1L3N (Cell Signaling Technology), 4059 (ProSci, Inc.), h5H1 (Advanced Cell Diagnostics), and 9A11. In some examples, the anti-PD-L1 antibody is SP142. In other examples, the anti-PD-L1 antibody is SP263.

[0274] In some examples, a tumor sample obtained from a subject has detectable PD-L1 expression levels in less than 1% of tumor cells in the tumor sample, in 1% or more tumor cells in the tumor sample, in between 1% and less than 5% of tumor cells in the tumor sample, in 5% or more tumor cells in the tumor sample, in between 5% and less than 50% of tumor cells in the tumor sample, or in 50% or more tumor cells in the tumor sample.

[0275] In some examples, a tumor sample obtained from a subject has detectable PD-L1 expression levels in tumor-infiltrating immune cells that comprise less than 1% of the tumor sample, more than 1% of the tumor sample, between 1% and less than 5% of the tumor sample, more than 5% of the tumor sample, between 5% and less than 10% of the tumor sample, or more than 10% of the tumor sample.

[0276] In some embodiments, a subject's esophageal cancer treated according to any of the methods provided herein has a PD-L1 positive tumor cell (TC) fraction or tumor-infiltrating immune cell (IC) fraction of less than 5% (<5%). In some embodiments, the esophageal cancer has a PD-L1 positive TC fraction of less than 1%. In other embodiments, a subject's esophageal cancer treated according to any of the methods provided herein has a PD-L1 positive TC fraction or IC fraction of 5% or more (≧5%). In some embodiments, PD-L1 is detected using the Ventana SP142 IHC assay, the Ventana SP263 IHC assay, the pharmDx 22C3 IHC assay, or the pharmDx 28-8 IHC assay.

[0277] In some examples, tumor samples may be scored for PD-L1 positivity in tumor-infiltrating immune cells and / or tumor cells according to the criteria for diagnostic evaluation set forth in Table 2 and / or Table 3, respectively.

[0278] Table 2. Tumor-infiltrating immune cell (IC) IHC diagnostic criteria TIFF2024529451000002.tif93170

[0279] Table 3. Tumor cell (TC) IHC diagnostic criteria TIFF2024529451000003.tif55170

[0280] VI. Evaluation of TIGIT expression The expression level of TIGIT can be assessed in a subject with cancer (e.g., esophageal cancer (e.g., metastatic esophageal cancer)) treated according to any of the methods, uses, and compositions for use described herein. The methods, uses, and compositions for use can include determining the expression level of TIGIT in a biological sample (e.g., a tumor sample) obtained from the subject. In other examples, the expression level of TIGIT in a biological sample (e.g., a tumor sample) obtained from the subject is determined before or after the start of treatment. TIGIT expression can be determined using any suitable approach. Any suitable tumor sample can be used, such as a formalin-fixed, paraffin-embedded (FFPE) tumor sample, an archived tumor sample, a fresh tumor sample, or a frozen tumor sample.

[0281] For example, TIGIT expression can be determined in terms of the percentage of tumor-infiltrating immune cells in a tumor sample that express a detectable level of TIGIT, as the percentage of tumor-infiltrating immune cells in a tumor sample that express a detectable level of TIGIT, and / or as the percentage of tumor cells in a tumor sample that express a detectable level of TIGIT. In any of the above examples, it should be understood that the percentage of a tumor sample that is comprised of tumor-infiltrating immune cells can be determined in terms of the percentage of tumor area covered by tumor-infiltrating immune cells in a section of a tumor sample obtained from a subject, for example, when assessed by IHC using an anti-TIGIT antagonist antibody. Any suitable anti-TIGIT antagonist antibody can be used. In some examples, the anti-TIGIT antagonist antibody is 10A7 (WO 2009 / 126688 A3; U.S. Patent No. 9,499,596).

[0282] VII. Anti-TIGIT antagonist antibodies The present invention provides anti-TIGIT antagonist antibodies useful for treating cancer in a subject (eg, a human) with cancer.

[0283] In some instances, the anti-TIGIT antagonist antibody is tiragolumab (CAS Registry Number: 1918185-84-8). Tiragolumab (Genentech) is also known as MTIG7192A.

[0284] In certain instances, the anti-TIGIT antagonist antibody comprises at least one, two, three, four, five, or six HVRs selected from the following: (a) HVR-H1 comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 1); (b) HVR-H2 comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 2); (c) HVR-H3 comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 3); (d) HVR-H4 comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 4). (e) HVR-L1 comprising the amino acid sequence of WASTRES (SEQ ID NO: 5); and / or (f) HVR-L3 comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 6), or a combination of one or more of the above HVRs with one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1-6.

[0285] In some examples, the anti-TIGIT antagonist antibody may comprise: (a) HVR-H1 comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 1); (b) HVR-H2 comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 2); (c) HVR-H3 comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 3); (d) HVR-L1 comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 4); (e) HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 5); and (f) HVR-L3 comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 6). In some instances, the anti-TIGIT antagonist antibody has an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence of EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 17), or EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTY an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the sequence: YRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 17), or the sequence: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 18);or a VH domain comprising the sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 18); and / or DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSTPFTFGPGTKVEIK (SEQ ID NO: 19), or a VL domain comprising the sequence DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK (SEQ ID NO: 19). In some instances, the anti-TIGIT antagonist antibody has a VH domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 17, or the sequence of SEQ ID NO: 17, and / or an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 19, or a VL domain comprising the sequence of SEQ ID NO: 19. In some instances, the anti-TIGIT antagonist antibody has a VH domain comprising the amino acid sequence of SEQ ID NO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 19. In some instances, the anti-TIGIT antagonist antibody comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 18, or a VH domain comprising the sequence of SEQ ID NO: 18;and / or an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 19, or a VL domain comprising the sequence of SEQ ID NO: 19. In some instances, the anti-TIGIT antagonist antibody has a VH domain comprising the amino acid sequence of SEQ ID NO: 18 and a VL domain comprising the amino acid sequence of SEQ ID NO: 19.

[0286] In some instances, the anti-TIGIT antagonist antibody comprises a heavy chain sequence and a light chain sequence, wherein (a) the heavy chain sequence has the amino acid sequence: EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY and (b) the light chain sequence comprises the amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24).

[0287] In some instances, the anti-TIGIT antagonist antibody further comprises at least one, two, three, or four of the following light chain variable region framework regions (FRs): FR-L1 comprising the amino acid sequence of DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 7); FR-L2 comprising the amino acid sequence of WYQQKPGQPPNLLIY (SEQ ID NO: 8); FR-L3 comprising the amino acid sequence of GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 9); and / or FR-L4 comprising the amino acid sequence of FGPGTKVEIK (SEQ ID NO: 10), or a combination of one or more of the above FRs with one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 7-20. In some instances, for example, the antibody comprises FR-L1 comprising the amino acid sequence DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 7); FR-L2 comprising the amino acid sequence WYQQKPGQPPNLLIY (SEQ ID NO: 8); FR-L3 comprising the amino acid sequence GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 9); and FR-L4 comprising the amino acid sequence FGPGTKVEIK (SEQ ID NO: 10).

[0288] In some instances, the anti-TIGIT antagonist antibody further comprises at least one, two, three, or four of the following heavy chain variable region FRs: FR-H1 comprising the amino acid sequence of X1VQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 11) (X1 is E or Q); FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 12); FR-H3 comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and / or FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14), or a combination of one or more of the above FRs with one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 11-14. The anti-TIGIT antagonist antibody may further comprise, for example, at least one, two, three, or four of the following heavy chain variable region framework regions FR: FR-H1 comprising the amino acid sequence EVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 15); FR-H2 comprising the amino acid sequence WIRQSPSRGLEWLG (SEQ ID NO: 12); FR-H3 comprising the amino acid sequence RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and / or FR-H4 comprising the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 14), or a combination of one or more of the above FRs with one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 12 to 15.In some embodiments, the anti-TIGIT antagonist antibody includes FR-H1 having the amino acid sequence EVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 15); FR-H2 having the amino acid sequence WIRQSPSRGLEWLG (SEQ ID NO: 12); FR-H3 having the amino acid sequence RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and FR-H4 having the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 14). In another example, the anti-TIGIT antagonist antibody may further comprise at least one, two, three, or four of the following heavy chain variable region framework regions FR: FR-H1 comprising the amino acid sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 16); FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 12); FR-H3 comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and / or FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14), or a combination of one or more of the above FRs with one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 12 to 14 and 16. In some embodiments, the anti-TIGIT antagonist antibody includes FR-H1 having the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 16); FR-H2 having the amino acid sequence WIRQSPSRGLEWLG (SEQ ID NO: 12); FR-H3 having the amino acid sequence RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and FR-H4 having the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 14).

[0289] In another aspect, an anti-TIGIT antagonist antibody is provided, the antibody comprising a VH as any of the above examples and a VL as any of the above examples, wherein one or both of the variable domain sequences comprises a post-translational modification.

[0290] In some embodiments, any one of the above anti-TIGIT antagonist antibodies can bind to rabbit TIGIT in addition to human TIGIT. In some embodiments, any one of the above anti-TIGIT antagonist antibodies can bind to both human TIGIT and cynomolgus monkey (cyno) TIGIT. In some embodiments, any one of the above anti-TIGIT antagonist antibodies can bind to human TIGIT, cynomolgus monkey TIGIT, and rabbit TIGIT. In some embodiments, any one of the above anti-TIGIT antagonist antibodies can bind to human TIGIT, cynomolgus monkey TIGIT, and rabbit TIGIT, but cannot bind to mouse TIGIT.

[0291] In some instances, the anti-TIGIT antagonist antibody has a K of about 10 nM or less. D binds to human TIGIT with a K of approximately 10 nM or less D binds to cynomolgus monkey TIGIT (e.g., K of about 0.1 nM to about 1 nM) D It binds to human TIGIT with a K of approximately 0.5 nM to approximately 1 nM. D binds to cynomolgus monkey TIGIT with a K of, for example, about 0.1 nM or less D binds to human TIGIT with a K of approximately 0.5 nM or less D binds to cynomolgus monkey TIGIT).

[0292] In some instances, the anti-TIGIT antagonist antibody specifically binds to TIGIT and inhibits or blocks TIGIT interaction with the poliovirus receptor (PVR) (e.g., the antagonist antibody inhibits intracellular signaling mediated by TIGIT binding to PVR). In some instances, the antagonist antibody inhibits or blocks binding of human TIGIT to human PVR with an IC50 value of 10 nM or less (e.g., 1 nM to about 10 nM). In some instances, the anti-TIGIT antagonist antibody specifically binds to TIGIT and inhibits or blocks TIGIT interaction with PVR without affecting PVR-CD226 interaction. In some instances, the antagonist antibody inhibits or blocks binding of cynomolgus monkey TIGIT to cynomolgus monkey PVR with an IC50 value of 50 nM or less (e.g., 1 nM to about 50 nM, e.g., 1 nM to about 5 nM). In some instances, the anti-TIGIT antagonist antibody inhibits and / or blocks the interaction between CD226 and TIGIT, hi some instances, the anti-TIGIT antagonist antibody inhibits and / or blocks the ability of TIGIT to disrupt CD226 homodimerization.

[0293] In some examples, the methods or uses described herein may include administering or using an isolated anti-TIGIT antagonist antibody that competes with any of the above-mentioned anti-TIGIT antagonist antibodies for binding to TIGIT. For example, the method may include administering an isolated anti-TIGIT antagonist antibody that competes with an anti-TIGIT antagonist antibody having the following six HVRs for binding to TIGIT: (a) HVR-H1 comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 1); (b) HVR-H2 comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 2); (c) HVR-H3 comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 3); (d) HVR-L1 comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 4); (e) HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 5); and (f) HVR-L3 comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 6). The methods described herein can also include administering an isolated anti-TIGIT antagonist antibody that binds to the same epitope as the anti-TIGIT antagonist antibody described above.

[0294] In some embodiments, the anti-TIGIT antagonist antibody exhibits Fc-mediated effector function, e.g., participates in antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the anti-TIGIT antagonist antibody is an antibody with intact Fc-mediated effector function (e.g., tiragolumab, vibostolimab, etiglimab, EOS084448, or TJ-T6) or enhanced effector function (e.g., SGN-TGT).

[0295] In other embodiments, the anti-TIGIT antagonist antibody is an antibody that lacks Fc-mediated effector functions (e.g., domvanalimab, BMS-986207, ASP8374, or COM902).

[0296] In some embodiments, the anti-TIGIT antagonist antibody is an IgG class antibody. In some embodiments, the anti-TIGIT antagonist antibody is an IgG1 class antibody, such as tiragolumab, vibostolimab, domvanalimab, BMS-986207, etigilimab, BGB-A1217, SGN-TGT, EOS084448 (EOS-448), TJ-T6, or AB308. In some embodiments, the antibody is a human monoclonal full-length IgG1 class antibody containing an Fc region.

[0297] In some embodiments, the anti-TIGIT antagonist antibody is a human monoclonal full-length IgG1 subclass antibody comprising a human IgG1 Fc region, a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 19.

[0298] In another embodiment, the anti-TIGIT antagonist antibody is an IgG4 class antibody, such as ASP8374 or COM902.

[0299] Anti-TIGIT antagonist antibodies (e.g., tiragolumab) useful in the present invention, including compositions containing such antibodies, may be used in combination with PD-1 axis-binding antagonists (e.g., PD-L1 binding antagonists (e.g., anti-PD-L1 antagonist antibodies, e.g., atezolizumab), PD-1 binding antagonists (e.g., anti-PD-1 antagonist antibodies, e.g., pembrolizumab), and PD-L2 binding antagonists (e.g., anti-PD-L2 antagonist antibodies)).

[0300] In some embodiments, the anti-TIGIT antagonist antibody functions to inhibit TIGIT signaling. In some embodiments, the anti-TIGIT antagonist antibody inhibits binding of TIGIT to its binding partner. Exemplary TIGIT binding partners include CD155 (PVR), CD112 (PVRL2 or nectin-2), and CD113 (PVRL3 or nectin-3). In some embodiments, the anti-TIGIT antagonist antibody can inhibit the binding between TIGIT and CD155. In some embodiments, the anti-TIGIT antagonist antibody can inhibit the binding between TIGIT and CD112. In some embodiments, the anti-TIGIT antagonist antibody inhibits the binding between TIGIT and CD113. In some embodiments, the anti-TIGIT antagonist antibody inhibits TIGIT-mediated cell signaling of immune cells. In some embodiments, the anti-TIGIT antagonist antibody inhibits TIGIT by depleting regulatory T cells (eg, FcγR).

[0301] In some embodiments, the anti-TIGIT antibody is a monoclonal antibody. In some embodiments, the anti-TIGIT antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the anti-TIGIT antibody is a humanized antibody. In some embodiments, the anti-TIGIT antibody is a human antibody. In some embodiments, the anti-TIGIT antibody described herein binds to human TIGIT. In some embodiments, the anti-TIGIT antibody is an Fc fusion protein.

[0302] In some embodiments, the anti-TIGIT antibody is selected from the group consisting of tiragolumab (MTIG7192A, RG6058 or RO7092284), vibostolimab (MK-7684), ASP8374 (PTZ-201), EOS884448 (EOS-448), SEA-TGT (SGN-TGT), BGB-A1217, BMS-986207 (ONO-4686), COM902 (CGEN-15137), IBI939, domvanalimab (AB154), M6223, AB308, AB154, TJ-T6, MG1131, NB6253, HLX301, HLX53, SL-9258 (TIGIT-Fc-LIGHT), STW264, and YBL-012. In some embodiments, the anti-TIGIT antibody is selected from the group consisting of tiragolumab (MTIG7192A, RG6058, or RO7092284), vibostolimab (MK-7684), ASP8374 (PTZ-201), EOS-448, and SEA-TGT (SGN-TGT). The anti-TIGIT antibody can be tiragolumab (MTIG7192A, RG6058, or RO7092284).

[0303] In some embodiments, the anti-TIGIT antibody comprises at least one, two, three, four, five, or six complementarity-determining regions (CDRs) of any of the anti-TIGIT antibodies disclosed herein. In some embodiments, the anti-TIGIT antibody comprises six CDRs of any of the anti-TIGIT antibodies disclosed herein. In some embodiments, the anti-TIGIT antibody comprises six CDRs of any one of antibodies selected from the group consisting of tiragolumab, ASP8374 (PTZ-201), BGB-A1217, BMS-986207 (ONO-4686), COM902 (CGEN-15137), M6223, IBI939, EOS884448 (EOS-448), domvanalimab (AB154), vibostolimab (MK-7684), and SEA-TGT (SGN-TGT).

[0304] In some embodiments, the anti-TIGIT antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain variable region (VH) sequence of any one of the anti-TIGIT antibodies disclosed herein, and the light chain comprises the light chain variable region (VL) of the same antibody. In some embodiments, the anti-TIGIT antibody comprises the VH and VL of an anti-TIGIT antibody selected from the group consisting of tiragolumab, ASP8374 (PTZ-201), BGB-A1217, BMS-986207 (ONO-4686), COM902 (CGEN-15137), M6223, IBI939, EOS884448 (EOS-448), domvanalimab (AB154), vibostolimab (MK-7684), and SEA-TGT (SGN-TGT).

[0305] In some embodiments, the anti-TIGIT antibody comprises the heavy and light chains of any of the anti-TIGIT antibodies disclosed herein. In some embodiments, the anti-TIGIT antibody comprises the heavy and light chains of an anti-TIGIT antibody selected from the group consisting of tiragolumab, ASP8374 (PTZ-201), BGB-A1217, BMS-986207 (ONO-4686), COM902 (CGEN-15137), M6223, IBI939, EOS884448 (EOS-448), domvanalimab (AB154), vibostolimab (MK-7684), and SEA-TGT (SGN-TGT).

[0306] VIII. Bispecific antibodies targeting PD-1 and LAG3 A. Exemplary Bispecific Antibodies that Bind PD-1 and LAG3 In one aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein said first antigen-binding domain that specifically binds to PD-1 is: (i) HVR-H1 comprising the amino acid sequence of GFSFSSY (SEQ ID NO: 25); (ii) HVR-H2 containing the amino acid sequence GGR, and (iii) a VH domain comprising HVR-H3 comprising the amino acid sequence of TGRVYFALD (SEQ ID NO: 26); and (i) HVR-L1 comprising the amino acid sequence of SESVDTSDNSF (SEQ ID NO: 27); (ii) HVR-L2 containing the amino acid sequence RSS, and (iii) a VL domain comprising HVR-L3 containing the amino acid sequence of NYDVPW (SEQ ID NO: 28) Includes:

[0307] In one embodiment, the bispecific antibody comprises an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain, which Fc domain has reduced or even abolished effector function, in particular the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, in particular Fcγ receptors.

[0308] In a further aspect, there is provided a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises an Fc domain that is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc domain, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor.

[0309] In another aspect, a bispecific antibody is provided, comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the second antigen-binding domain that specifically binds to LAG3 is: (i) HVR-H1 comprising the amino acid sequence of DYTMN (SEQ ID NO: 31); (ii) HVR-H2 comprising the amino acid sequence VISWDGGGTYYTDSVKG (SEQ ID NO: 32), and (iii) a VH domain comprising HVR-H3 comprising the amino acid sequence of GLTDTTLYGSDY (SEQ ID NO: 33); and (i) HVR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 34); (ii) HVR-L2 comprising the amino acid sequence of AASTLQS (SEQ ID NO: 35), and (iii) a VL domain comprising HVR-L3 containing the amino acid sequence of QQTYSSPLT (SEQ ID NO: 36) Includes:

[0310] In a further aspect, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the first antigen-binding domain that specifically binds to PD-1 comprises a VH domain comprising the amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYTMSWVRQAPGKGLEWVATISGGGRDIYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVLLTGRVYFALDSWGQGTLVTVSS (SEQ ID NO: 29) and a VL domain comprising the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKASESVDTSDNSFIHWYQQKPGQSPKLLIYRSSTLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNYDVPWTFGQGTKVEIK (SEQ ID NO: 30).

[0311] In another aspect, a bispecific antibody is provided, comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the second antigen-binding domain that specifically binds to LAG3 comprises a VH domain comprising the amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFIFDDYTMNWVRQAPGKGLEWVAVISWDGGGTYYTDSVKGRFTISRDDFKNTLYLQMNSLRAEDTAVYYCAKGLTDTTLYGSDYWGQGTLVTVSS (SEQ ID NO: 37) and a VL domain comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSSPLTFGGGTKVEIK (SEQ ID NO: 38).

[0312] In one embodiment, a bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the first antigen-binding domain that specifically binds PD-1 comprises a VH domain having at least 90% identity (e.g., having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more identity) to the amino acid sequence of SEQ ID NO:29, and a VL domain having at least 90% identity (e.g., having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more identity) to the amino acid sequence of SEQ ID NO:30. In one embodiment, a first antigen-binding domain that specifically binds to PD-1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO:29 and a VL domain comprising the amino acid sequence of SEQ ID NO:30.

[0313] In one embodiment, a bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the second antigen-binding domain that specifically binds LAG3 comprises a VH domain having at least 90% identity (e.g., having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more identity) to the amino acid sequence of SEQ ID NO: 37, and a VL domain having at least 90% identity (e.g., having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more identity) to the amino acid sequence of SEQ ID NO: 38. In one aspect, the second antigen-binding domain that specifically binds to LAG3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.

[0314] In one embodiment, a bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the first antigen-binding domain that specifically binds PD-1 comprises a VH domain having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity) to the amino acid sequence of SEQ ID NO: 29, and a VH domain having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity) to the amino acid sequence of SEQ ID NO: 30. The second antigen-binding domain that specifically binds to LAG3 comprises a VH domain that has at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more identity) to the amino acid sequence of SEQ ID NO: 37, and a VL domain that has at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more identity) to the amino acid sequence of SEQ ID NO: 38.

[0315] In another aspect, a bispecific antibody is provided, comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein: the first antigen-binding domain that specifically binds to PD-1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30; The second antigen-binding domain that specifically binds to LAG3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.

[0316] In a further aspect, a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3 is a human antibody, a humanized antibody, or a chimeric antibody. In particular, the bispecific antibody is a humanized antibody or a chimeric antibody.

[0317] In one embodiment, a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3 is bivalent, meaning that the bispecific antibody comprises one antigen-binding domain that specifically binds to PD-1 and one antigen-binding domain that specifically binds to LAG3 (1+1 format).

[0318] In one aspect, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises an Fc domain, a first Fab fragment comprising an antigen-binding domain that specifically binds to PD-1, and a second Fab fragment comprising an antigen-binding domain that specifically binds to LAG3. In a particular aspect, in one of the Fab fragments, the variable domains VL and VH are swapped with each other such that the VH domain is part of a light chain and the VL domain is part of a heavy chain. In a particular aspect, in the first Fab fragment comprising an antigen-binding domain that specifically binds to PD-1, the variable domains VL and VH are swapped with each other.

[0319] In certain aspects, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 39, a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 41, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 42. For example, in one aspect, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a first light chain comprising the amino acid sequence of SEQ ID NO: 40, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 41, and a second light chain comprising the amino acid sequence of SEQ ID NO: 42 (PD1-LAG3).

[0320] In a further aspect, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises an Fc domain, a first Fab fragment comprising the antigen-binding domain that specifically binds to PD-1, and a second Fab fragment comprising the antigen-binding domain that specifically binds to LAG3 fused to the C-terminus of the Fc domain. In particular, the Fab fragment comprising the antigen-binding domain that specifically binds to LAG3 is fused via its VH domain to the C-terminus of the Fc domain (trans 1+1 format).

[0321] In one embodiment, the bispecific antibody comprises a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 39, a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 40, a second heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 61, and a second light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 42. More specifically, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a first light chain comprising the amino acid sequence of SEQ ID NO: 40, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 61, and a second light chain comprising the amino acid sequence of SEQ ID NO: 42.

[0322] i. Fc domain modifications that reduce Fc receptor binding and / or effector function In certain aspects, bispecific antibodies are provided comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3, wherein the bispecific antibody comprises an Fc domain that comprises one or more amino acid modifications that reduce binding to an Fc receptor, particularly an Fcγ receptor, and reduce or abolish effector function.

[0323] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0324] The following sections describe preferred embodiments of bispecific antigen-binding molecules of the invention comprising Fc domain modifications that reduce Fc receptor binding and / or effector function. In one aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor. In particular, the Fc domain is an Fc domain of the human IgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0325] The Fc domain confers desirable pharmacokinetic properties to the bispecific antibodies of the invention, including a long serum half-life and a desirable tissue-to-blood distribution ratio, which contribute to favorable accumulation in target tissues. However, at the same time, it may cause undesirable targeting of the bispecific antibodies of the invention to cells expressing Fc receptors rather than cells bearing the desired antigen. Thus, in certain embodiments, the Fc domain of the bispecific antibodies of the invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to native IgG Fc domains, particularly IgG1 Fc domains or IgG4 Fc domains. More specifically, the Fc domain is an IgG1 Fc domain.

[0326] In one such embodiment, the Fc domain (or the bispecific antigen-binding molecule of the invention comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to an Fc receptor compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the invention comprising a native IgG1 Fc domain), and / or exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the invention comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or the bispecific antigen-binding molecule of the invention comprising said Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In a particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a specific embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcRIIB. In one embodiment, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the bispecific antigen-binding molecule of the invention comprising said Fc domain) exhibits a binding affinity for FcRn that is greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90%, compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the invention comprising a native IgG1 Fc domain).

[0327] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function compared to a non-engineered Fc domain. In certain embodiments, the Fc domain of a bispecific antigen-binding molecule of the present invention comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In another embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least two-fold, at least five-fold, or at least ten-fold. In one embodiment, a bispecific antigen-binding molecule of the present invention comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity to an Fc receptor compared to a bispecific antibody of the present invention comprising a non-engineered Fc domain. In a specific embodiment, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a specific embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcRIIB. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly C1q, is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn (i.e., preservation of the binding affinity of the Fc domain for the receptor) is achieved when the Fc domain (or a bispecific antigen-binding molecule of the invention comprising the Fc domain) exhibits a binding affinity for FcRn that is greater than about 70% of the binding affinity of an unengineered form of the Fc domain (or a bispecific antigen-binding molecule of the invention comprising this unengineered form of the Fc).The Fc domain, or a bispecific antigen-binding molecule of the present invention comprising the Fc domain, may exhibit greater than about 80%, or even greater than about 90%, of such affinity. In certain embodiments, the Fc domain of a bispecific antigen-binding molecule of the present invention is engineered to have reduced effector function compared to an unengineered Fc domain. Reduced effector function includes, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0328] Antibodies with reduced effector function include antibodies with substitutions of one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581). Specific antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0329] In one aspect of the invention, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331 and P329. In some aspects, the Fc domain comprises amino acid substitutions L234A and L235A ("LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and further amino acid substitutions selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a further particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor binding of a human IgG1 Fc domain, as described in PCT Application WO 2012 / 130831 A1, which also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function. Such antibodies may comprise the mutations L234A and L235A, or the mutations L234A, L235A, and P329G (numbering according to the EU index in Kabat et al.; Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0330] In one aspect, the bispecific antibody of the invention comprises (i) a homodimeric Fc region of the human IgG1 subclass, optionally with the mutations P329G, L234A and L235A, or (ii) a homodimeric Fc region of the human IgG4 subclass, optionally with the mutations P329G, S228P and L235E, or (iii) a homodimeric Fc region of the human IgG4 subclass, optionally with the mutations P329G, L234A, L235A, I253A, H310A and H435A, or optionally with the mutations P329G, L234A, L235A, I253A, H310A and H435A. or (iv) a homodimeric Fc region of the human IgG1 subclass having the mutations T366W and the other Fc region polypeptide having the mutations T366S, L368A, Y407V, and S354C; or (v) a homodimeric Fc region of the human IgG1 subclass having the mutations T366W and Y349C and the other Fc region polypeptide having the mutations T366S, L368A, Y407V, and S354C; or (v) a heterodimeric Fc region in which one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C, or (v) both Fc region polypeptides comprise the mutations P329G, L234A and L235A, and one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A and Y407V, or one The Fc region polypeptide comprises the mutations T366W and Y349C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C (all positions according to the EU index of Kabat).

[0331] In one aspect, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising the amino acid substitutions L235E, S228P, and P329G. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).

[0023] Thus, in one aspect, a bispecific antibody is provided comprising a heterodimeric Fc region of the human IgG4 subclass (all positions according to EU index of Kabat), wherein both Fc region polypeptides comprise the mutations P329G, S228P, and L235E, and wherein one Fc region polypeptide comprises the mutations T366W and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, or wherein one Fc region polypeptide comprises the mutations T366W and Y349C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C, or wherein one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C.

[0332] Antibodies with extended half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus (Guyer, R Let et al., J. Immunol. 117 (1976) 587-593, and Kim, J K et al., J. Immunol. 24 (1994) 242-92-434), are described in U.S. Patent Application Publication No. 2005 / 0014934. Such antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc variants include those containing a substitution at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan, A.R., and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0333] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare), and such Fc receptors can be obtained by recombinant expression. Suitable binding assays are described herein. Alternatively, the binding affinity of an Fc domain or a cell-activating bispecific antigen-binding molecule comprising an Fc domain to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor). The effector function of an Fc domain or a bispecific antibody of the present invention comprising an Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83, 70597063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 14991502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 13511361 (1987). Alternatively, non-radioactive assays can be used (e.g., ACTI for flow cytometry). TM non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model (such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998)).

[0334] The following sections describe preferred embodiments of bispecific antibodies of the invention comprising Fc domain modifications that reduce Fc receptor binding and / or effector function. In one aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor. In another aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the Fc domain comprises one or more amino acid substitutions that reduce effector function. In a specific aspect, the Fc domain is an Fc domain of the human IgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0335] ii. Fc domain modifications that promote heterodimerization The bispecific antigen-binding molecules of the present invention comprise different antigen-binding domains fused to one or the other of the two subunits of the Fc domain; thus, the two subunits of the Fc domain may be contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Therefore, to improve the yield and purity of the bispecific antibodies of the present invention during recombinant production, it may be advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecules of the present invention that promote the association of the desired polypeptides.

[0336] Thus, in a particular aspect, the present invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain. The site of greatest protein-protein interaction between the two subunits of a human IgG Fc domain is the CH3 domain of the Fc domain. Thus, in one aspect, the modification is within the CH3 domain of the Fc domain.

[0337] In certain embodiments, the modification comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain, a so-called "knob-into-hole" modification. Thus, the present invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding site that specifically binds to LAG3, wherein, according to the knob-into-hole method, the first subunit of the Fc domain comprises the knob and the second subunit of the Fc domain comprises the hole. In certain embodiments, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0338] This knob-into-hole technique is described, for example, in U.S. Pat. No. 5,731,168 and U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a protuberance ("knob") into the interface of a first polypeptide and a cavity ("hole") into the interface of a second polypeptide, such that the protuberance is positioned within the corresponding cavity ("hole"), promoting heterodimer formation and preventing homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0339] Thus, in one embodiment, in the CH3 domain of a first subunit of the Fc domain of a bispecific antigen-binding molecule of the present invention, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of the second subunit, and in the CH3 domain of a second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit that can be positioned within the protuberance in the CH3 domain of the first subunit. The protuberance and cavity can be created by modifying a nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In a particular embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0340] In yet a further embodiment, the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0341] However, other knobs-in-hole technologies may alternatively or additionally be used, as described in EP 1 870 459. In one embodiment, the multispecific antibody comprises the mutations R409D and K370E in the CH3 domain of the "knob chain" and the mutations D399K and E357K in the CH3 domain of the "hole chain" (numbering according to the Kabat EU index).

[0342] In one embodiment the bispecific antibody comprises a T366W mutation in the CH3 domain of the "knob chain", the mutations T366S, L368A and Y407V in the CH3 domain of the "hole" chain, and further comprises the mutations R409D and K370E in the CH3 domain of the "knob chain" and the mutations D399K and E357K in the CH3 domain of the "hole chain" (numbering according to Kabat EU index).

[0343] In one embodiment the bispecific antibody comprises the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains, or the multispecific antibody comprises the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains, and further comprises the mutations R409D and K370E in the CH3 domain of the "knob strand" and the mutations D399K and E357K in the CH3 domain of the "hole strand" (numbering according to Kabat EU index).

[0344] In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, e.g., as described in PCT Publication WO 2009 / 089004. Typically, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable.

[0345] Besides the "knob-into-hole" technique, other techniques for modifying the CH3 domains of the heavy chains of multispecific antibodies to force heterodimerization are known in the art. These techniques, in particular those described in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, and WO 2013 / 096291, are envisaged herein as alternatives to "knobs-into-holes" in combination with bispecific antibodies.

[0346] In one embodiment, to support heterodimerization of the first and second heavy chains of a multispecific antibody in a bispecific antibody, the approach described in EP 1 870 459 is used, which is based on the introduction of oppositely charged amino acids at specific amino acid positions in the CH3 / CH3 domain interface between the first and second heavy chains.

[0347] Thus, in this embodiment, in the tertiary structure of the multispecific antibody, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface between the respective antibody CH3 domains, and the amino acid sequence of each of the CH3 domains of the first heavy chain and the second heavy chain each comprises a series of amino acids located within said interface in the tertiary structure of the antibody, wherein a first amino acid of the series located at the interface of the CH3 domains of one heavy chain is substituted with a positively charged amino acid, and a second amino acid of the series located at the interface of the CH3 domains of the other heavy chain is substituted with a negatively charged amino acid. Bispecific antibodies of this embodiment are also referred to herein as "CH3(+ / -) engineered bispecific antibodies" (wherein the abbreviation "+ / -" represents amino acids of the opposite charge introduced into each CH3 domain).

[0348] In one aspect, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acids are selected from K, R and H and the negatively charged amino acids are selected from E or D.

[0349] In one aspect, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acids are selected from K and R and the negatively charged amino acids are selected from E or D.

[0350] In one aspect, the positively charged amino acid is K and the negatively charged amino acid is E in the CH3(+ / -) engineered bispecific antibody.

[0351] In one embodiment in the CH3(+ / -) engineered bispecific antibody, in the CH3 domain of one heavy chain the amino acid R at position 409 is substituted by D and the amino acid K at position 409 is substituted by E, and in the CH3 domain of the other heavy chain the amino acid D at position 399 is substituted by K and the amino acid E at position 357 is substituted by K (numbering according to Kabat EU index).

[0352] In one aspect, the approach described in WO 2013 / 157953 is used to support heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted by K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is substituted by D (numbering according to the Kabat EU index). In another embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted by K, and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is substituted by D (numbering according to the Kabat EU index).

[0353] In another embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted by K, and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain, the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index). Additionally, at least one of the following substitutions is contained in the CH3 domain of the other heavy chain: the amino acid Y at position 349 is substituted by E, the amino acid Y at position 349 is substituted by D, and the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index). In one embodiment, the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index).

[0354] In one aspect, the approach described in WO 2012 / 058768 is used to support heterodimerization of the first and second heavy chains of a multispecific antibody. In one aspect, in the CH3 domain of one heavy chain, the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to the Kabat EU index). In another embodiment, in addition to the substitutions mentioned above, in the CH3 domain of the other heavy chain, at least one of the amino acids at positions 411 (originally T), 399 (originally D), 400 (originally S), 405 (originally F), 390 (originally N) and 392 (originally K) is substituted (numbering according to the Kabat EU index). Preferred substitutions are as follows: - substitution of the amino acid T at position 411 with an amino acid selected from N, R, Q, K, D, E and W (numbering according to the Kabat EU index), - substitution of the amino acid D at position 399 with an amino acid selected from R, W, Y and K (numbering according to the Kabat EU index), - substitution of amino acid D at position 400 with an amino acid selected from E, D, R and K (numbering according to the Kabat EU index), - substitution of the amino acid F at position 405 with an amino acid selected from I, M, T, S, V and W (numbering according to the Kabat EU index), - substitution of the amino acid N at position 390 with an amino acid selected from R, K and D (numbering according to the Kabat EU index), - the amino acid K at position 392 is substituted with an amino acid selected from V, M, R, L, F and E (numbering according to the Kabat EU index).

[0355] In another aspect, a bispecific antibody is engineered according to WO 2012 / 058768), i.e., in the CH3 domain of one heavy chain, the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is substituted by V and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In another embodiment of a multispecific antibody, in the CH3 domain of one heavy chain, the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In the latter above-mentioned embodiment, in the CH3 domain of the other heavy chain, the amino acid K at position 392 is substituted by E, the amino acid T at position 411 is substituted by E, the amino acid D at position 399 is substituted by R, and the amino acid S at position 400 is substituted by R (numbering according to the Kabat EU index).

[0356] In one embodiment, the techniques described in WO 2011 / 143545 are used to support heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, amino acid modifications in the CH3 domains of both heavy chains are introduced at positions 368 and / or 409 (numbering according to the Kabat EU index).

[0357] In one embodiment, the technique described in WO 2011 / 090762 is used to support heterodimerization of the first and second heavy chains of a bispecific antibody. WO 2011 / 090762 relates to amino acid modification by "knobs-into-holes" (KiH) technology. In one embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with W, and in the CH3 domain of the other heavy chain, the amino acid Y at position 407 is substituted with A (numbering according to the Kabat EU index). In another embodiment, in the CH3 domain of one heavy chain, the amino acid T at position 366 is substituted with Y, and in the CH3 domain of the other heavy chain, the amino acid Y at position 407 is substituted with T (numbering according to the Kabat EU index).

[0358] In one aspect, the approach described in WO 2009 / 089004 is used to support heterodimerization of the first and second heavy chains of a bispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, the amino acid K or N at position 392 is substituted by a negatively charged amino acid (in one embodiment by E or D, in a preferred embodiment by D), and in the CH3 domain of the other heavy chain, the amino acid D at position 399, the amino acid E or D at position 356, or the amino acid E at position 357 is substituted by a positively charged amino acid (in one embodiment by K or R, in a preferred embodiment by K, and in a preferred embodiment the amino acid at position 399 or 356 is substituted by K) (numbering according to the Kabat EU index). In a further embodiment, in addition to the above substitutions, in the CH3 domain of one heavy chain, the amino acid K or R at position 409 is substituted by a negatively charged amino acid (in one embodiment by E or D, in a preferred embodiment by D) (numbering according to Kabat EU index). In a still further aspect, in addition to or instead of the above substitutions, in the CH3 domain of one heavy chain, the amino acid K at position 439 and / or the amino acid K at position 370 are / are substituted, independently of one another, by a negatively charged amino acid (in one embodiment by E or D, in a preferred embodiment by D) (numbering according to Kabat EU index).

[0359] In one aspect, the approach described in WO 2007 / 147901 is used to support heterodimerization of the first and second heavy chains of a multispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, the amino acid K at position 253 is substituted by E, the amino acid D at position 282 is substituted by K, and the amino acid K at position 322 is substituted by D, and in the CH3 domain of the other heavy chain, the amino acid D at position 239 is substituted by K, the amino acid E at position 240 is substituted by K, and the amino acid K at position 292 is substituted by D (numbering according to the Kabat EU index).

[0360] The C-terminus of the heavy chain of the bispecific antibody reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may also be a truncated C-terminus in which one or two of the C-terminal amino acid residues have been removed. In one preferred embodiment, the C-terminus of the heavy chain is PG, ending in a truncated C-terminus.

[0361] In one aspect of all aspects reported herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the Kabat EU index). In one embodiment of all aspects reported herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, numbering according to the Kabat EU index).

[0362] iii. Modifications in the Fab domain In one aspect, the invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD-1 and a second Fab fragment that specifically binds to LAG3, wherein either the variable domains VH and VL or the constant domains CH1 and CL are exchanged in one of the Fab fragments. The bispecific antibody is prepared according to crossmab technology.

[0363] Multispecific antibodies with domain replacement / swapping in one binding arm (CrossMabVH-VL or CrossMabCH-CL) are described in WO 2009 / 080252, WO 2009 / 080253, and Schaefer, W. et al., PNAS, 108 (2011) 11187-1191. These multispecific antibodies clearly reduce by-products caused by mismatches between a light chain for one antigen and an incorrect heavy chain for a second antigen (compared to approaches without such domain swapping).

[0364] In certain aspects, the invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD-1 and a second Fab fragment that specifically binds to LAG3, wherein in one of the Fab fragments the variable domains VL and VH are swapped for each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In certain aspects, the bispecific antibody is a bispecific antibody in which the variable domains VL and VH are swapped for each other in the first Fab fragment that comprises the antigen-binding domain that specifically binds to PD-1.

[0365] In another aspect, to further improve correct pairing, a bispecific antibody comprising a first Fab fragment that specifically binds to PD-1 and a second Fab fragment that specifically binds to LAG3 may contain differentially charged amino acid substitutions (so-called "charged residues"). These modifications may be introduced into the crossover or non-crossover CH1 and CL domains. These modifications are described, for example, in WO 2015 / 150447, WO 2016 / 020309, and PCT / EP2016 / 073408.

[0366] In a particular aspect, the invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD-1 and a second Fab fragment that specifically binds to LAG3, wherein in one of the Fab fragments, in the constant domain CL, the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU index), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index). In a particular embodiment, the bispecific antibody is a bispecific antibody, wherein in the constant domain CL of the second Fab fragment comprising an antigen-binding domain that specifically binds to TIM3, the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0367] In a particular aspect, the invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD-1 and a second Fab fragment that specifically binds to LAG3, wherein in one of the CL domains the amino acid at position 123 (EU numbering) is replaced by arginine (R) and the amino acid at position 124 (EU numbering) is replaced by lysine (K), and in one of the CH1 domains the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are replaced by glutamic acid (E). In a particular aspect, the bispecific antibody is a Fab fragment comprising an antigen-binding domain that specifically binds to LAG3, wherein the amino acid at position 123 (EU numbering) is replaced by arginine (R) and the amino acid at position 124 (EU numbering) is replaced by lysine (K), and in one of the CH1 domains the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are replaced by glutamic acid (E).

[0368] In a further aspect, the bispecific antibody comprises: a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen; wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other.

[0369] The antibody of a) does not contain the modifications reported in b), and the heavy and light chains of a) are isolated chains.

[0370] In the antibody of (b), in the light chain, the variable light domain VL is replaced by the variable heavy domain VH of said antibody, and in the heavy chain, the variable heavy domain VH is replaced by the variable light domain VL of said antibody.

[0371] and in the constant domain CHI of the first heavy chain of (a), the amino acid at position 147 or the amino acid at position 213 (numbering according to Kabat EU index) is substituted by a negative amino acid; or (ii) in the constant domain CL of the second light chain of (b), the amino acid at position 124 (numbering according to Kabat) is substituted by a positive amino acid; and in the constant domain CHI of the second heavy chain of (b), the amino acid at position 147 or the amino acid at position 213 (numbering according to Kabat EU index) is substituted by a negative amino acid.

[0372] In another aspect, (i) in the constant domain CL of the first light chain of (a) the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in one preferred embodiment, independently substituted by lysine (K) or arginine (R)); and in the constant domain CHI of the first heavy chain of (a) the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat numbering). and (ii) in the constant domain CL of the second light chain of (b), the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in one preferred embodiment, independently by lysine (K) or arginine (R)); and in the constant domain CHI of the second heavy chain of (b), the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0373] In one aspect, in the constant domain CL of the second heavy chain, the amino acids at positions 124 and 123 are substituted by K (numbering according to the Kabat EU index).

[0374] In one aspect, in the constant domain CL of the second heavy chain, the amino acid at position 123 is substituted by R and the amino acid at position 124 is substituted by K (numbering according to Kabat EU index).

[0375] In one aspect, in the constant domain CH1 of the second light chain, the amino acids at positions 147 and 213 are substituted by E (numbering according to the Kabat EU index).

[0376] In one aspect, in the constant domain CL of the first light chain the amino acids at positions 124 and 123 are substituted by K and in the constant domain CH1 of the first heavy chain the amino acids at positions 147 and 213 are substituted by E (numbering according to Kabat EU index).

[0377] In one aspect, in the constant domain CL of the first light chain the amino acid at position 123 is substituted by R and the amino acid at position 124 is substituted by K; and in the constant domain CH1 of the first heavy chain the amino acids at positions 147 and 213 are both substituted by E (numbering according to Kabat EU index).

[0378] In one aspect in the constant domain CL of the second heavy chain the amino acids at positions 124 and 123 are substituted by K, and in the constant domain CHI of the second light chain the amino acids at positions 147 and 213 are substituted by E, in the variable domain VL of the first light chain the amino acid at position 38 is substituted by K, and in the variable domain VH of the first heavy chain the amino acid at position 39 is substituted by E, in the variable domain VL of the second heavy chain the amino acid at position 38 is substituted by K and in the variable domain VH of the second light chain the amino acid at position 39 is substituted by E (numbering according to Kabat EU index).

[0379] In one aspect, the bispecific antibody comprises: a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen; wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other, and the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other.

[0380] The antibody of (a) does not comprise the modifications reported in (b), and the heavy and light chains of (a) are isolated chains. In the antibody of (b), within the light chain, the variable light domain VL is replaced by the variable heavy domain VH of said antibody, the constant light domain CL is replaced by the constant heavy domain CH1 of said antibody, and within the heavy chain, the variable heavy domain VH is replaced by the variable light domain VL of said antibody, and the constant heavy domain CH1 is replaced by the constant light domain CL of said antibody.

[0381] In one aspect, the bispecific antibody comprises: a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen; wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other.

[0382] The antibody of a) does not contain the modifications reported in b), and the heavy and light chains of a) are isolated chains. In the antibody of b), in the light chain, the constant light chain domain CL is replaced by the constant heavy chain domain CH1 of said antibody, and in the heavy chain, the constant heavy chain domain CH1 is replaced by the constant light chain domain CL of said antibody.

[0383] In one aspect, the bispecific antibody comprises: a) a full-length antibody that specifically binds to a first antigen and that consists of two antibody heavy chains and two antibody light chains; and b) one, two, three or four single-chain Fab fragments that specifically bind to a second antigen; a bispecific antibody comprising Here, the single-chain Fab fragment of b) is fused to the full-length antibody of a) via a peptide linker at the C-terminus or N-terminus of the heavy chain or light chain of the full-length antibody.

[0384] In one embodiment, one or two identical single-chain Fab fragments that bind to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of the heavy or light chain of the full-length antibody.

[0385] In one embodiment, one or two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of the heavy chain of the full-length antibody via a peptide linker.

[0386] In one embodiment, one or two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of the light chain of the full-length antibody via a peptide linker.

[0387] In one embodiment, two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of each heavy or light chain of the full-length antibody.

[0388] In one embodiment, two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of each heavy chain of the full-length antibody.

[0389] In one embodiment, two identical single-chain Fab (scFab) fragments that bind to a second antigen are fused to the full-length antibody via a peptide linker at the C-terminus of each light chain of the full-length antibody.

[0390] In one aspect, the bispecific antibody comprises: a) a full-length antibody that specifically binds to a first antigen and is composed of two antibody heavy chains and two antibody light chains; b) ba) an antibody heavy chain variable domain (VH), or bb) antibody heavy chain variable domain (VH) and antibody constant domain 1 (CH1) A first polypeptide consisting of a first polypeptide fused by the N-terminus of its VH domain to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker; c) ca) an antibody light chain variable domain (VL), or cb) antibody light chain variable domain (VL) and antibody light chain constant domain (CL) a second polypeptide consisting of The second polypeptide is fused via the N-terminus of the VL domain to the C-terminus of the other of the two heavy chains of the full-length antibody via a peptide linker. Including, Here, the antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide combine to form an antigen-binding domain that specifically binds to a second antigen, making it a trivalent antibody.

[0391] In one embodiment, the antibody heavy chain variable domain (VH) of the polypeptide of b) and the antibody light chain variable domain (VL) of the polypeptide of c) are linked and stabilized via an interchain disulfide bridge by introducing a disulfide bond between the following positions: (i) heavy chain variable domain position 44 to light chain variable domain position 100, or (ii) heavy chain variable domain position 105 to light chain variable domain position 43, or (iii) heavy chain variable domain position 101 to light chain variable domain position 100 (numbering always according to the Kabat EU index).

[0392] Techniques for introducing non-natural disulfide bridges for stabilization are described, for example, in WO 94 / 029350; Rajagopal, V., et al., Prot. Eng. (1997) 1453-1459; Kobayashi, H., et al., Nucl. Med. Biol. 25 (1998) 387-393; and Schmidt, M., et al., Oncogene 18 (1999) 1711-1721. In one embodiment, the optional disulfide bond between the variable domains of polypeptides b) and c) is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the optional disulfide bond between the variable domains of polypeptides b) and c) is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (numbering always according to the Kabat EU index). In one embodiment, trivalent bispecific antibodies are preferred that do not have any said disulfide stabilization between the variable domains VH and VL of the single-chain Fab fragments.

[0393] In one aspect, the bispecific antibody comprises: a) a first light chain and a first heavy chain of a full-length antibody that specifically binds to a first antigen, and b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to a second antigen, wherein the variable domains VL and VH are replaced with each other and / or the constant domains CL and CH1 are replaced with each other. Including, c) A triabody or tetrabody in which one to four antigen-binding domains that specifically bind to one or two additional antigens (i.e., bind to a third and / or fourth antigen) are fused via peptide linkers to the C-terminus or N-terminus of the light chain or heavy chain of a) and / or b).

[0394] The antibody of a) does not contain the modifications reported in b), and the heavy and light chains of a) are isolated chains.

[0395] In one embodiment the triabody or tetrabody comprises in c) one or two antigen-binding domains that specifically bind to one or two further antigens.

[0396] In one embodiment, the antigen binding domain is selected from the group consisting of an scFv fragment and an scFab fragment.

[0397] In one embodiment, the antigen-binding domain is an scFv fragment.

[0398] In one embodiment, the antigen binding domain is an scFab fragment.

[0399] In one embodiment the antigen binding domain is fused to the C-terminus of the heavy chain of a) and / or b).

[0400] In one embodiment, the triabody or tetrabody comprises in c) one or two antigen-binding domains that specifically bind to one further antigen.

[0401] In one aspect, the triabody or tetrabody comprises two identical antigen-binding domains in c) that specifically bind to a third antigen. In a preferred embodiment, such two identical antigen-binding domains are both fused to the C-terminus of the heavy chains of a) and b) via the same peptide linker. In a preferred embodiment, the two identical antigen-binding domains are either scFv or scFab fragments.

[0402] In one aspect, the triabody or tetrabody comprises two antigen-binding domains in c) that specifically bind to a fourth antigen. In one embodiment, the two antigen-binding domains are both fused to the C-terminus of the heavy chains of a) and b) via the same peptide linkage. In a preferred embodiment, the two antigen-binding domains are either scFv or scFab fragments.

[0403] In one aspect, the bispecific antibody comprises: a) two light chains and two heavy chains of an antibody that specifically binds to a first antigen (and comprises two Fab fragments); b) two additional Fab fragments of an antibody that specifically bind to a second antigen, said additional Fab fragments being fused via a peptide linker to either the C-terminus or the N-terminus of the heavy chain of a). a bispecific tetravalent antibody comprising Here, the following modifications have been made to the Fab fragment: (i) in both Fab fragments of a) or in both Fab fragments of b) the variable domains VL and VH are replaced with each other and / or the constant domains CL and CH1 are replaced with each other, or (ii) in a) both Fab fragments, the variable domains VL and VH are replaced by one another and the constant domains CL and CH1 are replaced by one another; and in b) both Fab fragments, the variable domains VL and VH are replaced by one another or the constant domains CL and CH1 are replaced by one another; or (iii) in a) both Fab fragments, the variable domains VL and VH are replaced by one another or the constant domains CL and CH1 are replaced by one another, and in b) both Fab fragments, the variable domains VL and VH are replaced by one another and the constant domains CL and CH1 are replaced by one another; or (iv) in a) both Fab fragments the variable domains VL and VH are replaced with each other, and in b) both Fab fragments the constant domains CL and CH1 are replaced with each other; or (v) In both Fab fragments of a), the constant domains CL and CH1 are replaced with each other, and in both Fab fragments of b), the variable domains VL and VH are replaced with each other.

[0404] In one embodiment, said additional Fab fragments are both fused to either the C-terminus of the heavy chain of a) or the N-terminus of the heavy chain of a) via a peptide linker.

[0405] In one embodiment, said additional Fab fragments are both fused to the C-terminus of the heavy chain of a) via a peptide linker.

[0406] In one embodiment, said additional Fab fragments are both fused to the N-terminus of the heavy chain of a) via a peptide linker.

[0407] In one embodiment, the following modifications are made in the Fab fragments: a) in both Fab fragments, or b) in both Fab fragments, the variable domains VL and VH are swapped for each other and / or the constant domains CL and CH1 are swapped for each other.

[0408] In one aspect, the bispecific antibody comprises: a) a (modified) heavy chain of a first antibody that specifically binds to a first antigen and comprises a first VH-CH1 domain pair, wherein the N-terminus of a second VH-CH1 domain pair of the first antibody is fused to the C-terminus of the heavy chain via a peptide linker; b) the two light chains of said first antibody of a) c) a (modified) heavy chain of a second antibody that specifically binds to a second antigen and comprises a first VH-CL domain pair, wherein the N-terminus of a second VH-CL domain pair of the second antibody is fused to the C-terminus of the heavy chain via a peptide linker; and d) two (modified) light chains of said second antibody of c), each of which comprises a CL-CH1 domain pair; It is a tetravalent antibody comprising:

[0409] In one aspect, the bispecific antibody comprises: a) the heavy and light chains of a first full-length antibody that specifically binds to a first antigen, and b) the heavy and light chains of a second full-length antibody that specifically binds to a second antigen, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker. Includes:

[0410] The antibody in a) does not contain the modifications reported in b), and the heavy and light chains are isolated chains.

[0411] In one aspect, the bispecific antibody comprises: a) a full-length antibody that specifically binds to a first antigen and that consists of two antibody heavy chains and two antibody light chains; and b) an Fv fragment that specifically binds to a second antigen, comprising a VH2 domain and a VL2 domain, both domains being linked to each other via disulfide bridges; Here, either the VH2 domain or the VL2 domain alone is fused via a peptide linker to the heavy or light chain of a full-length antibody that specifically binds to a first antigen.

[0412] In a bispecific antibody, a) the heavy and light chains are separate chains.

[0413] In one embodiment, the other of the VH2 domain or the VL2 domain is not fused via a peptide linker to the heavy or light chain of the full length antibody that specifically binds to the first antigen.

[0414] In all embodiments reported herein, the first light chain comprises a VL domain and a CL domain, and the first heavy chain comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain.

[0415] In one aspect, the bispecific antibody comprises: a) two Fab fragments that specifically bind to a first antigen; b) one CrossFab fragment that specifically binds to a second antigen and in which the CH1 and CL domains are exchanged with each other; c) an Fc region comprising the heavy chain of a first Fc region and the heavy chain of a second Fc region; a trivalent antibody comprising Here, the C-terminus of the CH1 domains of the two Fab fragments is linked to the N-terminus of the heavy chain Fc region polypeptide, and the C-terminus of the CL domain of the CrossFab fragment is linked to the N-terminus of the VH domain of one of the Fab fragments.

[0416] In one aspect, the bispecific antibody comprises: a) two Fab fragments that specifically bind to a first antigen; b) one CrossFab fragment that specifically binds to a second antigen and in which the CH1 and CL domains are exchanged with each other; c) an Fc region comprising the heavy chain of a first Fc region and the heavy chain of a second Fc region; a trivalent antibody comprising Here, the C-terminus of the CH1 domain of the first Fab fragment is linked to the N-terminus of one of the heavy chain Fc region polypeptides, the C-terminus of the CL domain of the CrossFab fragment is linked to the N-terminus of the other heavy chain Fc region polypeptide, and the C-terminus of the CH1 domain of the second Fab fragment is linked to the N-terminus of the VH domain of the first Fab fragment or the N-terminus of the VH domain of the CrossFab fragment.

[0417] In one aspect, the bispecific antibody comprises: a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; and b) a Fab fragment that specifically binds to a second antigen, comprising a heavy chain fragment and a light chain fragment comprising a VH2 domain and a VL2 domain, wherein in the light chain fragment the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of said antibody, and in the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of said antibody. Including, Here, the heavy chain Fab fragment is inserted between the CH1 domain of one of the heavy chains of the full-length antibody and the respective Fc region of the full-length antibody, and the N-terminus of the light chain Fab fragment is conjugated to the C-terminus of the light chain of the full-length antibody that pairs with the heavy chain of the full-length antibody into which the heavy chain Fab fragment is inserted.

[0418] In one aspect, the bispecific antibody comprises: a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; and b) a Fab fragment which specifically binds to a second antigen, comprising a VH2 domain and a VL2 domain comprising a heavy chain fragment and a light chain fragment, wherein in the light chain fragment the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of said antibody, and in the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of said antibody, and wherein the C-terminus of the heavy chain fragment of the Fab fragment is conjugated to the N-terminus of one of the heavy chains of a full-length antibody, and the C-terminus of the light chain fragment of the Fab fragment is conjugated to the N-terminus of the light chain of a full-length antibody which pairs with the heavy chain of the full-length antibody, whereas the heavy chain fragment of the Fab fragment is conjugated Includes:

[0419] B. Dosing of Bispecific Antibodies Binding to PD-1 and LAG3 The appropriate dosage of a bispecific antibody comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG3 of the present invention for the prevention or treatment of disease (when used alone or in combination with one or more other additional therapeutic agents) will vary depending on the type of disease being treated, the route of administration, the subject's weight, the type of fusion protein, the severity and course of the disease, whether the bispecific antibody is being administered for prophylactic or therapeutic purposes, previous or current therapeutic interventions, the subject's medical history and response to the fusion protein, and the judgment of the attending physician. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for the individual subject. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0420] Bispecific antibodies comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, as defined herein, are preferably administered to a subject at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of bispecific antibody may be an initial candidate dosage for administration to a subject, whether by one or more separate administrations or by continuous infusion, for example. Depending on the factors described above, a typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more. For repeated administrations over several days or more, treatment is typically continued until a desired suppression of disease symptoms occurs, depending on the condition. One exemplary dosage of a bispecific antibody ranges from about 0.005 mg / kg to about 10 mg / kg. In other examples, dosages may also include about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight, about 500 mg / kg body weight, to about 1000 mg / kg body weight, or any range derivable therebetween. Examples of ranges derivable from the numbers listed herein may be administered in the ranges of about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 μg / kg body weight to about 500 mg / kg body weight, etc., based on the above numbers. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the subject. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., the patient receives from about 2 to about 20, or for example, about 6, doses of the fusion protein). An initial higher loading dose, followed by one or more lower doses, may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0421] In one particular aspect, the bispecific antibody targeting PD-1 and LAG3 is administered to the subject at a fixed dose of about 600 mg every three weeks (Q3W), e.g., a fixed dose of 600 mg Q3W.

[0422] In another embodiment, the bispecific antibody targeting PD-1 and LAG3 is administered to the subject at a fixed dose of about 1200 mg every three weeks, e.g., a fixed dose of 1200 mg Q3W.

[0423] In another embodiment, the bispecific antibody targeting PD-1 and LAG3 is administered to the subject at a fixed dose of about 2100 mg every two weeks (Q2W), e.g., a fixed dose of 2100 mg Q2W.

[0424] IX. VEGF Antagonists VEGF antagonists include any molecule that can bind to VEGF, reduce VEGF expression levels, or neutralize, block, inhibit, abrogate, reduce, or interfere with the biological activity of VEGF. An exemplary human VEGF is set forth as UniProtKB / Swiss-Prot accession number P15692, Gene ID (NCBI):7422.

[0425] In some instances, the VEGF antagonist is an anti-VEGF antibody. In some embodiments, the anti-VEGF antibody is bevacizumab, also known as "rhuMab VEGF" or "AVASTIN®." Bevacizumab is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al. (Cancer Res. 57:4593-4599, 1997). It contains mutated human IgG1 framework regions and antigen-binding complementarity-determining regions from the murine anti-hVEGF monoclonal antibody A.4.6.1, which blocks the binding of human VEGF to its receptor. Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgG1, and approximately 7% of the sequence is derived from the murine antibody A4.6.1. Bevacizumab has a molecular weight of approximately 149,000 daltons and is glycosylated. Bevacizumab and other humanized anti-VEGF antibodies are further described in US Pat. No. 6,884,879, issued Feb. 26, 2005, the entire disclosure of which is incorporated herein by reference.

[0426] Additional preferred antibodies include the G6 or B20 series antibodies (e.g., G6-31, B20-4.1) described in PCT Application Publication No. WO 2005 / 012359. For additional preferred antibodies, see U.S. Patent Nos. 7,060,269, 6,582,959, 6,703,020; 6,054,297; WO 98 / 45332; WO 96 / 30046; WO 94 / 10202; EP 0 666 868; U.S. Patent Application Publication Nos. 2006 / 009360, 2005 / 0186208, 2003 / 0206899, 2003 / 0190317, 2003 / 0203409, and 2005 / 0112126; and Popkov et al. (Journal of Immunological Methods 288:149164, 2004). Other preferred antibodies include antibodies that bind to a functional epitope on human VEGF that includes residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104, or that includes residues F17, Y21, Q22, Y25, D63, 183, and Q89.

[0427] In other examples, the VEGF antagonist is an anti-VEGFR2 antibody or related molecule (e.g., ramucirumab, tanibirumab, aflibercept), an anti-VEGFR1 antibody or related molecule (e.g., icrucumab, aflibercept (VEGF Trap-Eye, EYLEA®), or dib-aflibercept (VEGF Trap, ZALTRAP®)), a bispecific VEGF antibody (e.g., MP-0250, vanucizumab (VEGF-ANG2), or the bispecific antibodies disclosed in US 2001 / 0236388), a bispecific antibody comprising a combination of two of an anti-VEGF arm, an anti-VEGFR1 arm, and an anti-VEGFR2 arm, an anti-VEGFA antibody (e.g., bevacizumab, sevacizumab), an anti-VEGFR2 antibody (e.g., cevacizumab), an anti-VEGF ... The VEGF antagonist may be a GFB antibody, an anti-VEGF C antibody (e.g., VGX-100), an anti-VEGF D antibody, or a non-peptide small molecule VEGF antagonist (e.g., pazopanib, axitinib, vandetanib, stivarga, cabozantinib, lenvatinib, nintedanib, orantinib, telatinib, dovitinib, cediranib, motesanib, surufatinib, apatinib, foretinib, famitinib, or tivozanib). In some examples, the VEGF antagonist may be a tyrosine kinase inhibitor, including a receptor tyrosine kinase inhibitor (e.g., a multi-targeted receptor tyrosine kinase inhibitor such as sunitinib or axitinib).

[0428] X.PD-1 axis binding antagonist PD-1 axis binding antagonists can include PD-L1 binding antagonists, PD-1 binding antagonists, and PD-L2 binding antagonists. Any suitable PD-1 axis binding antagonist can be used to treat a subject with cancer.

[0429] A. PD-L1 Binding Antagonists In some instances, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In other instances, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In yet other instances, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some instances, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. The PD-L1 binding antagonist can be, but is not limited to, an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, oligopeptide, or small molecule. In some instances, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 (e.g., GS-4224, INCB 086550, MAX-10181, INCB 090244, CA-170, or ABSK 041). In some instances, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and VISTA. In some instances, the PD-L1 binding antagonist is CA-170 (also known as AUPM-170). In some instances, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and TIM3. In some instances, the small molecule is a compound described in WO 2015 / 033301 and / or WO 2015 / 033299.

[0430] In some instances, the PD-L1 binding antagonist is an anti-PD-L1 antibody. A variety of anti-PD-L1 antibodies are contemplated and described herein. In any of the examples herein, the isolated anti-PD-L1 antibody can bind to human PD-L1, for example, human PD-L1 as set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7-1, or a variant thereof. In some instances, the anti-PD-L1 antibody can inhibit the binding between PD-L1 and PD-1 and / or between PD-L1 and B7-1. In some instances, the anti-PD-L1 antibody is a monoclonal antibody. In some instances, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some instances, the anti-PD-L1 antibody is a humanized antibody. In some instances, the anti-PD-L1 antibody is a human antibody. Exemplary anti-PD-L1 antibodies include atezolizumab, MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), SHR-1316, CS1001, embafolimab, TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, cosibelimab, lodapolimab, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, RC98, PDL-GEX, KD036, KY1003, YBL-007, and HS-636. Examples of anti-PD-L1 antibodies useful in the methods of the invention and methods for making them are described in WO 2010 / 077634 and U.S. Patent No. 8,217,149, each of which is incorporated by reference in its entirety.

[0431] In some instances, the anti-PD-L1 antibody (a) the HVR-H1, HVR-H2, and HVR-H3 sequences of GFTFSDSWIH (SEQ ID NO: 64), AWISPYGGSTYYADSVKG (SEQ ID NO: 65), and RHWPGGFDY (SEQ ID NO: 66), respectively; and (b) HVR-L1, HVR-L2, and HVR-L3 sequences of RASQDVSTAVA (SEQ ID NO: 67), SASFLYS (SEQ ID NO: 68), and QQYLYHPAT (SEQ ID NO: 69), respectively Includes:

[0432] In one embodiment, the anti-PD-L1 antibody is: (a) a heavy chain variable region (VH) comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 70); and (b) a light chain variable region comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 71) Includes:

[0433] In some instances, the anti-PD-L1 antibody comprises (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of SEQ ID NO: 9; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of SEQ ID NO: 10; or a VH of (a) and a VL of (b).

[0434] In one embodiment, the anti-PD-L1 antibody is: (a) Heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 62), and (b) Light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 63) Contains atezolizumab.

[0435] In some instances, the anti-PD-L1 antibody is avelumab (CAS Registry Number: 1537032-82-8). Avelumab, also known as MSB0010718C, is a human monoclonal IgG1 anti-PD-L1 antibody (Merck KGaA, Pfizer).

[0436] In some instances, the anti-PD-L1 antibody is durvalumab (CAS Registry Number: 1428935-60-7). Durvalumab, also known as MEDI4736, is an Fc-optimized human monoclonal IgG1 kappa anti-PD-L1 antibody (MedImmune, AstraZeneca) described in WO 2011 / 066389 and U.S. Patent Application Publication No. 2013 / 034559.

[0437] In some instances, the anti-PD-L1 antibody is MDX-1105 (Bristol Myers Squibb). MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007 / 005874.

[0438] In some instances, the anti-PD-L1 antibody is LY3300054 (Eli Lilly).

[0439] In some instances, the anti-PD-L1 antibody is STI-A1014 (Sorrento). STI-A1014 is a human anti-PD-L1 antibody.

[0440] In some instances, the anti-PD-L1 antibody is KN035 (Suzhou Alphamab), which is a single domain antibody (dAB) generated from a camel phage display library.

[0441] In some instances, the anti-PD-L1 antibody comprises a cleavable moiety or linker that, when cleaved (e.g., by proteases in the tumor microenvironment), activates the antibody antigen-binding domain so that it is able to bind its antigen, e.g., by removing non-binding steric moieties. In some instances, the anti-PD-L1 antibody is CX-072 (CytomX Therapeutics).

[0442] In some instances, the anti-PD-L1 antibody comprises six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and / or heavy chain and light chain variable domains of an anti-PD-L1 antibody described in US Patent Application Publication No. 20160108123, WO 2016 / 000619, WO 2012 / 145493, US Patent No. 9205148, WO 2013 / 181634, or WO 2016 / 061142.

[0443] In yet a further specific embodiment, the anti-PD-L1 antibody has reduced or minimal effector function. In a further specific embodiment, the minimal effector function is due to an "effector-less Fc mutation" or an aglycosylation mutation. In yet a further example, the effector-less Fc mutation is an N297A or D265A / N297A substitution in the constant region. In yet a further example, the effector-less Fc mutation is an N297A substitution in the constant region. In some examples, the isolated anti-PD-L1 antibody is aglycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of a glycosylation site from an antibody is conveniently accomplished by altering the amino acid sequence so that one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) is deleted. This alteration can be made by substituting the asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[0444] B. PD-1 Binding Antagonists In some instances, the PD-1 axis binding antagonist is a PD-1 binding antagonist. For example, in some instances, the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. In some instances, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In other instances, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In yet other instances, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. The PD-1 binding antagonist can be, but is not limited to, an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, or a small molecule. In some instances, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). For example, in some instances, the PD-1 binding antagonist is an Fc fusion protein. In some instances, the PD-1 binding antagonist is AMP-224. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342. In some instances, the PD-1 binding antagonist is a peptide or small molecule compound. In some instances, the PD-1 binding antagonist is AUNP-12 (PierreFabre / Aurigene). See, e.g., WO 2012 / 168944, WO 2015 / 036927, WO 2015 / 044900, WO 2015 / 033303, WO 2013 / 144704, WO 2013 / 132317, and WO 2011 / 161699. In some instances, the PD-1 binding antagonist is a small molecule that inhibits PD-1.

[0445] In some instances, the PD-1 binding antagonist is an anti-PD-1 antibody. A variety of anti-PD-1 antibodies may be utilized in the methods and uses disclosed herein. In any of the instances herein, the PD-1 antibody is capable of binding to human PD-1 or a variant thereof. In some instances, the anti-PD-1 antibody is a monoclonal antibody. In some instances, the anti-PD-1 antibody is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some instances, the anti-PD-1 antibody is a humanized antibody. In other instances, the anti-PD-1 antibody is a human antibody. Exemplary anti-PD-1 antagonist antibodies include nivolumab, pembrolizumab, MEDI-0680, PDR001 (spartalizumab), REGN2810 (cemiplimab), BGB-108, prorugolimab, canrelizumab, sintilimab, tislelizumab, toripalimab, dostarimab, retifanlimab, sasanlimab, penprimab, CS1003, HLX10, SCT-I10A, zimberelimab, balstilimab, genolimuzumab, BI 754091, cetrelimab, YBL-006, BAT1306, HX008, budicalimab, AMG404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103 and hAb21.

[0446] In some instances, the anti-PD-1 antibody is nivolumab (CAS Registry Number 946414-94-4). Nivolumab (Bristol-Myers Squibb / Ono), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168.

[0447] In some instances, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number 1374853-91-4). Pembrolizumab (Merck), also known as MK-3475, Merck 3475, lambrolizumab, SCH-900475, and KEYTRUDA®, is an anti-PD-1 antibody described in WO 2009 / 114335.

[0448] In some instances, the anti-PD-1 antibody is MEDI-0680 (AMP-514; Astra Zeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

[0449] In some instances, the anti-PD-1 antibody is PDR001 (CAS Registry Number 1859072-53-9; Novartis). PDR001 is a humanized IgG4 anti-PD-1 antibody that blocks the binding of PD-L1 and PD-L2 to PD-1.

[0450] In some instances, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD-1 antibody.

[0451] In some instances, the anti-PD-1 antibody is BGB-108 (BeiGene).

[0452] In some instances, the anti-PD-1 antibody is BGB-A317 (BeiGene).

[0453] In some instances, the anti-PD-1 antibody is JS-001 (Shanghai Junshi). JS-001 is a humanized anti-PD-1 antibody.

[0454] In some instances, the anti-PD-1 antibody is STI-A1110 (Sorrento). STI-A1110 is a human anti-PD-1 antibody.

[0455] In some instances, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD-1 antibody.

[0456] In some instances, the anti-PD-1 antibody is PF-06801591 (Pfizer).

[0457] In some instances, the anti-PD-1 antibody is TSR-042 (also known as ANB011; Tesaro / AnaptysBio).

[0458] In some instances, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).

[0459] In some instances, the anti-PD-1 antibody is ENUM 244C8 (Enumeral Biomedical Holdings). ENUM 244C8 is an anti-PD-1 antibody that inhibits PD-1 function without blocking the binding of PD-L1 to PD-1.

[0460] In some instances, the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings). ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits the binding of PD-L1 to PD-1.

[0461] In some instances, the anti-PD-1 antibody is one described in WO 2015 / 112800, WO 2015 / 112805, WO 2015 / 112900, U.S. Patent Application Publication Nos. 20150210769, WO 2016 / 089873, WO 2015 / 035606, WO 2015 / 085847, WO 2014 / 206107, WO 2012 / 145493, U.S. Pat. and WO 2014 / 194302. The anti-PD-1 antibodies may comprise six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and / or heavy chain and light chain variable domains of the anti-PD-1 antibodies described in WO 2015 / 05148, WO 2015 / 119930, WO 2015 / 119923, WO 2016 / 032927, WO 2014 / 179664, WO 2016 / 106160, and WO 2014 / 194302.

[0462] In still further specific embodiments, the anti-PD-1 antibody has reduced or minimal effector function. In even more specific embodiments, the minimal effector function is due to an "effector-less Fc mutation" or an aglycosylation mutation. In yet further examples, the effector-less Fc mutation is an N297A or D265A / N297A substitution in the constant region. In some examples, the isolated anti-PD-1 antibody is aglycosylated.

[0463] C. PD-L2 Binding Antagonists In some instances, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some instances, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its ligand binding partner. In certain embodiments, the PD-L2 binding ligand partner is PD-1. The PD-L2 binding antagonist can be, but is not limited to, an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, or a small molecule.

[0464] In some instances, the PD-L2 binding antagonist is an anti-PD-L2 antibody. In any of the examples herein, the anti-PD-L2 antibody is capable of binding to human PD-L2 or a variant thereof. In some instances, the anti-PD-L2 antibody is a monoclonal antibody. In some instances, the anti-PD-L2 antibody is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some instances, the anti-PD-L2 antibody is a humanized antibody. In other instances, the anti-PD-L2 antibody is a human antibody. In still further particular aspects, the anti-PD-L2 antibody has reduced or minimal effector function. In even more particular aspects, the minimal effector function results from an "effector-less Fc mutation" or an aglycosylation mutation. In still further instances, the effector-less Fc mutation is a N297A or D265A / N297A substitution in the constant region. In some instances, the isolated anti-PD-L2 antibody is aglycosylated.

[0465] XI. Pharmaceutical Compositions and Formulations Also provided herein are pharmaceutical compositions and formulations comprising a bispecific antibody targeting PD-1 and LAG3, and optionally a pharmaceutically acceptable carrier. The present disclosure also provides (i) pharmaceutical compositions and formulations comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-VEGF antibody (e.g., bevacizumab), and optionally a pharmaceutically acceptable carrier; and (ii) pharmaceutical compositions and formulations comprising an anti-TIGIT antagonist antibody, a bispecific antibody targeting PD-1 and LAG3, and optionally a pharmaceutically acceptable carrier. Pharmaceutical compositions and formulations of bispecific antibodies targeting PD-1 and LAG3 and / or other agents (e.g., dexamethasone) described herein can be prepared by mixing them with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized composition or an aqueous solution. In some embodiments, mosunetuzumab is formulated for subcutaneous administration. In some embodiments, mosunetuzumab is formulated for intravenous administration.

[0466] The pharmaceutical compositions and formulations described herein can be prepared by mixing the active ingredient (e.g., a bispecific antibody targeting PD-1 and LAG3, an anti-TIGIT antagonist antibody, and / or an anti-VEGF antibody) having the desired purity with one or more optional pharmaceutically acceptable carriers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), for example, in the form of a lyophilized formulation or aqueous solution.

[0467] An exemplary tiragolumab formulation comprises a histidine solution containing polysorbate 20, sucrose, L-methionine, and WFI. Tiragolumab may be provided in a 15 mL vial containing 10 mL of tiragolumab formulation at an approximate concentration of 60 mg / mL of tiragolumab antibody.

[0468] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, saccharides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include intercalating drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

[0469] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation containing a histidine acetate buffer.

[0470] The formulations herein may also contain more than one active ingredient as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an additional therapeutic agent (e.g., a chemotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, and / or an antihormonal agent, such as those mentioned hereinabove). Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0471] The active ingredient may be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, in microcapsules prepared, for example, by coacervation or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0472] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0473] Formulations to be used for in vivo administration are generally sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0474] XII. Manufactured Articles or Kits A. Kit containing a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody In another aspect, provided herein is an article of manufacture or kit comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody (e.g., tiragolumab). In some instances, the article of manufacture or kit further comprises a package insert containing instructions for using the anti-TIGIT antagonist antibody in combination with the bispecific antibody targeting PD-1 and LAG3 to treat or delay the progression of cancer in a subject. Any of the bispecific antibodies targeting PD-1 and LAG3 and / or anti-TIGIT antagonist antibodies described herein can be included in the article of manufacture or kit.

[0475] In another embodiment of the present invention, a kit is provided that includes a bispecific antibody targeting PD-1 and LAG3 for use in combination with an anti-TIGIT antagonist antibody to treat a subject with cancer according to any of the methods described herein. In some instances, the kit further includes an anti-TIGIT antagonist antibody. In some instances, the article of manufacture or kit further includes a package insert containing instructions for using the bispecific antibody targeting PD-1 and LAG3 in combination with the anti-TIGIT antagonist antibody (e.g., tiragolumab) to treat or delay the progression of cancer in a subject.

[0476] In some instances, the bispecific antibody targeting PD-1 and LAG3 and the anti-TIGIT antagonist antibody are contained in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some instances, the container holds the formulation, and a label on or associated with the container can indicate instructions for use. The article of manufacture or kit can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some instances, the article of manufacture further includes one or more additional agents (e.g., chemotherapeutic agents and anti-tumor agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0477] Any of the bispecific antibodies targeting PD-1 and LAG3 and / or anti-TIGIT antagonist antibodies described herein can be included in an article of manufacture or kit. Any of the articles of manufacture or kits can include instructions for administering the bispecific antibodies targeting PD-1 and LAG3 and / or anti-TIGIT antagonist antibodies to a subject according to any of the methods described herein, e.g., any of the methods described in Section III above.

[0478] B. Kit containing a bispecific antibody targeting PD-1 and LAG3 and an anti-VEGF antibody In another aspect, provided herein is an article of manufacture or kit comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-VEGF antibody (e.g., bevacizumab). In some instances, the article of manufacture or kit further comprises a package insert containing instructions for using the anti-VEGF antibody in combination with the bispecific antibody targeting PD-1 and LAG3 to treat or delay the progression of cancer in a subject. Any of the bispecific antibodies targeting PD-1 and LAG3 and / or anti-VEGF antibodies described herein can be included in the article of manufacture or kit.

[0479] In another embodiment of the invention, a kit is provided that includes a bispecific antibody targeting PD-1 and LAG3 for use in combination with an anti-VEGF antibody to treat a subject with cancer according to any of the methods described herein. In some instances, the kit further includes an anti-VEGF antibody. In some instances, the article of manufacture or kit further includes a package insert containing instructions for using the bispecific antibody targeting PD-1 and LAG3 in combination with the anti-VEGF antibody to treat or delay the progression of cancer in a subject.

[0480] In some instances, the bispecific antibody targeting PD-1 and LAG3 and the anti-VEGF antagonist antibody are contained in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some instances, the container holds the formulation, and a label on or associated with the container can provide instructions for use. The article of manufacture or kit can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some instances, the article of manufacture further includes one or more additional agents (e.g., chemotherapeutic agents and anti-tumor agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0481] Any of the bispecific antibodies targeting PD-1 and LAG3 and / or anti-VEGF antibodies described herein can be included in an article of manufacture or kit. Any of the articles of manufacture or kits can include instructions for administering the bispecific antibodies targeting PD-1 and LAG3 and / or anti-VEGF antibodies to a subject according to any of the methods described herein, e.g., any of the methods described in Section III above. [Example]

[0482] Example 1: A Phase Ib / II, Open-Label, Multicenter, Randomized Comprehensive Study Evaluating the Efficacy and Safety of Multiple Treatment Combinations in Patients with Melanoma Melanoma is a potentially fatal skin cancer and one of the fastest growing malignancies (Algazi et al. Cancer Manag Res. 2:197-211, 2010; Finn et al. BMC Med. 10:23, 2012). Currently, more than 300,000 people worldwide are diagnosed with melanoma each year, and 57,000 die from the disease. The clinical outcome of melanoma patients depends heavily on the stage at onset. Most individuals with more advanced melanoma have a poor prognosis (Finn et al. BMC Med. 10:23, 2012). Patients with lymph node metastasis (stage III disease) are at high risk for local and distant recurrence after surgery, with a 5-year survival rate of 32-93% in this patient population (Gershenwald et al. CA Cancer J Clin. 67:472-492, 2017). Although few patients present with metastatic disease (stage IV) at presentation, some develop metastases after initial definitive treatment. Immunotherapy and targeted therapies have improved the prognosis for these patients, with 5-year survival rates of approximately 50% (Larkin et al. N Engl J Med. 373:23-34, 2015; Wolchok et al. N Engl J Med. 377:1345-1356, 2017; Larkin et al. N Engl J Med. 381:1535-1546, 2019; Robert et al. Lancet Oncol. 20:1239-1251, 2019; Long et al. J Clin Oncol. 38(Suppl 15):10013, 2020). Despite recent advances in treatment, melanoma remains a serious health problem with high medical need and incidence rates that have been steadily increasing over the past 30 years (Bataille. Expert Rev Dermatol. 4:533-539, 2009).

[0483] BO43328 is a Phase 1b / 2, open-label, multicenter, randomized, comprehensive trial in patients with resectable Stage III (Cohort 1) or Stage IV (Cohort 2) melanoma. The trial is designed with flexibility to open new treatment arms as new therapies become available, close existing treatment arms that have shown minimal clinical activity or unacceptable toxicity, modify the patient population (e.g., with respect to prior anticancer therapy or biomarker status), and introduce additional cohorts of patients with other types of melanoma.

[0484] A. Overview of Study Design This study will evaluate the efficacy, safety, and pharmacokinetics of the treatment combination in cancer immunotherapy (CIT)-naïve patients with resectable stage III melanoma (Cohort 1) and patients with stage IV melanoma (Cohort 2). The specific objectives of the study and corresponding endpoints for Cohort 1 (see Table 5) and Cohort 2 (see Table 6) are outlined below.

[0485] Table 5. Cohort 1 objectives and corresponding endpoints TIFF2024529451000004.tif255170TIFF2024529451000005.tif138170ADA=Anti-drug antibodies;ASTCT=American Society for Transplantation Cytology;CLND=Complete lymph node dissection;CR=Complete response;CRS=Cytokine release syndrome;EFS=Event-free survival;NCI CTCAE v5.0=National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0;ORR=Objective response rate;OS=Overall survival;pCR=Pathological complete response;PK=Pharmacokinetics;pnCR=Pathological near complete response;pPR=Pathological partial response;PR=Partial response;pRR=Pathological response rate;RECIST v1.1=Response Evaluation Criteria in Solid Tumors, version 1.1;RFS=Recurrence-free survival.

[0486] Table 6. Cohort 2 objectives and corresponding endpoints TIFF2024529451000006.tif255168TIFF2024529451000007.tif54170ADA=Anti-drug antibodies; ASTCT=American Society for Transplantation; CR=Complete response; CRS=Cytokine release syndrome; DOR=Duration of response; iRECIST=Modified RECIST v1.1 for Immune-Based Therapeutics; NCI CTCAE v5.0=National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0; ORR=Objective response rate; OS=Overall survival; PFS=Progression-free survival; PK=Pharmacokinetics; PR=Partial response; RECIST v1.1=Response Evaluation Criteria in Solid Tumors, version 1.1. NOTE: Overall response at a single time point will be assessed by the investigator using RECIST v1.1.

[0487] The study is enrolling two cohorts in parallel: Cohort 1 will enroll patients with resectable stage III melanoma with biopsy-available, measurable lymph node metastases according to RECIST v1.1 (Response Evaluation Criteria in Solid Tumors, version 1.1), no history of in-transit metastases within the past 6 months, and no prior systemic chemotherapy with PD-1 / PD-L1 and / or CTLA-4 blocking agents or other agents.

[0488] Cohort 2 will enroll patients with stage IV melanoma who have experienced disease progression during or after one or more, but not more than two, lines of therapy for metastatic disease. Up to two lines of checkpoint inhibitor therapy (monotherapy or combination) will be allowed. Patients with BRAF-mutated disease may have received additional lines of targeted therapy (either before, intermittently, or after checkpoint inhibitor therapy) or may have received targeted therapy and checkpoint inhibitor therapy simultaneously as a single combination.

[0489] Treatment allocation In Cohort 1, patients will be randomly assigned to either the control arm (nivolumab plus ipilimumab (Nivo+Ipi)) or the experimental arm consisting of RO7247669 (a bispecific antibody that binds PD-1 and LAG3), atezolizumab in combination with tiragolumab (Atezo+Tira), or RO7247669 in combination with tiragolumab (RO7247669+Tira). Patients will be stratified by geographic region (Australia vs. rest of the world) and baseline LDH (below the upper limit of normal (ULN) vs. above the ULN). Details of the treatment regimen are shown in Table 7 and Figure 1.

[0490] In Cohort 2, patients will be enrolled in the experimental arm consisting of the combination of RO7247669 and tiragolumab (RO7247669+Tira). Enrollment will begin with a safety run-in phase of six patients.

[0491] Approximately 61-191 patients will be enrolled during the trial, including approximately 6 patients enrolled in the safety run-in phase of Cohort 2. Enrollment into the experimental arms will occur in two phases: a pilot phase and an expansion phase. Approximately 15-20 patients will be enrolled in each treatment arm during the pilot phase. If clinical activity (pathological response in Cohort 1) is observed in the experimental arm during the pilot phase, approximately 20 additional patients may be enrolled in that arm during the expansion phase.

[0492] Sponsors may decide to postpone or pause enrollment within a particular treatment arm. Experimental arms with insufficient clinical activity or unacceptable toxicity will not be expanded. Additional patients may be enrolled to ensure balance between treatment arms with respect to demographic and baseline characteristics, including potential predictive biomarkers, to allow for further subgroup analyses.

[0493] Randomization depends on the number of available experimental groups (e.g., if groups are added or enrollment in groups is paused pending analysis of results from the preliminary phase), with the stipulation that there is no more than a 35% chance of being assigned to the control group. Randomization takes into account group-specific exclusion criteria. Patients are ineligible for a particular group if they meet any of the exclusion criteria outlined for that group.

[0494] Details of the treatment regimen are shown in Table 7.

[0495] Table 7. Treatment regimens TIFF2024529451000008.tif38170 a Sponsors may decide to delay or halt enrollment within a given treatment arm; therefore, not all experimental arms may begin enrollment at the same time. b During the safety run-in phase, patients will be assigned to available treatment arms, with the treatment allocation ratio determined by the number of experimental arms open for enrollment. c The randomization rate depends on the number of experimental groups open for randomization (e.g., if groups are added or randomization to groups is suspended pending analysis of results from the preliminary phase), with the stipulation that the chance of being assigned to the control group is no more than 35%. b If clinical activity is observed in the experimental arm during the preliminary phase, approximately 20 additional patients will be enrolled in that arm during the expansion phase. Experimental arms with minimal clinical activity or unacceptable toxicity will not undergo expansion. e Enrollment will be paused in the RO7247669, Atezo+Tira, and RO7247669+Tira arms of Cohort 1 to allow for safety evaluation in a minimum of six patients. f After evaluating the safety of the treatment combination in Cohort 2, enrollment in the RO7247669 + Tira arm will begin in Cohort 1.

[0496] In Cohort 1, patients in the control and experimental groups received 6 weeks of neoadjuvant treatment. After completion of neoadjuvant treatment, or if treatment was discontinued due to toxicity, and in the absence of disease progression, patients underwent surgery (complete lymph node dissection (CLND)) at week 7. At the investigator's discretion, outside of this study, patients then began adjuvant therapy or observation at week 13 (Figure 2).

[0497] Due to the possibility of an initial increase in the size of metastatic lymph nodes caused by immune cell infiltration associated with CIT-induced T-cell responses (called pseudoprogression), suspected clinical or radiological progression according to RECIST v1.1 may not represent actual disease progression. In the absence of unacceptable toxicity, patients who meet the criteria for disease progression according to RECIST v1.1 while receiving CIT-based therapy will be permitted to continue study treatment until surgery. Progression will be confirmed by biopsy or repeat radiological evaluation by an additional expert reviewer before discontinuing study treatment and / or surgery. All patients are expected to proceed to surgery if there is no distant metastasis and the surgeon determines the disease is completely resectable.

[0498] In Cohort 2, patients will receive study treatment until unacceptable toxicity or loss of clinical benefit, as determined by the investigator after an integrated assessment of radiological and biochemical data, local biopsy results (if available), and clinical status (e.g., symptomatic worsening, such as pain secondary to disease). In the setting of a T-cell response (called pseudoprogression) due to atezolizumab and other CITs, radiological progression by RECIST v1.1 may not represent true disease progression due to the potential for an initial increase in tumor burden caused by immune cell infiltration. In the absence of unacceptable toxicity, patients who meet the criteria for disease progression by RECIST v1.1 while receiving CIT combination therapy will be permitted to continue treatment if they meet all of the following criteria: Evidence of clinical benefit as determined by the investigator after review of all available data. Absence of symptoms and signs of clear disease progression (including laboratory values, e.g., new or worsening hypercalcemia). No decline in ECOG performance status attributable to disease progression. · Absence of tumor progression in critical anatomic locations (e.g., leptomeningeal disease) that cannot be managed by protocol-permitted medical interventions.

[0499] Patients who are eligible to receive treatment beyond progression will be informed by the investigator that they may forgo other treatment options known to provide clinical benefit while continuing study treatment. Patients have the right to voluntarily withdraw from the study at any time for any reason. In addition, investigators have the right to withdraw patients from the study for medical conditions that the investigator or sponsor determines may jeopardize the patient's safety if they continue in the study.

[0500] If subsequent tumor assessments rule out pseudoprogression and confirm disease progression, patients will discontinue study treatment.

[0501] Safety evaluation phase (Cohort 1) To evaluate the toxicity of experimental treatments in the neoadjuvant setting, enrollment will be halted after approximately six patients have been enrolled to allow for safety evaluation. Safety evaluations will be based on safety data from at least six patients who have received at least one dose of treatment (i.e., one dose of each agent for a given combination) and completed safety follow-up evaluations leading up to surgery. Of note, timely performance of surgery (CLND) is an indicator of treatment tolerability. If, at any time during or after the safety evaluation of six patients, 30% or more of the patients experience one or more of the following events considered at least possibly related to the study treatment, enrollment in that combination will be suspended while the sponsor evaluates the benefit-risk profile of the treatment: Treatment-related adverse events of grade 3 or higher that do not improve to grade 2 or higher within 2 weeks (with or without treatment). Treatment-related adverse events causing a delay in surgery of more than two weeks. Treatment-related serious adverse events. Treatment-related adverse events requiring permanent discontinuation of study drug. Deaths other than those unquestionably related to disease progression or unrelated causes such as accidents.

[0502] If no new safety signals are detected, enrollment will resume in that treatment arm.

[0503] Safety Run-in Phase (Cohort 2) To assess the safety and tolerability of this novel combination, which is being clinically tested for the first time, an early safety run-in phase will be conducted in Cohort 2. Approximately 6 patients with metastatic disease will be treated with the novel combination (i.e., RO7247669 + Tira) and safety and tolerability will be assessed for a minimum of 28 days.

[0504] In Cohort 2, at least six patients must complete the initial safety run-in phase. If the RO7247669 + Tira combination is determined to be tolerable, preliminary enrollment will begin, and enrollment in the RO7247669 + Tira arm of Cohort 1 can begin. Patients in the safety run-in phase will be enrolled and treated sequentially, with at least one week between the first patient and the remaining patients.

[0505] Safety evaluations are based on safety data from at least six patients who received at least one dose of treatment (i.e., one dose of each drug) and completed at least 28 days of safety follow-up assessments. If 30% or more of patients experience one or more of the following events considered at least possibly related to the study treatment, enrollment in the combination will be suspended while the sponsor evaluates the benefit-risk profile of the treatment: Treatment-related adverse events of grade 3 or higher that do not improve to grade 2 or higher within 2 weeks (with or without treatment). Treatment-related serious adverse events. Treatment-related adverse events requiring permanent discontinuation of study drug. Deaths other than those unquestionably related to disease progression or unrelated causes such as accidents.

[0506] If no new safety signals are detected, the combination will also begin in Cohort 1.

[0507] B. Exam Completion and Exam Period The study completion date will be defined as the date the last patient completes their last visit, including survival follow-up visits conducted by telephone or in clinic. The total duration of the study, from screening of the first patient to study completion, is expected to be approximately 5 years.

[0508] C. Rationale for Study Design Patient Population Rationale Cohort 1 will enroll patients with resectable stage III melanoma with measurable lymph node metastases that can be biopsied (per RECIST v1.1) and no history of in-transit metastases within the past six months. Patients must not have received prior immunotherapy f...

Claims

1. A medicament for treating a subject having cancer, comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, and a second antigen-binding domain that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38, and the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks, a medicament.

2. The medicament according to claim 1, wherein the cancer is a solid tumor.

3. The medicament according to claim 1 or 2, wherein the cancer is locally advanced or metastatic.

4. The medicament according to claim 1 or 2, wherein the cancer is skin cancer, liver cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, or esophageal cancer.

5. The medicament according to claim 4, wherein the skin cancer is melanoma.

6. The medicament according to claim 4, wherein the skin cancer is untreated unresectable or metastatic melanoma.

7. The melanoma is (a) stage III melanoma with measurable lymph node metastasis; (b) unresectable stage III melanoma; or (c) stage IV melanoma and optionally, the melanoma is not mucosal melanoma or uveal melanoma, according to claim 5.

8. The medicament according to claim 4, wherein the liver cancer is hepatocellular carcinoma (HCC).

9. A medicament for treating a subject having melanoma, comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, and a second antigen-binding domain that specifically binds to LAG3, comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38, and the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks, wherein the melanoma is (a) unresectable stage III melanoma; or (b) stage IV melanoma, a medicament.

10. A medicament for treating a subject having liver cancer, comprising a bispecific antibody targeting PD-1 and LAG3, The bispecific antibody includes a first antigen-binding domain that specifically binds to PD-1, which includes a VH domain containing the amino acid sequence of SEQ ID NO: 29 and a VL domain containing the amino acid sequence of SEQ ID NO: 30, and a second antigen-binding domain that specifically binds to LAG3, which includes a VH domain containing the amino acid sequence of SEQ ID NO: 37 and a VL domain containing the amino acid sequence of SEQ ID NO:

38. The bispecific antibody is administered to a subject at a fixed dose of 600 mg every three weeks. Pharmaceutical.

11. The pharmaceutical according to claim 10, wherein the liver cancer is HCC.

12. The pharmaceutical according to claim 11, wherein the HCC is locally advanced, metastatic, and / or unresectable.

13. The pharmaceutical according to claim 10 or 11, wherein the subject has not previously received a systemic anti-cancer therapy.

14. The pharmaceutical according to claim 10 or 11, wherein bevacizumab is further administered to the subject at a dose of about 15 mg / kg every three weeks.

15. The pharmaceutical according to claim 1 or 2, wherein the bispecific antibody targeting PD-1 and LAG3 includes an Fc domain that is an IgG1 Fc domain or an IgG4 Fc domain.

16. The bispecific antibody targeting PD-1 and LAG3 (a) an Fc domain of a human IgG1 subclass having amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index); and / or (b) an Fc domain containing a modification that promotes the association of the first subunit and the second subunit of the Fc domain The pharmaceutical according to claim 1 or 2, comprising.

17. The pharmaceutical according to claim 1 or 2, wherein the bispecific antibody includes a first heavy chain containing the amino acid sequence of SEQ ID NO: 39, a first light chain containing the amino acid sequence of SEQ ID NO: 40, a second heavy chain containing the amino acid sequence of SEQ ID NO: 41, and a second light chain containing the amino acid sequence of SEQ ID NO:

42.

18. The pharmaceutical according to claim 1 or 2, wherein the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in the tumor.

19. Use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a pharmaceutical for treating a subject having cancer. The bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1, which comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, and a second antigen-binding domain that specifically binds to LAG3, which comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38, the bispecific antibody is administered to a subject at a fixed dose of 600 mg every three weeks, Use.

20. The use according to claim 19, wherein the cancer is a solid tumor.

21. The use according to claim 19 or 20, wherein the cancer is locally advanced or metastatic.

22. The use according to claim 19 or 20, wherein the cancer is skin cancer, liver cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, or esophageal cancer.

23. The use according to claim 22, wherein the skin cancer is melanoma.

24. The use according to claim 22, wherein the skin cancer is untreated unresectable or metastatic melanoma.

25. The melanoma is (a) stage III melanoma with measurable lymph node metastases; (b) unresectable stage III melanoma; or (c) stage IV melanoma and optionally, the melanoma is not mucosal melanoma or uveal melanoma, according to the use of claim 23.

26. The use according to claim 22, wherein the liver cancer is hepatocellular carcinoma (HCC).

27. Use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a medicament for treating a subject having melanoma, the bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1, which comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, and a second antigen-binding domain that specifically binds to LAG3, which comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38, the bispecific antibody is administered to a subject at a fixed dose of 600 mg every three weeks, the melanoma is (a) unresectable stage III melanoma; or (b) stage IV melanoma, Use.

28. Use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a medicament for treating a subject having liver cancer, The bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1, which comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30, and a second antigen-binding domain that specifically binds to LAG3, which comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 37 and a VL domain comprising the amino acid sequence of SEQ ID NO:

38. The bispecific antibody is administered to a subject at a fixed dose of 600 mg every three weeks. Use. **Claim 29** The use according to claim 19 or 20, wherein the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG1 Fc domain or an IgG4 Fc domain. **Claim 30** The bispecific antibody targeting PD-1 and LAG3 (a) an Fc domain of a human IgG1 subclass having amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index); and / or (b) an Fc domain comprising a modification that promotes the association of the first subunit and the second subunit of the Fc domain The use according to claim 19 or 20, comprising. **Claim 31** The use according to claim 19 or 20, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 39, a first light chain comprising the amino acid sequence of SEQ ID NO: 40, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 41, and a second light chain comprising the amino acid sequence of SEQ ID NO:

42. **Claim 32** The use according to claim 19 or 20, wherein the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in tumors.

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