HK40109234A - Methods and compositions for treating cancer - Google Patents

Description

Methods and compositions for treating cancer

sequence list

This application contains a sequence list, which has been submitted electronically in XML format and incorporated herein by full reference. The XML copy was created on July 27, 2022, named 50474-304WO2_Sequence_Listing_7_27_22, and is 68,756 bytes in size.

Technical Field

This invention relates to methods and compositions for treating a subject’s cancer by administering a bispecific antibody (PD1-LAG3) targeting programmed cell death protein 1 (PD-1) and lymphocyte activation gene-3 (LAG3) to the subject, along with an optional anti-TIGIT antagonist antibody (e.g., tireliumab) or a VEGF antagonist (e.g., bevacizumab).

Background Technology

Cancer is characterized by the uncontrolled growth of subpopulations of cells. 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 diagnoses and over 8 million cancer deaths annually. Therefore, cancer care represents a significant and ever-increasing social burden.

There is an urgent need for treatments for common and difficult-to-treat cancers.

Melanoma is a malignant tumor of melanocytes. This potentially deadly form of skin cancer is one of the fastest-growing malignancies. Currently, more than 300,000 people are diagnosed with melanoma globally each year, and 57,000 die from the disease. The prognosis is poor for most people with advanced melanoma. Patients with lymph node metastasis (stage III) have a high risk of local and distant recurrence after surgery, and the 5-year survival rate for this patient group is 32%–93%. A very small number of patients present with metastatic disease (stage IV), but some develop metastases after their initial definitive treatment. Immunotherapy and targeted therapy have improved outcomes for those patients, with a 5-year survival rate of approximately 50%. Melanoma remains a serious health problem with high medical demand, and its incidence has steadily increased over the past 30 years. Therefore, there remains a pressing need for new treatments in this population.

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 diagnosis, most patients with primary liver cancer present with advanced disease, a stage where curative therapies are not recommended. The WHO estimates that more than one million people will die from liver cancer by 2030, highlighting this as a significant global public health issue.

Therefore, there is an unmet need in this field for developing effective immunotherapies and methods for administering these immunotherapies to treat cancers including melanoma and liver cancer.

Summary of the Invention

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

In some respects, cancer is a solid tumor.

In some respects, the cancer is locally advanced or metastatic.

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

In another aspect, this disclosure provides a method for treating a subject with melanoma, the method comprising administering to the subject one or more dosing cycles a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to 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. In some aspects, the subject does not have ocular melanoma.

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

In some respects, liver cancer is hepatocellular carcinoma (HCC). In others, HCC is locally advanced, metastatic, and/or unresectable.

In some respects, the participants had not previously received systemic anticancer therapy.

In some aspects, the length of each of the one or more dosing cycles is 21 days. In some aspects, the method includes administering a bispecific antibody to the subject on day 1 of each of the one or more dosing cycles.

In some respects, the method involves administering bispecific antibodies intravenously to the subject.

In some aspects, the method further includes administering bevacizumab to the subject every three weeks at a dose of approximately 15 mg/kg. In some aspects, the length of each of the one or more dosing cycles is 21 days and the method includes administering bevacizumab to the subject on day 1 of each of the one or more dosing cycles. In some aspects, bevacizumab is administered intravenously.

In some respects, the subjects had not previously been treated for metastatic or unresectable disease.

In some respects, the subjects had not previously been treated with anticancer therapies, including immunomodulators.

In some respects, the subjects had not previously been treated with anti-LAG3 therapy.

In some respects, the bispecific antibody targeting PD-1 and LAG3 includes a first antigen-binding domain that specifically binds to PD-1, the first antigen-binding domain 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.

In some respects, the bispecific antibody targeting PD-1 and LAG3 includes a second antigen-binding domain that specifically binds to LAG3, the first antigen-binding domain comprising: 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 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.

In some aspects, the first antigen-binding domain comprises: 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 the second antigen-binding domain comprises: 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.

In some respects, bispecific antibodies are full-length antibodies.

In some aspects, the bispecific antibody targeting PD-1 and LAG3 includes an Fc domain as IgG, optionally wherein the IgG Fc domain is an IgG1 Fc domain or an IgG4 Fc domain. In some aspects, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor, optionally wherein the Fc receptor is an Fcγ receptor.

In some respects, bispecific antibodies targeting PD-1 and LAG3 comprise (a) an Fc domain of a human IgG1 subclass with amino acid mutations L234A, L235A, and P329G (according to Kabat EU index number); and/or (b) an Fc domain containing a modified Fc domain that promotes association between the first and second subunits of the Fc domain.

In some respects, the first subunit of the Fc domain contains amino acid substitutions of S354C and T366W (according to Kabat EU index number), and the second subunit of the Fc domain contains amino acid substitutions of Y349C, T366S, and Y407V (according to Kabat EU index number).

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain, a first Fab fragment containing a first antigen-binding domain, and a second Fab fragment containing a second antigen-binding domain. In some aspects, in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3, variable domains VL and VH are substituted for each other, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain, optionally wherein the variable domains VL and VH are substituted for each other in the first Fab fragment. In some respects, in the constant domain CL of one segment of the Fab fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number). Optionally, in the constant domain CL of the second Fab fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

In some aspects, the bispecific antibody comprises a first heavy chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:39; a first light chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:40; a second heavy chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:41; and a second light chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:42. In some aspects, the bispecific antibody comprises 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.

In some respects, bispecific antibodies achieve at least 90% LAG3 receptor occupancy (RO) in tumors.

In some respects, the subjects are human.

In another aspect, this 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 to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks.

In some respects, cancer is a solid tumor.

In some respects, the cancer is locally advanced or metastatic.

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

In another aspect, this 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 to PD-1 and a second antigen-binding domain that specifically binds to 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. In some aspects, the patient does not have ocular melanoma.

In another aspect, this disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method for treating a subject with hepatocellular carcinoma (HCC), 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 wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks. In some aspects, the HCC is hepatocellular carcinoma (HCC). In some aspects, the HCC is locally advanced, metastatic, and/or unresectable.

In some respects, the participants had not previously received systemic anticancer therapy.

In some aspects, the length of each of the one or more dosing cycles is 21 days. In some aspects, the method includes administering a bispecific antibody to the subject on day 1 of each of the one or more dosing cycles.

In some respects, the method involves administering bispecific antibodies intravenously to the subject.

In some aspects, the method further includes administering bevacizumab to the subject every three weeks at a dose of approximately 15 mg/kg. In some aspects, the length of each of the one or more dosing cycles is 21 days and the method includes administering bevacizumab to the subject on day 1 of each of the one or more dosing cycles. In some aspects, bevacizumab is administered intravenously.

In some respects, the subjects had not previously been treated for metastatic or unresectable disease.

In some respects, the subjects had not previously been treated with anticancer therapies, including immunomodulators.

In some respects, the subjects had not previously been treated with anti-LAG3 therapy.

In some respects, the bispecific antibody targeting PD-1 and LAG3 includes a first antigen-binding domain that specifically binds to PD-1, the first antigen-binding domain 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.

In some respects, the bispecific antibody targeting PD-1 and LAG3 includes a second antigen-binding domain that specifically binds to LAG3, the first antigen-binding domain comprising: 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 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.

In some aspects, the first antigen-binding domain comprises: 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 the second antigen-binding domain comprises: 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.

In some respects, bispecific antibodies are full-length antibodies.

In some aspects, the bispecific antibody targeting PD-1 and LAG3 includes an Fc domain as IgG, optionally wherein the IgG Fc domain is an IgG1 Fc domain or an IgG4 Fc domain. In some aspects, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor, optionally wherein the Fc receptor is an Fcγ receptor.

In some respects, bispecific antibodies targeting PD-1 and LAG3 comprise (a) an Fc domain of a human IgG1 subclass with amino acid mutations L234A, L235A, and P329G (according to Kabat EU index number); and/or (b) an Fc domain containing a modified Fc domain that promotes association between the first and second subunits of the Fc domain.

In some respects, the first subunit of the Fc domain contains amino acid substitutions of S354C and T366W (according to Kabat EU index number), and the second subunit of the Fc domain contains amino acid substitutions of Y349C, T366S, and Y407V (according to Kabat EU index number).

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain, a first Fab fragment containing a first antigen-binding domain, and a second Fab fragment containing a second antigen-binding domain. In some aspects, in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3, variable domains VL and VH are substituted for each other, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain, optionally wherein the variable domains VL and VH are substituted for each other in the first Fab fragment. In some respects, in the constant domain CL of one segment of the Fab fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number). Optionally, in the constant domain CL of the second Fab fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

In some aspects, the bispecific antibody comprises a first heavy chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:39; a first light chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:40; a second heavy chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:41; and a second light chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:42. In some aspects, the bispecific antibody comprises 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.

In some respects, bispecific antibodies achieve at least 90% LAG3 receptor occupancy (RO) in tumors.

In some respects, the subjects are human.

In another aspect, this disclosure provides the 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 to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and wherein the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks.

In some respects, cancer is a solid tumor.

In some respects, the cancer is locally advanced or metastatic.

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

In another aspect, this disclosure provides the use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a medicament for treating a subject with melanoma, 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, wherein the bispecific antibody is administered 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. In some aspects, the subject does not have ocular melanoma.

On the other hand, this disclosure provides the use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a medicament for treating a subject with hepatocellular carcinoma (HCC), 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, wherein the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks. In some aspects, the HCC is HCC. In some aspects, the HCC is locally advanced, metastatic, and/or unresectable.

In some respects, the participants had not previously received systemic anticancer therapy.

In some respects, the length of each of the one or more dosing cycles is 21 days. In some respects, the bispecific antibody is administered to the subject on day 1 of each of the one or more dosing cycles.

In some cases, bispecific antibodies are administered intravenously to the subject.

In some respects, bevacizumab is administered to the subject at a dose of approximately 15 mg/kg every three weeks. In some respects, the length of each of one or more dosing cycles is 21 days and bevacizumab is administered to the subject on day 1 of each of one or more dosing cycles. In some respects, bevacizumab is administered intravenously.

In some respects, the subjects had not previously been treated for metastatic or unresectable disease.

In some respects, the subjects had not previously been treated with anticancer therapies, including immunomodulators.

In some respects, the subjects had not previously been treated with anti-LAG3 therapy.

In some respects, the bispecific antibody targeting PD-1 and LAG3 includes a first antigen-binding domain that specifically binds to PD-1, the first antigen-binding domain 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.

In some respects, the bispecific antibody targeting PD-1 and LAG3 includes a second antigen-binding domain that specifically binds to LAG3, the first antigen-binding domain comprising: 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 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.

In some aspects, the first antigen-binding domain comprises: 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 the second antigen-binding domain comprises: 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.

In some respects, bispecific antibodies are full-length antibodies.

In some aspects, the bispecific antibody targeting PD-1 and LAG3 includes an Fc domain as IgG, optionally wherein the IgG Fc domain is an IgG1 Fc domain or an IgG4 Fc domain. In some aspects, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor, optionally wherein the Fc receptor is an Fcγ receptor.

In some respects, bispecific antibodies targeting PD-1 and LAG3 comprise (a) an Fc domain of a human IgG1 subclass with amino acid mutations L234A, L235A, and P329G (according to Kabat EU index number); and/or (b) an Fc domain containing a modified Fc domain that promotes association between the first and second subunits of the Fc domain.

In some respects, the first subunit of the Fc domain contains amino acid substitutions of S354C and T366W (according to Kabat EU index number), and the second subunit of the Fc domain contains amino acid substitutions of Y349C, T366S, and Y407V (according to Kabat EU index number).

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain, a first Fab fragment containing a first antigen-binding domain, and a second Fab fragment containing a second antigen-binding domain. In some aspects, in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3, variable domains VL and VH are substituted for each other, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain, optionally wherein the variable domains VL and VH are substituted for each other in the first Fab fragment. In some respects, in the constant domain CL of one segment of the Fab fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number). Optionally, in the constant domain CL of the second Fab fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

In some aspects, the bispecific antibody comprises a first heavy chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:39; a first light chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:40; a second heavy chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:41; and a second light chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:42. In some aspects, the bispecific antibody comprises 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.

In some respects, bispecific antibodies achieve at least 90% LAG3 receptor occupancy (RO) in tumors.

In some respects, the subjects are human.

Attached Figure Description

Figure 1 is a flowchart illustrating 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 = randomization; Tira = tisreliumab.

Figure 2 is a schematic diagram of the study protocol, illustrating an overview of the study schedule and activities in cohort 1 of a phase Ib/II clinical trial for patients with melanoma. CLND = Complete lymph node dissection; Comp. = Complete; CT = Computed tomography; Discon. = Discontinued; M = Month; R = Randomization; Q3M = Every 3 months; SFU = Survival follow-up; Tx = Treatment; W = Week.

Figure 3 is a schematic diagram illustrating the minimal physiological-based pharmacokinetic (mPBPK) model of pembrolizumab. Jt = Pembrolizumab flow rate into the tumor compartment; kint = internalization rate constant of the pembrolizumab-PD-1 complex; koff = dissociation rate constant; kon = binding rate constant; kdeg = degradation rate of PD-1; Ksyn = synthesis rate of PD-1; Lt = lymph flow rate from the tumor compartment; L1 = lymph flow rate from the compact tissue compartment; L2 = lymph flow rate from the leaking tissue compartment; mAb = pembrolizumab concentration; Qt = tumor plasma flow rate; RC = pembrolizumab-PD-1 complex concentration; Rmax = total PD-1 concentration; Vleakage = volume of tissue compartment with leaking vascular structures; Vlymph = volume of lymphatic compartment; Vp = volume of plasma compartment; Vcompact = volume of tissue compartment with compact vascular structures; Vtumor = volume of tumor compartment; σcompact = vascular reflection coefficient of compact tissue; σleakage = vascular reflection coefficient of leaking tissue; σLy = lymphatic reflection coefficient.

Figure 4 is a schematic diagram showing the additional LAG3 receptor added to the PD1-LAG3 mPBPK model.

Figure 5 is a table showing an overview of adverse events in safety-assessable patients in the dose escalation (A1, Q2W) portion of the NP41300 study.

Figure 6 is a table showing an overview of adverse events in safety-assessable patients in the dose escalation (A1, Q2W) portion of the NP41300 study.

Figure 7 is a set of box plots showing the predicted C- _valences after the first and third administrations of PD1-LAG3 at doses of 600 mg or 1200 mg in Q2W (every 2 weeks) or Q3W (every 3 weeks) dosing regimens. The lower and upper hinges correspond to the first and third quartiles (25th and 75th percentiles). The upper whisker extends from the hinge to the maximum value at a distance of no more than 1.5 * IQR (where IQR is the interquartile range, or the distance between the first and third quartiles). The lower whisker extends from the hinge to the minimum value at a distance of at most 1.5 * IQR. Data beyond the whisker ends are referred to as “peripheral” points and are plotted separately. Simulations were performed using a population pharmacokinetic model (a nonlinear mixed-effects modeling approach). For each dosing regimen, simulations were performed on 500 individuals using a set of covariates guided (with replacements) from the original analysis dataset.

Figure 8 is a graph showing simulated PD1 and LAG3 binding within the dose range of RO7247669 administered at Q3W after 3 cycles.

Figure 9 is a flowchart illustrating the study design of the BP43963 trial in patients with melanoma.

Figure 10 is a flowchart illustrating the study design of the GO42216 clinical trial. CIT = Cancer Immunotherapy; HCC = Hepatocellular Carcinoma; R = Randomization.

Figure 11 is a flowchart illustrating the detailed study design of the GO42216 clinical trial. Bev = bevacizumab; HCC = hepatocellular carcinoma; Q2W = every 2 weeks; Q3W = every 3 weeks; R = randomization.

Detailed Implementation

This invention provides methods and compositions for treating cancer. It also provides compositions, uses, and kits relating to such combination and/or dosing regimens.

I. Definition

The following abbreviations are used in this article:

As used herein, the term "about" refers to a common range of error for a corresponding value that is readily known to those skilled in the art. References to a value or parameter "about" herein include (and describe) aspects relating to that value or parameter itself. For example, a description of "about X" includes a description of "X".

Unless otherwise specified, the term “TIGIT” or “T-cell immune receptor with Ig and ITIM domains” as used herein refers to any natural TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). TIGIT is also referred to in the art as DKFZp667A205, FLJ39873, V-set and protein 9 containing the immunoglobulin domain, protein 3 containing the V-set and transmembrane domain, VSIG9, VSTM3, and WUCAM. The 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 produced by cell processing (e.g., processed human TIGIT without the signal sequence 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. The amino acid sequence of the exemplary human TIGIT can be found with UniProt accession number Q495A1.

As used herein, “tisrelimumab” is a fully human IgG1/κMAb-derived compound from open monoclonal antibody (OMT) rats that binds to TIGIT and contains the heavy chain sequence of SEQ ID NO:23 and the light chain sequence of SEQ ID NO:24. Tirelimumab contains two N-linked glycosylation sites (N306) in its Fc domain. Tirelimumab is also described in: WHO Drug Information (International Nonproprietary Names for Medicinal Substances), Proposed INN: List 117, Volume 31, Issue 2, published on July 7, 2017 (see page 343).

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 signaling via PVR, PVRL2, and/or PVRL3, thereby restoring functional responses (e.g., proliferation, cytokine production, target cell killing) induced by T cells from a dysfunctional state to antigen stimulation. For example, an anti-TIGIT antagonist antibody can block signaling via PVR without affecting PVR-CD226 interaction. Those skilled in the art will understand that in some cases, an anti-TIGIT antagonist antibody can antagonize one TIGIT activity without affecting another. For example, an anti-TIGIT antagonist antibody used in certain methods or uses described herein is an anti-TIGIT antagonist antibody that antagonizes TIGIT activity in response to one of the PVR interaction, PVRL3 interaction, or PVRL2 interaction, for example, without affecting or minimally affecting any other TIGIT interaction. In one respect, as measured by radioimmunoassay (RIA), the binding degree of the anti-TIGIT antagonist antibody to unrelated non-TIGIT proteins is less than about 10% of the binding degree of the antibody to TIGIT. In some respects, the dissociation constant (K_{_D} ) of the anti-TIGIT antagonist antibody bound to TIGIT 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 to 10 ^{^-13} M, e.g., 10 ^{^-9} M to 10 ^{^-13} M). In some respects, the anti-TIGIT antagonist antibody binds to conserved TIGIT epitopes between TIGITs from different species or to epitopes on TIGITs that allow cross-species reactivity. In some respects, anti-TIGIT binding antibodies possess intact Fc-mediated effector function (e.g., tirelumab, vibostolimab, etigilimab, EOS084448, or TJ-T6). In some respects, anti-TIGIT binding antibodies possess enhanced Fc-mediated effector function (e.g., SGN-TGT). In other respects, anti-TIGIT binding antibodies lack Fc-mediated effector function (e.g., domvanalimab, BMS-986207, ASP8374, or COM902). In some respects, anti-TIGIT binding antibodies are IgG1 class antibodies (e.g., tirelumab, vibostolimab, domvanalimab, BMS-986207, etigilimab, BGB-A1217, SGN-TGT, EOS084448 (EOS-448), TJ-T6, or AB308). In other respects, anti-TIGIT binding antibodies are IgG4 class antibodies (e.g., ASP8374 or COM902). In another respect, anti-TIGIT antagonist antibodies are tireliumab.

The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction between a PD-1 axis binding partner and one or more of its binding partners to eliminate T cell dysfunction caused by signal transduction along the PD-1 signaling axis, resulting in the restoration or enhancement of 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 cases, PD-1 axis binding antagonists include either PD-L1 binding antagonists or PD-1 binding antagonists. In a preferred aspect, the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1 and/or B7-1). In some cases, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 with its binding partners. In one specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some cases, PD-L1 binding antagonists include anti-PD-L1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction 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 scenario, a PD-L1 binding antagonist reduces negative co-stimulatory signals mediated by PD-L1 signaling, either mediated by cell surface proteins expressed on T lymphocytes or by signal transduction mediated by PD-L1, thereby reducing dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some scenarios, the PD-L1 binding antagonist binds to PD-L1. In some scenarios, 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, envafolimab, TQB2450, ZKAB001, LP-002, CX-072, and IMC-001. The following are listed: 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 respects, anti-PD-L1 antibodies are atezolizumab, MDX-1105, MEDI4736 (dvorumab), or MSB0010718C (averumab). In one specific respect, the PD-L1 binding antagonist is MDX-1105. In another specific respect, the PD-L1 binding antagonist is MEDI4736 (dvorumab). In another specific aspect, the PD-L1 binding antagonist is MSB0010718C (avermab). In other aspects, 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 cases can be administered orally. Other exemplary PD-L1 binding antagonists include AVA-004, MT-6035, VXM10, LYN192, GB7003, and JS-003. In a preferred aspect, the PD-L1 binding antagonist is atezolizumab.

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling generated by the interaction of PD-1 with one or more of its binding partners (such as PD-L1 and/or PD-L2). PD-1 (programmed cell death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." Exemplary human PD-1 is shown in UniProtKB/Swiss-Prot accession number Q15116. In some cases, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 with one or more of its binding partners. In a specific aspect, 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 and their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signaling generated by the interaction of PD-1 with PD-L1 and/or PD-L2. In one case, a PD-1 binding antagonist reduces negative co-stimulatory signals mediated by PD-1 signaling, either mediated by cell surface proteins expressed on T lymphocytes, thereby reducing dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some cases, the PD-1 binding antagonist binds to PD-1. In some cases, 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 (spartazumab), REGN2810 (cimipril), BGB-108, palolizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dotalimab, refulimab, sasanlimab, penexazol, CS1003, HLX10, SCT-I10A, and cepalim Monoclonal antibodies, including batitimab, genolumab, BI 754091, cilimab, YBL-006, BAT1306, HX008, buglimab, AMG 404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103, and hAb21. In one specific aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In yet another specific aspect, the PD-1 binding antagonist is a PD-L2 fusion protein, such as AMP-224. In another specific aspect, the PD-1 binding antagonist is MED1-0680. In another specific aspect, the PD-1 binding antagonist is PDR001 (spartazolizumab). In another specific aspect, the PD-1 binding antagonist is REGN2810 (cimiprimab). In another specific aspect, the PD-1 binding antagonist is BGB-108. In another specific aspect, the PD-1 binding antagonist is palolizumab. In another specific aspect, the PD-1 binding antagonist is camrelizumab. In another specific aspect, the PD-1 binding antagonist is sintilimab. In another specific aspect, the PD-1 binding antagonist is tislelizumab. In another specific aspect, the PD-1 binding antagonist is toripalimab. Other additional exemplary PD-1 binding antagonists include BION-004, CB201, AUNP-012, ADG104, and LBL-006.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling generated by the interaction of PD-L2 with one or more binding partners, such as PD-1. PD-L2 (programmed cell death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1LG2," "CD273," "B7-DC," "Btdc," and "PDL2." Exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot accession number Q9BQ51. In some cases, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 with one or more of its binding partners. In a specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. Exemplary PD-L2 antagonists include anti-PD-L2 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signaling generated by the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In one aspect, PD-L2 binding antagonists reduce negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes, which, through PD-L2-mediated signaling, reduce dysfunction of dysfunctional T cells (e.g., enhance effector responses to antigen recognition). In some aspects, PD-L2 binding antagonists bind to PD-L2. In some aspects, PD-L2 binding antagonists are immunoadhesins. In other aspects, PD-L2 binding antagonists are anti-PD-L2 antagonist antibodies.

The terms “programmed cell death ligand 1” and “PD-L1” refer herein to the natural sequence human PD-L1 polypeptide. The natural sequence PD-L1 polypeptide is provided under Uniprotocol accession number Q9NZQ7. For example, the natural sequence PD-L1 may have the amino acid sequence described in Uniprotocol accession number Q9NZQ7-1 (isomer 1) (SEQ ID NO: 22). In another example, the natural sequence PD-L1 may have the amino acid sequence described in Uniprotocol accession number Q9NZQ7-2 (isomer 2). In yet another example, the natural sequence PD-L1 may have the amino acid sequence described in Uniprotocol accession number Q9NZQ7-3 (isomer 3). PD-L1 is also referred to in the art as “programmed cell death 1 ligand 1”, “PDCD1LG1”, “CD274”, “B7-H”, and “PDL1”.

When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is typically used (e.g., the EU index reported by Kabat et al. above). The “EU index described by Kabat” refers to the residue numbering of human IgG1 EU antibodies.

For the purposes of this article, “atezolizumab” is an Fc-engineered, humanized, non-glycosylated IgG1κ immunoglobulin that binds to PD-L1 and contains 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) at position 297 on the heavy chain (N297A), using the EU number of the amino acid residues in the Fc region, which results in a non-glycosylated antibody with minimal binding to the Fc receptor. Atezolizumab is also described in: WHO Drug Information (International Nonproprietary Names for Medicinal Substances), Proposed INN: List 112, Volume 28, Issue 4, published on January 16, 2015 (see page 485).

The term "cancer" refers to a disease caused by the uncontrolled division of abnormal cells in a part of the body. In one case, 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 gland carcinoma). In another case, cancer is liver cancer (e.g., hepatocellular carcinoma (HCC), such as locally advanced or metastatic HCC and/or unresectable HCC). Cancer includes solid tumor cancers and non-solid tumor cancers, as well as locally advanced or metastatic cancers (e.g., locally advanced or metastatic tumors). 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, urothelial carcinoma (UC), including locally advanced and metastatic UC (mUC); bladder cancer (e.g., muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC), such as BCG-refractory NMIBC); MIBC urothelial bladder cancer (UBC); kidney or renal cell carcinoma (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 squamous NSCLC or non-squamous NSCLC, including locally advanced unresectable NSCLC (e.g., stage IIIB NSCLC) or recurrent or metastatic NSCLC (e.g., stage IV NSCLC)); lung adenocarcinoma or squamous cell carcinoma (e.g., upper lung cancer). Squamous cell carcinoma of the skin (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 and head and neck squamous cell carcinoma (HNSCC)); ovarian cancer (OC); esophageal cancer; peritoneal cancer; hepatocellular carcinoma; gastric cancer (GC) (e.g., gastroesophageal junction (GEJ) cancer 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), which is estrogen receptor negative (ER-), progesterone receptor negative (PgR-), and HER2 negative (HER2-))); prostate cancer, such as castration-resistant prostate. Cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric cancer or stomach 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, such as metastatic and/or recurrent PD-L1 positive cervical cancer); ovarian cancer; liver cancer; colon cancer; rectal cancer; colorectal cancer (CRC; e.g., CRC with low microsatellite stability (MSS) and low microsatellite instability (MSI-Low)); endometrial cancer or uterine cancer; salivary gland cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, including superficial spreading melanoma, malignant lentigines melanoma, acral melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma). Chikin'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 small non-cleaved cell NHL; large-volume disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloid leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal angiogenesis associated with phakomatoses, edema (such as edema associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and related metastases.

In some cases, cancers (e.g., skin cancer (e.g., melanoma) or liver cancer (e.g., HCC)) are tumors with a tumor microenvironment containing CD8+ T cells expressing LAG3.

In some cases, cancer may be unresectable (e.g., unresectable locally advanced or metastatic cancer).

Other examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific 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)). Other examples include metastatic esophageal cancer (e.g., metastatic ESCC, metastatic EAC, or metastatic ENEC). In one case, the cancer is colorectal cancer (CRC). As used herein, the terms “colorectal cancer,” “CRC,” “colon cancer,” or “intestinal cancer” refer to cancer that develops from the large intestine, such as the colon or rectum (e.g., colorectal adenocarcinoma). Other examples of cancer include, but are not limited to, hematologic malignancies such as mature B-cell carcinoma, excluding Hodgkin lymphoma but including non-Hodgkin lymphoma (NHL), such as diffuse large B-cell lymphoma (DLBCL), which may be recurrent. Sexually resistant or refractory DLBCL or Richter 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, primary mediastinal cavity (thymus) large B-cell lymphoma (PMLBCL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), transformed LL, Waldenström's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt lymphoma (BL), precursor B-cell lymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma. Lymphoma/leukemia (unclassifiable), diffuse red pulp small B-cell lymphoma of the spleen, variant hairy cell leukemia, heavy chain disease (α heavy chain disease, γ heavy chain disease, μ heavy chain disease), plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, mucosa-associated lymphoid tissue extranodal marginal zone lymphoma (MALT lymphoma), lymph node marginal zone lymphoma, pediatric lymph node marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicular center lymphoma, T-cell/histiocytic rich large B-cell lymphoma, primary CNS DLBCL, primary cutaneous DLBCL (leg type), EBV-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, lymphomatoid granuloma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma caused by HHV8-associated multicentric Kassman disease, primary exudative lymphoma Tumors: B-cell lymphoma (unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma), and B-cell lymphoma (unclassifiable, with features intermediate between DLBCL and classical Hodgkin's lymphoma). Other examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies, including B-cell lymphomas. More specific examples of such cancers include, but are not limited to, multiple myeloma (MM); low-grade malignant/follicular NHL; small lymphocytic (SL) NHL; intermediate-grade malignant/follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL; giant mass NHL; AIDS-related lymphoma; and acute lymphoblastic leukemia (ALL); chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD).

The term "B-cell proliferative disorder" or "B-cell malignancy" refers to a disorder associated with some degree of abnormal B-cell proliferation and includes, for example, lymphoma, leukemia, myeloma, and myelodysplastic syndromes. In some cases, a B-cell proliferative disorder is a lymphoma, such as non-Hodgkin 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 transformed), MCL, high-grade B-cell lymphoma, or PMLBCL). In another embodiment, a B-cell proliferative disorder is a leukemia, such as chronic lymphocytic leukemia (CLL). In one embodiment, a B-cell proliferative disorder is relapsed and/or refractory FL.

The term "tumor" refers to all proliferative cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "proliferative disorder," "proliferative lesion," and "tumor" are not mutually exclusive in this document.

As used herein, “tumor cell” means any tumor cell present in a tumor or its sample. Using methods known in the art and/or those described herein, tumor cells can be distinguished from other cells that may be present in a tumor sample, such as stromal cells and tumor-infiltrating immune cells.

"Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Therefore, as a therapeutic concept, tumor immunity is "treated" when this evasion is mitigated and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

As used in this article, “metastasis” refers to the spread of cancer from its primary site to other parts of the body. Cancer cells can detach from the primary tumor, infiltrate lymphatic vessels and blood vessels, circulate in the bloodstream, and grow in distant lesions in normal tissues elsewhere in the body (metastasis). Metastasis can be local or distant. Metastasis is a continuous process that depends on tumor cells detaching from the primary tumor, crossing the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Excitatory and inhibitory molecular pathways within tumor cells regulate this behavior, and interactions between tumor cells and distant host cells are also important.

As used herein, “treatment” includes effective cancer therapy with an effective dose of a therapeutic agent (e.g., a bispecific antibody targeting PD-1 and LAG3). Treatment in this article specifically includes adjuvant therapy, neoadjuvant therapy, therapy for non-metastatic cancer (e.g., therapy for locally advanced cancer), and therapy for metastatic cancer. Treatment can be first-line (e.g., in patients who may have been previously untreated or have not received prior systemic therapy), or second-line or subsequent treatment.

In this article, “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, tireliumab, or a VEGF antagonist, e.g., an anti-VEGF antibody (e.g., bevacizumab)) that achieves the therapeutic outcome. In some examples, the effective amount of a therapeutic agent or combination of therapeutic agents is the amount of that 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).

As used in this article, "complete remission" and "CR" refer to the disappearance of all target lesions.

As used in this article, "partial remission" and "PR" refer to a reduction of at least 30% in the SLD of the target lesion, with reference to the sum of the longest diameters (SLD) at baseline before treatment.

As used here, “disease progression” and “PD” refer to an increase of at least 20% in the target lesion’s SLD, with reference to the minimum sum (lowest point) of the study (including baseline). The appearance of one or more new lesions can also be considered PD.

As used in this article, “disease stable” or “SD” means, with the minimum sum as a reference, that the disease has neither sufficiently decreased to meet the PR nor sufficiently increased to meet the PD.

As used in this article, “disease control rate” and “DCR” refer to the percentage of patients with advanced or metastatic cancer who have achieved CR, PR, or stable disease (SD). For example, DCR can be defined as the proportion of patients with SD for ≥12 weeks or CR or PR, as determined by researchers according to RECIST v1.1.

As used herein, “overall response rate,” “objective response rate,” and “ORR” are interchangeable and refer to the sum of the complete response (CR) rate and the partial response (PR) rate. For example, an objective response can be defined as a CR or PR according to the Remedial Evaluation Criteria for Solid Tumors (RECIST) v.1.1, as determined by an investigator’s assessment and confirmed by a repeat assessment ≥4 weeks after the initial recording. In another example, ORR can be defined as the proportion of patients who have two consecutive CRs or PRs at ≥4 weeks intervals, as determined by an investigator according to RECIST v1.1.

As used herein, “pathological response rate” and “pRR” can be used interchangeably to refer to the proportion of patients with pathological complete response (pCR, e.g., no live tumors in the treated tumor bed), pathological near-complete response (pnCR, e.g., <10% of the treated tumor bed is occupied by live tumor cells), and pathological partial response (pPR, e.g., <50% of the treated tumor bed is occupied by live tumor cells) (e.g., at the time of surgery).

As used herein, “progression-free survival” and “PFS” refer to the length of time during and after treatment when cancer does not worsen. PFS may include the amount of time a patient has achieved complete remission (CR) or partial remission (PR), and the amount of time a patient has experienced disease stabilization. For example, PFS may be defined as the time from the first study treatment to the first progression or death from any cause (whichever occurs first), as determined by the researchers according to RECIST v.1.1. In another example, PFS may be defined as the time from study enrollment to the first progression or death from any cause (whichever occurs first), as determined by the researchers according to RECIST v.1.1.

As used in this article, “overall survival” and “OS” refer to the length of time a patient remains alive from the date of diagnosis or the start of treatment for a disease (e.g., cancer). For example, OS can be defined as the time from the first investigational treatment to death from any cause.

As used herein, the terms “duration of response” and “DOR” refer to the length of time from the time of recording to the time of tumor response until disease progression or death (whichever occurs first). For example, DOR can be defined as the time from the first recorded objective response to the time of first recorded disease progression or death from any cause (whichever occurs first), as determined by the investigator according to RECIST v1.1.

As used herein, the term "chemotherapeutic agent" refers to a compound that can be used to treat cancer. Examples of chemotherapeutic agents include EGFR inhibitors (including small molecule inhibitors such as erlotinib (Genentech/OSIPharm.); PD 183805 (CI 1033, 2-acrylamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-dihydrochloride, Pfizer Inc.); ZD1839, gefitinib 4-(3'-chloro-4'-fluoroaniline)-7-methoxy-6-(3-morpholinopropoxy)quinazolin, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimidino[5,4-d]pyrimidin-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(SU 5271; Pfizer); and dual EGFR/HER2 tyrosine kinase inhibitors, such as lapatinib (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, oral selective inhibitors of ErbB2 receptor tyrosine kinases (Pfizer and OSI); dual HER inhibitors that preferentially bind to EGFR but simultaneously inhibit cells overexpressing HER2 and EGFR, such as EKB-569 (available from W Yeth); PKI-166 (Novartis); pan-HER inhibitors, such as cananetinib (CI-1033; Pharmacia); Raf-1 inhibitors, such as the antisense agent ISIS-5132 (ISIS Pharmaceuticals) that inhibits Raf-1 signaling; non-HER-targeting tyrosine kinase inhibitors, such as imatinib mesylate (Glaxo SmithKline); multi-targeting tyrosine kinase inhibitors, such as sunitinib (Pfizer); VEGF receptor tyrosine kinase inhibitors, such as vatalani (PTK787/ZK222584, Novartis/Schering AG); MAPK extracellular kinase I inhibitor CI-1040 (Pharmacia); Quinazolines, such as PD 153035, 4-(3-chloroaniline)quinazoline; Pyridopyrimidines; Pyrimidines; Pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; Pyrrolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; Curcumin (difluoroformylmethane, 4,5-bis(4-fluoro) Aniline (phthalimide); tyrosine containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., molecules that bind to HER-encoded nucleic acids); quinoxalines (US Patent No. 5,804,396); tyrosine phosphorylation inhibitors (US Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors, such as CI-1033 (Pfizer); Affinitac ( ISIS 3521; Isis/Lilly); PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); cermatinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); and rapamycin (sirolimus); proteasome inhibitors, such as bortezomib ( Millennium Pharm.); Disulfiram; Epigallocatexin; Halosporamide A; Carfilzomib; 17-AAG (Geraldycin); Rhizocarpine; Lactate Dehydrogenase A (LDH-A); Fulvestrant (AstraZeneca); Letrozole (Novartis), Finazine (Novartis); Oxaliplatin (Sanofi); 5-FU (5-fluorouracil); Formicoyltetrahydrofolate; Lonafanib (SCH 66336); Sorafenib (Bayer Labs); AG1478, Alkylating agents such as thiotepa and cyclophosphamide; Alkyl sulfonates, such as busulfan, indomethacin, and piperosulfan; azacyclopropane derivatives, such as benzotipane, carboquinone, metopepane, and urotepiperazine; ethylamines and methylmelamines, including hexamethylmelamine, triethylmelamine, triethylphosphamide, triethylthiophosphamide, and tris(hydroxymethylmelamine); anomeric lactones (especially bratasine and bratasineone); camptothecin (including topotecan and irinotecan); lichenin; carlistatin; CC-1065 (including its synthetic analogues adorelasin, carzelecanin, and bizelecanin); nostocins (especially nostocin 1 and nostocin 8); corticosteroids. Alcohols (including prednisone and prednisolone); cyproterone acetate; 5α-reductases (including finasteride and dutasteride); vorinostat, romidesin, ubiquistat, valproic acid, moxistat; interleukin, talc, ducamycin (including synthetic analogs KW-2189 and CB1-TM1); eleutheroscin; hydatidine; styrastatin; nitrogen mustards, such as chlorambucil, chlorpheniramine, chlorphosphamide, estradiol, ifosfamide, methomyl, methoxynitroglycerin, melphalan, neonitrogen, benzyl mustard cholesterol, prednisone, troprophosphatide, uramustine; nitrosoureas, such as carmustine. Chlorouracil, formustine, lomustine, nimustine, and lanimustine; antibiotics, such as enediyne antibiotics (e.g., chachimycin, especially chachimycin γ1 and chachimycin ω1); danendomycin, including danendomycin A; bisphosphonates, such as clophosphonates; esmicin; and new antitumor chromophores and associated chromophores (enediyne antibiotic chromophores), aclarubicin, actinomycin, amiodarone, azoserine, actinomycin, carrubicin, erythromycin, carcinomacin, chromomycin, actinomycin D, detoxin, 6-azido-5-oxo-L-leucine, morpholino-doxorubicin, cyanomoxanone Adriamycin (including phyto-doxorubicin, 2-pyrrolidine-doxorubicin, and deoxydoxorubicin), epirubicin, isopycin, idarubicin, maceralomycin, mitomycin C, mycophenolic acid, nogamycin, olivomycin, pepromycin, methylmitomycin, puromycin, triamcinolone acetonide, rhodopsin, streptozotocin, streptozotocin, tuberculin, ubenimex, fenestrated statin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, methotrexate, pteroxate, trimethopterin; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidine, etc. Thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azouridine, carmoflurane, vidarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, fluorouridine; androgens, such as capprotestone, drotalonone propionate, cyclothionol, meandrolone, testosterone; antiadrenergic drugs, such as aminoglutethimide, mitotane, troostanistan; folic acid supplements, such as folinic acid; acetoglucan lactone; aldehyde phosphoramide glycoside; aminolevulinic acid; enuracil; acridine; betributazone; pyrazinamide; edarazonatase; desphosphonamide; colchicine; iminoquinone; enolanide; eletate; epothilone; ethoxycycline; gallium nitrate; hydroxychloroquine Urea; Lentinan; Clonidamine; Maytansine alkaloids, such as maytansine and anthraquinone; Mitoguanidine hydrazone; Mitoantrone; Mopiperol; Diamine nitroacetate; Pentostatin; Methamidomus acid; Pirarubicin; Loxoantrone; Podophyllotoxin; 2-Ethylhydrazine; Methylbenzylhydrazine; Polysaccharide iron complex (JHS Natural Products); Razosen; Rhizomycin; Schizotypalin; Germonyl spiroamine; Alternaria spp. ketoacid; Triaminoquinone; 2,2',2”-Trichlorotriethylamine; Trichoderma toxins (especially T-2mycin, Veracrulin A, Erythromycin A, and Serpentin); Uranium; Vincaine; Dacarbazine Mannitol mustard; dibromomannitol; dibromoeutherol; piperobromane; gascitocin; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP-16); ifosfamide; mitoxantrone; novalong; teniposide; edastraxed; daunorubicin; aminopterin; capecitabine ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutical salts, acids, prodrugs, and derivatives of any of the above.

Chemotherapy agents also include: (i) anti-hormonal agents that modulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, raloxifene hydrochloride, LY117018, onapristone, and (toremifine citrate); (ii) aromatase inhibitors that inhibit aromatase. Preparations that regulate the production of estrogen by the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, (medroxyprogesterone acetate), (exemestane; Pfizer), formestanie, fadrozole, (vorozole), (letrozole; Novartis), and (anastrozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; Buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, Premarin, fluorometholone, all trans-retinoic acid, fenretinide, and trisatabolin (a 1,3-dioxane cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, especially those that inhibit gene expression in signaling pathways associated with abnormal cell proliferation, such as, for example, PKC-α, Ralf, and H-Ras; (vii) ribozymes, such as VEGF expression inhibitors (e.g., and HER2 expression inhibitors). Preparations; (viii) vaccines, such as gene therapy vaccines, and (ix) growth inhibitors, including vinblastine (e.g., vincristine and vinblastine), (vinorelbine), taxanes (e.g., paclitaxel, albumin-bound paclitaxel, and docetaxel), topoisomerase II inhibitors (e.g., doxorubicin, epirubicin, donomycin, etoposide, and bleomycin) and DNA alkylating agents (e.g., tamoxigenin, prednisone, dacarbazine, dichloromethyldiethylamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C); and (x) pharmaceutical salts, acids, prodrugs, and derivatives of any of the above.

As used herein, the term "cytotoxic agent" means any agent that is harmful to cells (e.g., causing cell death, inhibiting proliferation, or otherwise impairing cell function). Cytotoxic agents include, but are not limited to, radioisotopes (e.g., radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and Lu); chemotherapeutic agents; enzymes and fragments thereof, such as lysozymes; and toxins, such as small molecule toxins or enzyme-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, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, LDH-A inhibitors, fatty acid biosynthesis inhibitors, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and cancer metabolism inhibitors. In one instance, the cytotoxic agent is a platinum-based chemotherapeutic agent (e.g., carboplatin or cisplatin). In one instance, the cytotoxic agent is an EGFR antagonist, such as N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazoline-4-amine (e.g., erlotinib). In one instance, the cytotoxic agent is a RAF inhibitor, such as a BRAF and/or CRAF inhibitor. In one instance, the RAF inhibitor is vemurafenib. In one instance, the cytotoxic agent is a PI3K inhibitor.

The terms "patient" or "subject" refer to a human patient or subject. For example, a patient or subject can be an adult.

The term "antibody" in this article specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity. In one case, the antibody is a full-length monoclonal antibody.

As used herein, the term IgG “isotype” or “subclass” refers to any subclass of immunoglobulins defined by the chemical and antigenic characteristics of the immunoglobulin constant region.

Antibodies (immunoglobulins) can be classified into different categories based on the amino acid sequence of their heavy chain constant domains. Immunoglobulins are mainly divided into five classes: IgA, IgD, IgE, IgG, and IgM, and some of them can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known and are generally described in references such as Abbas et al., Cellular and Mol. Immunology, 4th edition (W.B. Saunders, Co., 2000). Antibodies can be part of a larger fusion molecule formed by the covalent or non-covalent binding of an antibody to one or more other proteins or peptides.

The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably in this document and refer to an antibody in its essentially complete form, rather than an antibody fragment as defined below. The term refers to an antibody containing the Fc region.

The term "Fc region" used herein is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain can comprise the full-length heavy chain, or said antibodies can comprise cleaved variants of the full-length heavy chain. This could be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the amino acid sequence of the heavy chain including the Fc region is represented herein as lacking a C-terminal lysine (Lys447). In one aspect, a heavy chain including the Fc region as specified herein is included in an antibody disclosed herein, the heavy chain containing an additional C-terminal glycine-lysine dipeptide (G446 and K447). In another aspect, a heavy chain including the Fc region as specified herein is included in an antibody disclosed herein, the heavy chain containing an additional C-terminal glycine residue (G446). In another aspect, a heavy chain including the Fc region as specified herein is included in an antibody disclosed herein, the heavy chain containing an additional C-terminal lysine residue (K447). In one embodiment, the Fc region contains a single amino acid substitution of N297A from the heavy chain. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th edition. U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD, 1991).

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabeled part. Naked antibodies may be present in pharmaceutical compositions.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or produced during the production of the monoclonal antibody formulation, such variants are typically present in small quantities), the individual antibodies comprising this group are identical and/or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies according to the invention can be prepared 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.

As used herein, the term "hypervariant region" or "HVR" refers to the regions within the variable domain of an antibody that are hypervariable in sequence and determine antigen-binding specificity, such as the "complementarity-determining region" ("CDR").

Typically, an antibody contains 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 in this document include:

(a) Hypervariable rings present 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 Edition, 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)).

Unless otherwise specified, the CDR is determined according to the method described by Kabat et al., citing above. Those skilled in the art will understand that the CDR name may also be determined according to the method described by Chothia, citing above, the method described by McCallum, citing above, or any other scientifically accepted naming system.

"Frame" or "FR" refers to the variable domain residues other than the complementarity-determining region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences usually appear in the VH (or VL) as follows: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

The terms “Kabat-described variable domain residue numbering” or “Kabat-described amino acid position numbering” and their variations refer to the numbering system proposed in the aforementioned Kabat et al. literature for heavy chain or light chain variable domains. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, corresponding to shortening or insertion of the FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion fragment (residue 52a according to Kabat numbering) after residue 52 of H2, and insertion residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat numbering). The Kabat number of residues for a given antibody can be determined by comparing the antibody sequence with homologous regions of a “standard” Kabat-numbered sequence.

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 means an antibody capable of specifically binding to at least two different antigenic determinants, for example, two binding sites, each formed by an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), binding to different antigens or different epitopes on the same antigen. Such bispecific antibodies are in a 1+1 form. Other forms of bispecific antibodies are 2+1 (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 (containing 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 of which is specific to a different antigen.

As used herein, “PD-L1 positive tumor cell score” is, in the context of an immunohistochemical (IHC) assay, such as an IHC assay using antibodies SP142, SP263, 22C3, or 28-8 to stain for PD-L1, the percentage of viable tumor cells, after staining, that are partially or completely stained (excluding cytoplasmic staining), relative to all viable tumor cells present in the sample. Therefore, the PD-L1 positive tumor cell score can be calculated using the PD-L1 IHC SP142 (Ventana) assay, for example, by the formula PD-L1 positive tumor cell score = (number of PD-L1 positive tumor cells) / (total number of PD-L1 positive and PD-L1 negative tumor cells), where PD-L1 staining of the cytoplasm of tumor cells and all non-tumor cells (e.g., tumor-infiltrating immune cells, normal cells, necrotic cells, and debris) is excluded from assessment and scoring. It should be understood that any given diagnostic PD-L1 antibody may correspond to a specific IHC assay protocol and/or scoring terminology that can be used to derive a PD-L1 positive tumor cell score. For example, a PD-L1 positive tumor cell score can be derived from a tumor cell sample stained with SP263, 22C3, SP142, or 28-8 using detection on Benchmark ULTRA, EnVision Flex on AutostainerLink 48, detection and amplification on Benchmark ULTRA, or EnVision Flex on AutostainerLink 48.

As used herein, the “Ventana SP142 IHC assay” was performed according to the Ventana PD-L1 (SP142) assay package insert (Tucson, AZ: Ventana Medical Systems, Inc.), which is incorporated herein by reference in its entirety.

As used herein, the “Ventana SP263 IHC assay” was performed according to the Ventana PD-L1 (SP263) assay package insert (Tucson, AZ: Ventana Medical Systems, Inc.), which is incorporated herein by reference in its entirety.

As used herein, the “pharmDx 22C3 IHC assay” was performed according to the PD-L1 IHC 22C3 pharmDx package insert (Carpinteria, CA: Dako, Agilent Pathology Solutions), which is incorporated herein by reference in its entirety.

As used herein, the “pharmDx 28-8 IHC assay” was performed according to the PD-L1 IHC 28-8 pharmDx package insert (Carpinteria, CA: Dako, Agilent Pathology Solutions), which is incorporated herein by reference in its entirety.

The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information concerning the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings related to the use of such therapeutic products.

As used herein, “in combination with” means administering another treatment modality in addition to one treatment modality, such as a regimen that includes administering a bispecific antibody targeting programmed cell death protein 1 (PD-1) and lymphocyte activation gene-3 (LAG3) along with an anti-TIGIT antagonist antibody (e.g., tislelizumab) or a VEGF antagonist (e.g., an anti-VEGF antibody (e.g., bevacizumab)). Thus, “in combination with” means administering another treatment modality before, during, or after administering one treatment modality to the patient.

A drug administered "concurrently" with one or more other drugs is administered on the same day as treatment with one or more other drugs within the same treatment cycle, and optionally concurrently with one or more other drugs. For example, in cancer treatment administered every 3 weeks, the concurrently administered drugs are each administered on day 1 of the 3-week cycle.

As used herein, the term “adverse event” or “AE” refers to any adverse and unexpected indication (including abnormal laboratory findings), symptom, or illness that is temporarily associated with the use of a medical treatment or procedure that may or may not be considered related to the medical treatment or procedure. Adverse events can be classified by “grade” as defined in the National Cancer Institute Common Terminology Standard for Adverse Events v4.0 or v5.0 (NIH CTCAE). In some respects, AEs are low-grade AEs, such as Grade 1 or Grade 2 AEs. Grade 1 includes asymptomatic or mildly symptomatic AEs. Grade 2 includes moderate AEs that limit age-appropriate assistive activities of daily living (e.g., preparing meals, grocery shopping, or clothing) and indicate a local or non-invasive intervention. In other cases, AEs are high-grade AEs, such as Grade 3, Grade 4, or Grade 5 AEs. In some cases, AEs are Grade 3 or Grade 4 AEs. Grade 3 includes serious or medically significant AEs that are not immediately life-threatening and indicate hospitalization or prolonged hospitalization. Grade 4 includes AEs with life-threatening consequences and indicate the need for urgent intervention. Level 5 includes AEs that result in or are related to death.

As used herein, the term “treatment-related AE” refers to an AE determined by the investigator to be an outcome of a treatment (e.g., PD-1 axis binding antagonist therapy (e.g., atezolizumab therapy) and/or anti-TIGIT antagonist antibody therapy (e.g., tislelizumab therapy)).

As used in the present application, the term "valence" indicates the presence of a specified number of binding domains in an antigen-binding molecule. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" respectively indicate the presence of two, four, and six binding domains in an antigen-binding molecule. The bispecific antibody according to the invention is at least "bivalent" and can be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). In one particular aspect, the antibody of the invention has two or more binding sites and is bispecific. That is, the antibody can be bispecific even when more than two binding sites are present (i.e., the antibody is trivalent or multivalent).

"Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies. For a review of some antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun in The harmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046. A bispecific antibody is an antibody fragment having two antigen-binding sites; the bispecific antibody may be bivalent or bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9,129-134 (2003); and Hollinger et al., Proc Natl Acad SciUSA 90,6444-6448 (1993). Trisomic and tetrasomic antibodies are also described in Hudson et al., Nat Med 9,129-134 (2003). A single-domain antibody is an antibody fragment containing all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516B1). Alternatively, the antibody fragment comprises a single-chain polypeptide characterized by having a VH domain, i.e., capable of assembling together with a VL domain to a functional antigen-binding site; or having a VL domain, i.e., capable of assembling together with a VH domain to a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody. The antibody fragment can be prepared using various techniques, as described herein, including but not limited to the proteolytic digestion of a complete antibody and production from recombinant host cells (e.g., E. coli or bacteriophages).

Papain digestion of an intact antibody produces two identical antigen-binding fragments called “Fab” fragments. Each “Fab” fragment contains a variable domain of the heavy chain and a variable domain of the light chain, as well as a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term “Fab fragment” refers to an antibody fragment containing a light chain fragment with a VL domain and a constant domain (CL) of the light chain, and a VH domain and a first constant domain (CH1) of the heavy chain. The Fab’ fragment differs from the Fab fragment in that the Fab’ fragment has residues added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine residues from the antibody hinge region. Fab’-SH is a Fab’ fragment in which the cysteine residues of the constant domain have free thiol groups. Pepsin treatment produces the F(ab’) ₂ fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

The terms "cross-Fab fragment,""xFabfragment," or "cross-Fab fragment" refer to Fab fragments in which the variable or constant regions of the heavy and light chains are exchanged. Two different chain compositions are possible for exchangeable Fab molecules and are included in the bispecific antibodies of this invention: On one hand, the variable regions of the Fab heavy and light chains are exchanged, i.e., the exchangeable Fab molecule comprises a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). This exchangeable Fab molecule is also called CrossFab _(VLVH) . On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the exchangeable Fab molecule comprises 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 exchangeable Fab molecule is also called CrossFab _(CLCH1) .

A “single-chain Fab fragment” or “scFab” is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: 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; and wherein the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab molecules can be further stabilized by generating interchain disulfide bonds via the insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

"Exchangeable single-chain Fab fragments" or "x-scFab" are polypeptides composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL together form an antigen-binding domain that specifically binds to the antigen, and wherein the linker is a polypeptide of at least 30 amino acids. Furthermore, these x-scFab molecules can be further stabilized by forming interchain disulfide bonds via the insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat number).

A “single-chain variable fragment (scFv)” is a fusion protein of the heavy chain variable region ( _VH ) and light chain variable region ( _VL ) of an antibody, linked by a short linker peptide of ten to approximately 25 amino acids. The linker is typically rich in glycine for flexibility and rich in serine or threonine for solubility, and can link the N-terminus of _VH to the C-terminus of _VL , or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original antibody. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Alternatively, antibody fragments may contain a single-chain polypeptide characterized by having a VH domain, which can assemble with the VL domain to a functional antigen-binding site; or having a VL domain, which can assemble with the VH domain to a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

A single-domain antibody is an antibody fragment composed of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of an antibody heavy chain from camels (nanobodies or _VHH fragments). Additionally, the term single-domain antibody includes autologous human heavy chain variable domains (aVH) or _VNAR fragments derived from sharks. Fibronectin can be engineered to bind antigen scaffolds. Adnectin consists of a backbone of the native amino acid sequence of the 10th domain of a 15-repetitive unit of human type III fibronectin (FN3). The three loops at one end of the β-segment can be engineered to enable Adnectin to specifically recognize target therapeutic targets. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764, and US6818418B1. Peptide aptamers are composite recognition molecules composed of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide ring inserted at the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microsomes are derived from naturally occurring microproteins containing 3-4 cysteine bridges and 25-50 amino acids in length; examples include KalataBI, cone snail toxins, and knottin. Microproteins have rings that can be engineered to include up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

The terms “bispecific antibody containing a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3,” “bispecific antibody that specifically binds to PD-1 and LAG3,” “bispecific antigen-binding molecule that is specific to PD-1 and LAG3,” or “anti-PD-1/anti-LAG3 antibody” are used interchangeably herein and refer to a bispecific antibody that can bind to PD-1 and LAG3 with sufficient affinity, such that the antibody can be used as a diagnostic and/or therapeutic agent targeting PD-1 and LAG3.

The term "PD-1," also known as programmed cell death protein 1, is a type I membrane protein composed of 288 amino acids, 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 has 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 in the immunoreceptor tyrosine repressive motif and the immunoreceptor tyrosine transconverting motif, indicating that PD-1 negatively regulates TCR signaling. This is consistent with the binding of SHP-1 phosphatases and SHP-2 phosphatases to the cytoplasmic tail of PD-1 after ligand binding. Although PD-1 is not expressed on naïve T cells, it is upregulated after T cell receptor (TCR)-mediated activation and has been observed on both activated and exhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772). These exhausted T cells exhibit a dysfunctional phenotype and are unable to respond appropriately. Despite the relatively broad expression pattern of PD-1, its most important role is likely as a co-inhibitory receptor on T cells (Chinai et al., Trendsin Pharmacological Sciences 36 (2015), 587-595). Therefore, current therapeutic approaches focus on blocking the interaction between PD-1 and its ligands to enhance T cell responses. The terms “programmed cell death 1,” “programmed cell death 1,” “protein PD-1,” “PD-1,” “PD1,” “PDCD1,” “hPD-1,” and “hPD-1” are used interchangeably and include variants, isotypes, species homologs of human PD-1, and analogs that share at least one common epitope with PD-1. The amino acid sequence of human PD-1 is shown in UniProt (www.uniprot.org) accession number Q15116 (SEQ ID NO:55).

Unless otherwise stated, as used herein, the terms “LAG3” or “Lag-3” or “lymphocyte activation gene-3” or “CD223” refer to any naturally occurring LAG3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers “full-length”, unprocessed LAG3, as well as any form of LAG3 produced by intracellular processing. The term also covers naturally occurring variants of LAG3, such as splice variants or allelic variants. In a preferred embodiment, the term “LAG3” refers to human LAG3. An exemplary processed (signal-free) LAG3 amino acid sequence is shown in SEQ ID NO:56. An exemplary extracellular domain (ECD) LAG3 amino acid sequence is shown in SEQ ID NO:57.

The terms "anti-LAG3 antibody" and "LAG3-binding antibody" refer to antibodies that bind to LAG3 with sufficient affinity, making them usable as diagnostic and/or therapeutic agents targeting LAG3. In one aspect, for example, as measured by radioimmunoassay (RIA), the binding degree of the anti-LAG3 antibody to unrelated non-LAG3 proteins is less than about 10% of the binding degree of the antibody to LAG3. In some embodiments, the dissociation constant (K_{_D}) of the LAG3-binding 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 lower, e.g., 10 ^{^-8} M to 10^{^-13} M, e.g., 10 ^{^-9} M to 10 ^{^-13} M). In some aspects, the anti-LAG3 antibody binds to epitopes of LAG3 that are conserved in LAG3 from different species. In a preferred embodiment, "anti-LAG3 antibody,""antibody specifically binding to human LAG3," and "antibody binding to human LAG3" refer to antibodies that specifically bind to the human LAG3 antigen or its extracellular domain (ECD) having a _KD value of 1.0 × ^10⁻⁸ mol/L or less, in one embodiment a _KD value of 1.0 × ^10⁻⁹ mol/L or less, and in another embodiment a _KD value from 1.0 ^{× 10⁻⁹} mol/L to 1.0 × ^10⁻¹³ mol/L. In this context, for example using the LAG3 extracellular domain, binding affinity is determined using standard binding assays (such as surface plasmon resonance technology (GE-HealthcareUppsala, Sweden)). The term "anti-LAG3 antibody" also encompasses bispecific antibodies capable of binding to both LAG3 and a second antigen.

The "mortar and pestle structure" technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., ProtEng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a protrusion ("mortar") at the interface of a first polypeptide and a corresponding cavity ("pot") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote the formation of a heterodimer and inhibit the formation of a homodimer. 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 compensating 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 prepared by altering the nucleic acid encoding the polypeptide (e.g., through site-specific mutagenesis or via peptide synthesis). In a specific embodiment, the clathrate-modified subunit of one of the two subunits containing the Fc domain has an amino acid substitution of T366W, while the mortar-modified subunit of the other of the two subunits containing the Fc domain has amino acid substitutions of T366S, L368A, and Y407V. In another specific embodiment, the clathrate-modified subunit of the Fc domain further has an amino acid substitution of S354C, while the mortar-modified subunit of the Fc domain further has an amino acid substitution of Y349C. The introduction of these two cysteine residues results in the formation of a disulfide bond between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

The term "effective function" refers to those biological activities attributable to the Fc region of an antibody that vary with 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 phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

"Activated Fc receptors" are Fc receptors that, upon binding to the Fc region of an antibody, induce signal transduction events that stimulate receptor-carrying cells to perform effector functions. Activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). The specific activated Fc receptor is human FcγRIIIa (see UniProt accession number P08637, version 141).

The term "peptide linker" refers to a peptide that contains one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic adaptor peptides are, for example, ( _G4S ) _n , ( _SG4 ) _n , or _G4 ( _SG4 ) _n peptide adaptors, where "n" is typically a number between 1 and 10, typically between 2 and 4, especially 2, i.e., peptides selected from the group consisting of: GGGGS (SEQ ID NO:46), GGGGSGGGGS (SEQ ID NO:47), SGGGGSGGGG (SEQ ID NO:48), and GGGGSGGGGSGGG (SEQ ID NO:49), but also including sequences GSPGSSSSGS (SEQ ID NO:50), (G4S)3 (SEQ ID NO:51), (G4S)4 (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), _GGSG (SEQ ID NO:58), GGSG (SEQ ID NO: ₅₉ ), GGSG (SEQ ID NO:50), GGSG (SEQ ID NO:50), GGSG (SEQ ID NO:51), GGSG (SEQ ID NO:52), GGSGSGS (SEQ ID NO:53), GGSGSGNGS (SEQ ID NO:54), GGSGSGSG (SEQ ID NO:55), GGSGSG (SEQ ID NO:56), GGSG (SEQ ID NO:57), GGSG (SEQ ID NO:58), GGSG (SEQ ID NO:59), GGSG (SEQ ID NO:50 ... (SEQ ID NO:57), GGSGNGSG (SEQ ID NO:58), GGNGSGSG (SEQ ID NO:59), and GGNGSG (SEQ ID NO:60). Of particular interest are the peptide linkers (G4S) (SEQ ID NO:46), ( _G4S ) ₂ or GGGGSGGGGS (SEQ ID NO:47), (G4S) ₃ (SEQ ID NO:51), and ( _G4S ) ₄ (SEQ ID NO:53), and more particularly ( _G4S ) ₂ (SEQ ID NO:47) or GGGGSGGGGS (SEQ ID NO:47).

"Fused to" or "connected to" means that components (e.g., antigen-binding domains and Fc domains) are connected by peptide bonds, either directly or via one or more peptide linkers.

"VEGF antagonists" or "VEGF-specific antagonists" refer to molecules that can bind to VEGF, reduce VEGF expression levels, or neutralize, block, inhibit, eliminate, reduce, or interfere with VEGF biological activity, including but not limited to the binding of VEGF to one or more VEGF receptors, VEGF signaling, and VEGF-mediated angiogenesis and endothelial cell survival or proliferation. For example, molecules that can neutralize, block, inhibit, eliminate, reduce, or interfere with VEGF biological activity can exert their effects by binding to one or more VEGF receptors (VEGFRs) (e.g., VEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF receptors (mbVEGFR), or soluble VEGF receptors (sVEGFR)). Such antagonists are also referred to as "VEGFR inhibitors" in this document. As VEGF-specific antagonists that can be used in the methods of the present invention, they include peptides that specifically bind to VEGF, anti-VEGF antibodies and their antigen-binding fragments, receptor molecules and derivatives that specifically bind to VEGF to isolate it from one or more receptors, fusion proteins (e.g., VEGF-Trap (Regeneron)), and VEGF ₁₂₁ -Peregrine. VEGF-specific antagonists also include antagonist variants of VEGF peptides, antisense nucleobase oligomers complementary to at least one fragment of a nucleic acid molecule encoding a VEGF peptide; small RNAs complementary to at least one fragment of a nucleic acid molecule encoding a VEGF peptide; ribozymes targeting VEGF; peptide bodies targeting VEGF; and VEGF aptamers. VEGF antagonists also include peptides that bind to VEGFR, anti-VEGFR antibodies (e.g., bevacizumab) and their antigen-binding fragments, and derivatives that bind to VEGFR to block, inhibit, eliminate, reduce, or interfere with VEGF biological activity (e.g., VEGF signaling), or fusion proteins. VEGF-specific antagonists also include non-peptide small molecules that bind to VEGF or VEGFR and are capable of blocking, inhibiting, eliminating, reducing, or interfering with VEGF biological activity. Therefore, the term "VEGF biological activity" specifically includes VEGF-mediated VEGF biological activity. In some embodiments, the VEGF antagonist reduces or inhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the expression level or biological activity of VEGF. In some embodiments, the VEGF inhibited by the VEGF-specific antagonist is VEGF(8-109), VEGF(1-109), or VEGF ₁₆₅ .

As used herein, VEGF antagonists may include, but are not limited to: anti-VEGFR2 antibodies and related molecules (e.g., ramucirumab, tanibirumab, aflibercept); anti-VEGFR1 antibodies and related molecules (e.g., icrucumab, aflibercept (VEGF Trap-Eye), and ziv-aflibercept (VEGF Trap); bispecific VEGF antibodies (e.g., MP-0250, vanucizumab (VEGF-ANG2), and the bispecific antibodies disclosed in US2001/0236388); bispecific antibodies including combinations of two of the anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms; and anti-VEGFA antibodies (e.g., bevacizumab, sevacizumab). b) Anti-VEGFB antibodies, anti-VEGFC antibodies (e.g., VGX-100), anti-VEGFD antibodies, and non-peptide small molecule VEGF antagonists (e.g., pazopanib, axitinib, vandetanib, Stivarga, cabozantinib, lenvatinib, nintedanib, orantinib, telatinib, dovitinib, cediranib, motesanib, sulfatinib, apatinib, foretinib, famitinib, and tivozanib). In some examples, VEGF antagonists can be tyrosine kinase inhibitors, including receptor tyrosine kinase inhibitors (e.g., multi-target receptor tyrosine kinase inhibitors, such as sunitinib or axitinib).

"Anti-VEGF antibody" is an antibody that binds to VEGF with sufficient affinity and specificity. In some embodiments, the antibody will have a sufficiently high binding affinity to VEGF; for example, the antibody may bind hVEGF with a K_{_D} value between 100 nM and 1 pM. Antibody affinity can be determined, for example, by surface plasmon resonance-based assays (such as those described in PCT application publication WO2005/012359), enzyme-linked immunosorbent assays (ELISA), and competitive assays (e.g., radioimmunoassay (RIA)).

In some embodiments, anti-VEGF antibodies can be used as therapeutic agents targeting and interfering with VEGF activity in diseases or conditions in which it is involved. Furthermore, other bioactivity assays can be performed on the antibody, for example, to evaluate its effectiveness as a therapeutic agent. Such assays are known in the art and depend on the intended use of the target antigen and the antibody. Examples include HUVEC inhibition assays; tumor cell growth inhibition assays (e.g., as described in WO 89/06692); antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (US Patent No. 5,500,362); and agonist activity or hematopoietic activity assays (see WO 95/27062). Anti-VEGF antibodies generally do not bind to other VEGF homologs (such as VEGF-B or VEGF-C) or other growth factors (such as P1GF, 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 generated from hybridoma ATCC HB 10709. In another embodiment, the anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (Cancer Res. 57:4593-4599, 1997), including but not limited to an antibody called bevacizumab (BV).

The anti-VEGF antibody “bevacizumab (BV)”, also known as “rhuMAb VEGF” or a recombinant humanized anti-VEGF monoclonal antibody derived from Presta et al. (Cancer Res. 57:4593-4599, 1997), comprises a mutated human IgG1 framework region and an antigen-binding complementarity-determining region derived from mouse anti-hVEGF monoclonal antibody A.4.6.1 that blocks the binding of human VEGF to its receptor. Approximately 93% of the amino acid sequence of bevacizumab, including the majority of the framework region, is derived from human IgG1, and approximately 7% of the sequence is derived from mouse 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 U.S. Patent No. 6,884,879, issued February 26, 2005, the entire disclosure of which is expressly incorporated herein by reference.

II. Treatment methods and compositions for cancer

A. Methods including bispecific antibodies targeting PD-1 and LAG3.

In one aspect, this disclosure provides a method for treating a subject with cancer, the method comprising administering to the subject one or more 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 specifically binding to PD-1 and a second antigen-binding domain specifically binding to 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 fixed dose of 600 mg).

Section VIII below provides exemplary bispecific antibodies targeting PD-1 and LAG3. A specific example of a bispecific antibody targeting PD-1 and LAG3 is PD1-LAG3 as defined herein.

Recently, the combination of renalalimab and nivolumab has provided a clinical proof-of-concept for dual inhibition of PD-1 and LAG3 in patients with previously untreated metastatic or unresectable melanoma (Tawbi et al., N Engl J Med, 386(1):24-34, 2022). By targeting both PD-1 and LAG3 on dysfunctional tumor-specific T lymphocytes, bispecific antibodies targeting PD-1 and LAG3 aim to restore an effective anti-tumor immune response and provide even greater survival benefits to cancer patients compared to currently available checkpoint inhibitors. By preferentially targeting dysfunctional T cells that co-express PD-1/LAG-3 and potentially reducing targeting of Tregs expressing LAG3 in the tumor microenvironment, bispecific antibodies targeting PD-1 and LAG3 may avoid reactivation of Treg-mediated immunosuppression while restoring an anti-tumor immune response.

In some aspects, the length of each of the one or more dosing cycles is 21 days. In some aspects, the method includes administering (e.g., intravenous) a bispecific antibody to the subject on day 1 of each of the one or more dosing cycles.

Cancer can be a solid tumor (e.g., a solid tumor with a tumor microenvironment containing CD8+ T cells expressing LAG3). For example, in some aspects, 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 cell carcinoma (e.g., renal cell carcinoma (RCC)), 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. Cancer may be locally advanced or metastatic.

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

In some respects, the participants had not previously received systemic anticancer therapy.

In some respects, the subjects had not previously received anticancer therapy that included immunomodulators, for example, not received anticancer agents containing checkpoint inhibitors (CPIs), such as anti-programmed death-ligand 1 (PD-L1)/PD-1 agents or anti-cytotoxic T-lymphocyte-associated antigen (CTLA-4) agents. In other respects, the subjects had previously received immunomodulators (e.g., CPIs) as adjuvant or neoadjuvant therapy.

In some respects, the subjects had not previously been treated for metastatic or unresectable disease.

In some respects, the subjects had not previously been treated with anti-LAG3 therapy.

In some respects, bispecific antibodies achieve at least 90% LAG3 receptor occupancy (RO) in tumors, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RO in tumors.

In some respects, the subjects are human.

B. Methods including bispecific antibodies targeting PD-1 and LAG3, and bevacizumab.

In some aspects, the method further includes administering (e.g., intravenous) a VEGF antagonist, such as an anti-VEGF antibody (e.g., bevacizumab), to the subject. Exemplary VEGF antagonists are provided in Section IX below.

In some respects, VEGF antagonists are administered before bispecific antibodies targeting PD-1 and LAG3. In other respects, VEGF antagonists are administered after bispecific antibodies targeting PD-1 and LAG3. In yet another respect, VEGF antagonists and bispecific antibodies targeting PD-1 and LAG3 are administered simultaneously.

In some respects, the method involves administering a VEGF antagonist (e.g., bevacizumab) to the subject every three weeks at a dose of approximately 15 mg/kg (e.g., a dose of 15 mg/kg).

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

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

In other respects, the method further includes administering a VEGF antagonist (e.g., bevacizumab) to the subject every two weeks at a dose of approximately 10 mg/kg (e.g., a dose of 10 mg/kg). In some respects, the length of each of the one or more dosing cycles is 28 days and the method includes administering a VEGF antagonist (e.g., bevacizumab) to the subject on day 1 and day 15 of each of the one or more dosing cycles.

On the other hand, this disclosure provides a bispecific antibody targeting PD-1 and LAG3 (e.g., a bispecific antibody targeting PD-1 and LAG3 for use in a method for treating a subject with 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 wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks. A specific example of a bispecific antibody targeting PD-1 and LAG3 is PD1-LAG3 as defined herein.

In another aspect, this disclosure provides the 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 to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and wherein the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks.

C. Methods including bispecific antibodies targeting PD-1 and LAG3 and anti-TIGIT antagonist antibodies.

In some aspects, the method further includes administering (e.g., intravenous) an anti-TIGIT antagonist antibody (e.g., tisreliumab) to the subject.

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

In some respects, anti-TIGIT antagonist antibodies are administered prior to bispecific antibodies targeting PD-1 and LAG3. In other respects, anti-TIGIT antagonist antibodies are administered following bispecific antibodies targeting PD-1 and LAG3. In yet another respect, anti-TIGIT antagonist antibodies and bispecific antibodies targeting PD-1 and LAG3 are administered simultaneously.

In some respects, the method involves administering an anti-TIGIT antagonist antibody (e.g., tisreliumab) to the subject every three weeks at a fixed dose of approximately 600 mg (e.g., a fixed dose of 600 mg).

In some respects, the length of each of the one or more dosing cycles is 21 days and the method includes administering an anti-TIGIT antagonist antibody to the subject on approximately day 1 (e.g., day 1) of each of the one or more dosing cycles.

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

III. Treatment methods and compositions for liver cancer

A. Methods including bispecific antibodies targeting PD-1 and LAG3.

In one aspect, this disclosure provides a method for treating a subject with liver cancer, the method comprising administering to the subject one or more 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 specifically binding to PD-1 and a second antigen-binding domain specifically binding to 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 fixed dose of 600 mg).

Section VIII below provides exemplary bispecific antibodies targeting PD-1 and LAG3. A specific example of a bispecific antibody targeting PD-1 and LAG3 is PD1-LAG3 as defined herein.

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

In some aspects, the length of each of the one or more dosing cycles is 21 days. In some aspects, the method includes administering (e.g., intravenous) a bispecific antibody to the subject on day 1 of each of the one or more dosing cycles.

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

In some aspects, the length of each of the one or more dosing cycles is 28 days. In some aspects, the method includes administering (e.g., intravenous) a bispecific antibody to the subject on day 1 and day 15 of each of the one or more dosing cycles.

In some respects, liver cancer is hepatocellular carcinoma (HCC). In other respects, liver cancer (e.g., HCC) has a tumor microenvironment that includes CD8+ T cells expressing LAG3. HCC can be, for example, locally advanced, metastatic, and/or unresectable.

In some respects, the participants had not previously received systemic anticancer therapy.

In some respects, the subjects had not previously received anticancer therapy that included immunomodulators, for example, not received anticancer agents containing checkpoint inhibitors (CPIs), such as anti-programmed death-ligand 1 (PD-L1)/PD-1 agents or anti-cytotoxic T-lymphocyte-associated antigen (CTLA-4) agents. In other respects, the subjects had previously received immunomodulators (e.g., CPIs) as adjuvant or neoadjuvant therapy.

In some respects, the subjects had not previously been treated for metastatic or unresectable disease.

In some respects, the subjects had not previously been treated with anti-LAG3 therapy.

In some respects, bispecific antibodies achieve at least 90% LAG3 receptor occupancy (RO) in tumors, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% RO in tumors.

In some respects, the subjects are human.

B. Methods including bispecific antibodies targeting PD-1 and LAG3, and bevacizumab.

In some aspects, the method further includes administering (e.g., intravenous) a VEGF antagonist, such as an anti-VEGF antibody (e.g., bevacizumab), to the subject. Exemplary VEGF antagonists are provided in Section IX below.

In some respects, anti-TIGIT antagonist antibodies are administered prior to bispecific antibodies targeting PD-1 and LAG3. In other respects, anti-TIGIT antagonist antibodies are administered following bispecific antibodies targeting PD-1 and LAG3. In yet another respect, anti-TIGIT antagonist antibodies and bispecific antibodies targeting PD-1 and LAG3 are administered simultaneously.

In some respects, the method involves administering a VEGF antagonist (e.g., bevacizumab) to the subject every three weeks at a dose of approximately 15 mg/kg (e.g., a dose of 15 mg/kg).

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

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

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

In other respects, the method further includes administering a VEGF antagonist (e.g., bevacizumab) to the subject every two weeks at a dose of approximately 10 mg/kg (e.g., a dose of 10 mg/kg). In some respects, the length of each of the one or more dosing cycles is 28 days and the method includes administering a VEGF antagonist (e.g., bevacizumab) to the subject on day 1 and day 15 of each of the one or more dosing cycles.

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

IV. Treatment methods and compositions for melanoma

A. Methods including anti-TIGIT antagonist antibodies and bispecific antibodies targeting PD-1 and LAG3.

In one aspect, this 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 PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain specifically binding to programmed cell death protein 1 (PD-1) and a second antigen-binding domain specifically binding to lymphocyte activation gene 3 (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.

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

In a particular aspect, the method includes administering an anti-TIGIT antagonist antibody to the subject at a fixed dose of about 600 mg every three weeks (e.g., a fixed dose of 600 mg); and (b) administering a bispecific antibody at a fixed dose of about 600 mg every three weeks (e.g., a fixed dose of 600 mg).

In another aspect, this disclosure provides a method for treating a subject with melanoma, the method comprising administering to the subject one or more dosing cycles a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to LAG3 (e.g., a bispecific antibody targeting PD-1 and LAG3 provided in Section VIII below, and particularly PD1-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 (unresectable stage III or IV) according to the American Joint Committee on Cancer (AJCC) staging system).

In some aspects, the length of each of the one or more dosing cycles is 21 days. In some aspects, the method includes administering an anti-TIGIT antagonist antibody and a bispecific antibody to the subject on approximately day 1 (e.g., day 1) of each of the one or more dosing cycles.

In some respects, the method includes administering a bispecific antibody to the subject prior to the anti-TIGIT antagonist antibody. In other respects, the method includes administering an anti-TIGIT antagonist antibody to the subject prior to the bispecific antibody.

In some respects, the method involves intravenous administration of a bispecific antibody and an anti-TIGIT antagonist antibody to the subject.

i. Neoadjuvant therapy

In some respects, one or more dosing cycles are administered as neoadjuvant therapy.

In some respects, anti-TIGIT antagonist antibodies and bispecific antibodies targeting PD-1 and LAG3 are used as neoadjuvant therapies.

In some respects, melanoma is stage III melanoma with measurable lymph node metastasis.

In some respects, no migration or metastasis occurred in the subjects within the six months prior to the start of treatment.

In some respects, the subjects had not previously received cancer immunotherapy.

In some respects, melanoma is neither mucosal melanoma nor uveal melanoma.

In some respects, the first dosing cycle is initiated before surgery.

In some respects, at least one dosing cycle (e.g., one, two, three, four, or more than four dosing cycles) or at least two dosing cycles (e.g., two, three, four, or more than four dosing cycles) are completed before surgery. In some respects, two dosing cycles are completed before surgery.

In some cases, the surgery is performed about a week after the last dosing cycle.

In some respects, the surgery is a complete lymph node dissection (CLND).

In some respects, the treatment-induced pathological response rate (pRR) increased compared to the reference pRR. In some respects, the reference pRR is the pRR of the subject population that received the control therapy. In some respects, the control therapy is a therapy that includes an anti-TIGIT antagonist antibody but does not include a bispecific antibody targeting PD-1 and LAG3; a therapy that includes a bispecific antibody targeting PD-1 and LAG3 but does not include an anti-TIGIT antagonist antibody; or a therapy that includes ipilimumab and nivolumab.

In some respects, treatment resulted in an increase in event-free survival (EFS) compared to a reference EFS; an increase in relapse-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 respects, the reference EFS, RFS, OS, or ORR is the EFS, RFS, OS, or ORR of a population of subjects who received a control therapy. In some respects, the control therapy is a therapy that includes an anti-TIGIT antagonist antibody but does not include a bispecific antibody targeting PD-1 and LAG3; a therapy that includes a bispecific antibody targeting PD-1 and LAG3 but does not include an anti-TIGIT antagonist antibody; or a therapy that includes ipilimumab and nivolumab.

ii. Treatment of stage IV melanoma

In some respects, melanoma is stage IV melanoma.

In some respects, (a) the subject has received no more than two first-line systemic therapies; or (b) the melanoma is a BRAF-mutant melanoma and the subject has received no more than three first-line systemic therapies.

In some respects, this treatment resulted in an increased overall response rate (ORR) compared to a reference ORR. In some respects, the reference ORR is the ORR of a population of subjects who received treatment comprising (a) a bispecific antibody targeting PD-1 and LAG3 but excluding an anti-TIGIT antagonist antibody; and/or (b) a treatment comprising an anti-TIGIT antagonist antibody but excluding a bispecific antibody targeting PD-1 and LAG3.

In some respects, treatment resulted in increased progression-free survival (PFS) compared to reference PFS; increased duration of response (DOR) compared to reference DOR; increased OS compared to reference OS; and increased disease control rate (DCR, e.g., disease stability for 12 weeks or longer, complete response (CR), or partial response (PR)) compared to reference DCR. In some respects, reference PFS, OS, DOR, or DCR is the PFS, OS, DOR, or DCR of a population of subjects who received a control therapy. In some respects, the control therapy is a therapy that includes an anti-TIGIT antagonist antibody but does not include a bispecific antibody targeting PD-1 and LAG3; a therapy that includes a bispecific antibody targeting PD-1 and LAG3 but does not include an anti-TIGIT antagonist antibody; or a therapy that includes ipilimumab and nivolumab.

In some respects, the subjects are human.

B. Methods including bispecific antibodies targeting PD-1 and LAG3.

In another aspect, this disclosure features a method for treating a subject with melanoma, the method comprising administering to the subject a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to LAG3. In some embodiments, the bispecific antibody is administered to the subject in a dosing regimen comprising one or more dosing cycles. In some embodiments, the one or more dosing cycles are administered as neoadjuvant therapy.

In some respects, the method involves administering a bispecific antibody to the subject every three weeks at a fixed dose of approximately 2100 mg (e.g., a fixed dose of 2100 mg).

In one specific aspect, the method involves administering a bispecific antibody to the subject every three weeks at a fixed dose of approximately 600 mg (e.g., a fixed dose of 600 mg).

In some aspects, the length of each of the one or more dosing cycles is 21 days. In some aspects, the method includes administering the bispecific antibody to the subject on approximately day 1 (e.g., day 1) of each of the one or more dosing cycles.

In some respects, the method involves administering bispecific antibodies intravenously to the subject.

In some respects, melanoma is stage III melanoma with measurable lymph node metastasis.

In some respects, no migration or metastasis occurred in the subjects within the six months prior to the start of treatment.

In some respects, the participants had not previously received cancer immunotherapy.

In some respects, melanoma is neither mucosal melanoma nor uveal melanoma.

In some respects, the first dosing cycle is initiated before surgery.

In some respects, at least one dosing cycle (e.g., one, two, three, four, or more than four dosing cycles) or at least two dosing cycles (e.g., two, three, four, or more than four dosing cycles) are completed before surgery. In some respects, two dosing cycles are completed before surgery.

In some cases, the surgery is performed about a week after the last dosing cycle.

In some respects, the surgery is a complete lymph node dissection (CLND).

In some respects, the treatment-induced pathological response rate (pRR) increased compared to the reference pRR. In some respects, the reference pRR is the pRR of the subject population that received a control therapy. In some respects, the control therapy is a therapy that includes ipilimumab and nivolumab.

In some respects, treatment resulted in an increase in event-free survival (EFS) compared to the reference EFS; an increase in relapse-free survival (RFS) compared to the reference RFS; an increase in overall survival (OS) compared to the reference OS; and/or an increase in overall response rate (ORR) compared to the reference ORR. In some respects, the reference EFS, RFS, OS, or ORR is the EFS, RFS, OS, or ORR of a population of subjects who received a control therapy. In some respects, the control therapy is a therapy including ipilimumab and nivolumab.

In some respects, the subjects are human.

C. Methods including anti-TIGIT antagonist antibodies and PD-1 axis-binding antagonists

In another aspect, this disclosure features a method for treating a subject with melanoma, the method comprising administering to the subject one or more dosing cycles of an anti-TIGIT antagonist antibody and a PD-1 axis binding antagonist, wherein the one or more dosing cycles are administered as neoadjuvant therapy. In another aspect, this disclosure features a method for treating a subject with melanoma, the method comprising administering to the subject an anti-TIGIT antagonist antibody and a PD-1 axis binding antagonist, wherein the anti-TIGIT antagonist antibody and the PD-1 axis binding antagonist are administered as neoadjuvant therapy.

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

In some respects, the length of each of one or more dosing cycles is 21 days.

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

In some aspects, the method includes administering a PD-1 axis binding antagonist to the subject prior to the anti-TIGIT antagonist antibody.

In some respects, the method involves intravenous administration of a PD-1 axis binding antagonist and an anti-TIGIT antagonist antibody to the subject.

In some respects, melanoma is stage III melanoma with measurable lymph node metastasis.

In some respects, no migration or metastasis occurred in the subjects within the six months prior to the start of treatment.

In some respects, the participants had not previously received cancer immunotherapy.

In some respects, melanoma is neither mucosal melanoma nor uveal melanoma.

In some respects, the first dosing cycle is initiated before surgery.

In some respects, at least one dosing cycle (e.g., one, two, three, four, or more than four dosing cycles) or at least two dosing cycles (e.g., two, three, four, or more than four dosing cycles) are completed before surgery. In some respects, two dosing cycles are completed before surgery.

In some cases, the surgery is performed about a week after the last dosing cycle.

In some respects, the surgery is a complete lymph node dissection (CLND).

In some respects, the treatment-induced pathological response rate (pRR) increased compared to the reference pRR. In some respects, the reference pRR is the pRR of the population of subjects who received the control therapy. In some respects, the control therapy is a therapy that includes an anti-TIGIT antagonist but not a PD-1 axis binding antagonist; a therapy that includes a PD-1 axis binding antagonist but not an anti-TIGIT antagonist antibody; or a therapy that includes ipilimumab and nivolumab.

In some respects, treatment resulted in an increase in event-free survival (EFS) compared to a reference EFS; an increase in relapse-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 respects, the reference EFS, RFS, OS, or ORR is the EFS, RFS, OS, or ORR of a population of subjects who received a control therapy. In some respects, the control therapy is a therapy that includes an anti-TIGIT antagonist but does not include a PD-1 axis binding antagonist; a therapy that includes a PD-1 axis binding antagonist but does not include an anti-TIGIT antagonist antibody; or a therapy that includes ipilimumab and nivolumab.

In some respects, the subjects are human.

D. Medications used in methods for treating melanoma.

i. Bispecific antibodies targeting PD-1 and LAG3

Further examples of bispecific antibodies targeting PD-1 and LAG3 and their dosing regimens are provided in Section VIII below. A specific example of a bispecific antibody targeting PD-1 and LAG3 is PD1-LAG3 as defined herein.

ii. Anti-TIGIT antagonist antibodies

Exemplary anti-TIGIT antagonist antibodies and their dosing regimens are provided in Section VII below.

iii. PD-1 axis binding antagonists

Exemplary PD-1 axis binding antagonists and their dosing regimens are provided in Section X below.

Assessment of V.PD-L1 expression

PD-L1 expression in subjects treated with any of the methods and compositions described herein can be assessed. The methods and compositions may include determining the level of PD-L1 expression 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 instances, the level of PD-L1 expression in a biological sample (e.g., a tumor sample) obtained from the subject has been determined before or after treatment initiation. PD-L1 expression can be determined using any suitable method. For example, PD-L1 expression can be determined as described in U.S. Patent Applications Nos. 15/787,988 and 15/790,680. Any suitable tumor sample can be used, such as formalin-fixed and paraffin-embedded (FFPE) tumor samples, archived tumor samples, fresh tumor samples, or frozen tumor samples.

For example, PD-L1 expression can be determined as the percentage of tumor-infiltrating immune cells expressing a detectable level of PD-L1 in a tumor sample, as the percentage of tumor-infiltrating immune cells expressing a detectable level of PD-L1 in the tumor sample, and/or as the percentage of tumor cells expressing a detectable level of PD-L1 in the tumor sample. It should be understood that in any of the foregoing examples, the percentage of tumor-infiltrating immune cells in a tumor sample can be the percentage of the tumor area covered by tumor-infiltrating immune cells in a slice of the tumor sample obtained from the subject, for example, as assessed 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 (CellSignaling 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.

In some instances, tumor samples obtained from subjects showed detectable PD-L1 expression levels in less than 1% of tumor cells, 1% or more of tumor cells, 1% to less than 5% of tumor cells, 5% or more of tumor cells, 5% to less than 50% of tumor cells, or 50% or more of tumor cells.

In some instances, tumor samples obtained from subjects had detectable PD-L1 expression levels in tumor-infiltrating immune cells that comprised less than 1% of the tumor sample, more than 1% of the tumor sample, 1% to less than 5% of the tumor sample, more than 5% of the tumor sample, 5% to less than 10% of the tumor sample, or more than 10% of the tumor sample.

In some respects, the esophageal cancer of a subject treated according to any of the methods provided herein has a PD-L1 positive tumor cell (TC) score of <5% or a tumor-infiltrating immune cell (IC) score. In some respects, the esophageal cancer has a PD-L1 positive TC score of <1%. In other respects, the esophageal cancer of a subject treated according to any of the methods provided herein has a PD-L1 positive TC score of ≥5% or an IC score. In some respects, 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.

In some embodiments, tumor samples can be scored based on PD-L1 positivity in tumor-infiltrating immune cells and/or tumor cells according to the diagnostic assessment criteria shown in Tables 2 and/or 3, respectively.

Table 2. Diagnostic criteria for tumor-infiltrating immune cells (IC) using IHC

Table 3. Diagnostic criteria for tumor cell (TC) IHC

VI. Evaluation of TIGIT Expression

TIGIT expression levels can be assessed in subjects with cancer (e.g., esophageal cancer, e.g., metastatic esophageal cancer) who have been treated according to any of the methods, uses, and compositions provided herein. These methods, uses, and compositions may include determining TIGIT expression levels in a biological sample (e.g., a tumor sample) obtained from the subject. In other instances, TIGIT expression levels in a biological sample (e.g., a tumor sample) obtained from the subject have been determined before or after treatment initiation. TIGIT expression can be determined using any suitable method. Any suitable tumor sample can be used, such as formalin-fixed and paraffin-embedded (FFPE) tumor samples, archived tumor samples, fresh tumor samples, or frozen tumor samples.

For example, TIGIT expression can be determined as the percentage of tumor-infiltrating immune cells expressing a detectable level of TIGIT in a tumor sample, as the percentage of tumor-infiltrating immune cells expressing a detectable level of TIGIT in the tumor sample, and/or as the percentage of tumor cells expressing a detectable level of TIGIT in the tumor sample. It should be understood that in any of the foregoing examples, the percentage of tumor-infiltrating immune cells in a tumor sample can be the percentage of the tumor area covered by tumor-infiltrating immune cells in a slice of the tumor sample obtained from the subject, for example, as assessed by IHC using an anti-TIGIT antagonist antibody. Any suitable anti-TIGIT antagonist antibody can be used. In some instances, the anti-TIGIT antagonist antibody is 10A7 (WO 2009/126688A3; US Patent No.: 9499596).

VII. Anti-TIGIT antagonist antibodies

The present invention provides an anti-TIGIT antagonist antibody that can be used to treat cancer in subjects (e.g., humans) with cancer.

In some cases, the anti-TIGIT antagonist antibody is tisrelimumab (CAS Registry No.: 1918185-84-8). Tirelimumab (Genentech) is also known as MTIG7192A.

In some cases, anti-TIGIT antagonist antibodies comprise at least one, two, three, four, five, or six HVRs selected from the following: (a) HVR-H1, containing the amino acid sequence SNSAAWN (SEQ ID NO:1); (b) HVR-H2, containing the amino acid sequence KTYYRFKWYSDYAVSVKG (SEQ ID NO:2); (c) HVR-H3, containing the amino acid sequence ESTTYDLLAGPFDY (SEQ ID NO:3); and (d) HVR-L1, containing the amino acid sequence KSSQTVLYSSNNKKY. The amino acid sequence of LA (SEQ ID NO:4), (e) HVR-L2, which contains the amino acid sequence of WASTRES (SEQ ID NO:5); and/or (f) HVR-L3, which contains the amino acid sequence of QQYYSTPFT (SEQ ID NO:6), or a combination of one or more of the above HVRs and one or more variants thereof, which variants have at least about 90% sequence identity with any of SEQ ID NO:1-6 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity).

In some cases, anti-TIGIT antagonist antibodies may include (a) HVR-H1, which contains the amino acid sequence SNSAAWN (SEQ ID NO:1); (b) HVR-H2, which contains the amino acid sequence KTYYRFKWYSDYAVSVKG (SEQ ID NO:2); (c) HVR-H3, which contains the amino acid sequence ESTTYDLLAGPFDY (SEQ ID NO:3); (d) HVR-L1, which contains the amino acid sequence KSSQTVLYSSNNKKYLA (SEQ ID NO:4); (e) HVR-L2, which contains the amino acid sequence WASTRES (SEQ ID NO:5); and (f) HVR-L3, which contains the amino acid sequence QQYYSTPFT (SEQ ID NO:6). In some cases, anti-TIGIT antagonist antibodies have a VH domain containing 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) with respect to the sequence of EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 17), or EVQLQQSGPGLVKPSQTLSLTCAISGDSVS The sequence of SNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO:17), or with QVQLQQSGPGLVKPSQTL The sequence of SLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO:18) has at least 90% sequence identity. The amino acid sequence having monochromaticity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 18); and/or a VL domain containing a sequence identical to DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYW The sequence of ASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK (SEQ ID NO:19) has an amino acid sequence with at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or the sequence of DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK (SEQ ID NO:19). In some cases, 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) with the sequence of SEQ ID NO:17, or the sequence of SEQ ID NO:17; and/or a VL 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) with the sequence of SEQ ID NO:19, or the sequence of SEQ ID NO:19. In some cases, 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 cases, 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) with the sequence of SEQ ID NO:18, or the sequence of SEQ ID NO:18; and/or a VL 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) with the sequence of SEQ ID NO:19, or the sequence of SEQ ID NO:19. In some cases, 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.

In some cases, anti-TIGIT antagonist antibodies comprise heavy and light chain sequences, wherein: (a) the heavy chain contains the following amino acid sequence: EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:23); and (b) the light chain contains the following amino acid sequence:

DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:24).

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

In some cases, the anti-TIGIT antagonist antibody also comprises at least one, two, three, or four of the following heavy chain variable regions FR: FR-H1, which contains the amino acid sequence _X1 VQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO:11), wherein _X1 is E or Q; FR-H2, which contains the amino acid sequence WIRQSPSRGLEWLG (SEQ ID NO:12); FR-H3, which contains the amino acid sequence RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO:13); and/or FR-H4, which contains 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, the variants being associated with SEQ ID NO:14. Any of NO:11-14 has at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity). The anti-TIGIT antagonist antibody may further comprise at least one, two, three, or four of the following heavy chain variable regions FR: FR-H1 containing the amino acid sequence EVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO:15); FR-H2 containing the amino acid sequence WIRQSPSRGLEWLG (SEQ ID NO:12); FR-H3 containing the amino acid sequence RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO:13); and/or FR-H4 containing 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, the variant having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with any of SEQ ID NO:12-15. In some cases, anti-TIGIT antagonist antibodies include FR-H1, which contains the amino acid sequence EVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO:15); FR-H2, which contains the amino acid sequence WIRQSPSRGLEWLG (SEQ ID NO:12); FR-H3, which contains the amino acid sequence RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO:13); and FR-H4, which contains the amino acid sequence WGQGTLVTVSS (SEQ ID NO:14). In another instance, for example, the anti-TIGIT antagonist antibody may further comprise at least one, two, three, or four of the following heavy chain variable regions FR: FR-H1, containing the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO:16); FR-H2, containing the amino acid sequence WIRQSPSRGLEWLG (SEQ ID NO:12); FR-H3, containing the amino acid sequence RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO:13); and/or FR-H4, containing 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, the variant having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with any one of SEQ ID NO:12-14 and 16. In some cases, anti-TIGIT antagonist antibodies include FR-H1, which contains the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO:16); FR-H2, which contains the amino acid sequence WIRQSPSRGLEWLG (SEQ ID NO:12); FR-H3, which contains the amino acid sequence RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO:13); and FR-H4, which contains the amino acid sequence WGQGTLVTVSS (SEQ ID NO:14).

On the other hand, an anti-TIGIT antagonist antibody is provided, wherein the antibody comprises VH as provided above and VL as provided above, wherein one or both variable domain sequences include post-translational modifications.

In some cases, any of the aforementioned anti-TIGIT antagonist antibodies can bind to both human TIGIT and rabbit TIGIT. In some cases, any of the aforementioned anti-TIGIT antagonist antibodies can bind to both human TIGIT and cynomolgus monkey (cynomolgus monkey) TIGIT. In some cases, any of the aforementioned anti-TIGIT antagonist antibodies can bind to human TIGIT, cyno TIGIT, and rabbit TIGIT. In some cases, any of the aforementioned anti-TIGIT antagonist antibodies can bind to human TIGIT, cyno TIGIT, and rabbit TIGIT, but not to mouse TIGIT.

In some cases, anti-TIGIT antagonist antibodies bind to human TIGIT with a _KD of about 10 nM or less and to cyno TIGIT with a _KD of about 10 nM or less (e.g., binding to human TIGIT with a _KD of about 0.1 nM to about 1 nM, and binding to cyno TIGIT with a _KD of about 0.5 nM to about 1 nM, e.g., binding to human TIGIT with a _KD of about 0.1 nM or less, and binding to cyno TIGIT with a _KD of about 0.5 nM or less).

In some cases, anti-TIGIT antagonist antibodies specifically bind to TIGIT and inhibit or block the interaction between TIGIT and the poliovirus receptor (PVR) (e.g., the antagonist antibody inhibits intracellular signaling mediated by TIGIT-PVR binding). In some cases, the antagonist antibody inhibits or blocks the 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 cases, anti-TIGIT antagonist antibodies specifically bind to TIGIT and inhibit or block the interaction between TIGIT and PVR without affecting the PVR-CD226 interaction. In some cases, the antagonist antibody inhibits or blocks the binding of cyno TIGIT to cyno PVR with an IC50 value of 50 nM or less (e.g., 1 nM to about 50 nM, such as 1 nM to about 5 nM). In some cases, anti-TIGIT antagonist antibodies inhibit and/or block the interaction between CD226 and TIGIT. In some cases, anti-TIGIT antagonist antibodies inhibit and/or block the ability of TIGIT to disrupt CD226 homodimerization.

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

In some respects, anti-TIGIT antagonist antibodies exhibit Fc-mediated effector functions, such as involvement in antibody-dependent cytotoxicity (ADCC). In other respects, anti-TIGIT antagonist antibodies are antibodies with intact Fc-mediated effector functions (e.g., tirelimumab, vimbrolizumab, etigilimab, EOS084448, or TJ-T6) or enhanced effector functions (e.g., SGN-TGT).

In other respects, anti-TIGIT antagonist antibodies are antibodies lacking Fc-mediated effector function (e.g., domvanalimab, BMS-986207, ASP8374, or COM902).

In some respects, anti-TIGIT antagonist antibodies are IgG antibodies. In other respects, anti-TIGIT antagonist antibodies are IgG1 antibodies, such as tireliumab, vimbrolizumab, domvanalimab, BMS-986207, etigilimab, BGB-A1217, SGN-TGT, EOS084448 (EOS-448), TJ-T6, or AB308. In some respects, the antibody is a full-length human monoclonal IgG1 antibody containing the Fc region.

In some respects, anti-TIGIT antagonist antibodies are human monoclonal full-length IgG1 subclass antibodies containing a human IgG1 Fc region, a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:17, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:19.

In other respects, anti-TIGIT antagonist antibodies are IgG4 class antibodies, such as ASP8374 or COM902.

Anti-TIGIT antagonist antibodies (e.g., tirelinumab) that can be used in this invention include compositions containing such antibodies that can bind to PD-1 axis antagonists (e.g., PD-L1 binding antagonists (e.g., anti-PD-L1 antagonist antibodies, such as atezolizumab), PD-1 binding antagonists (e.g., anti-PD-1 antagonist antibodies, such as pembrolizumab) and PD-L2 binding antagonists (e.g., anti-PD-L2 antagonist antibodies)).

In some embodiments, anti-TIGIT antagonist antibodies inhibit TIGIT signaling. In some embodiments, anti-TIGIT antagonist antibodies inhibit the 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, anti-TIGIT antagonist antibodies can inhibit the binding between TIGIT and CD155. In some embodiments, anti-TIGIT antagonist antibodies can inhibit the binding between TIGIT and CD112. In some embodiments, anti-TIGIT antagonist antibodies inhibit the binding between TIGIT and CD113. In some embodiments, anti-TIGIT antagonist antibodies inhibit TIGIT-mediated cell signaling in immune cells. In some embodiments, anti-TIGIT antagonist antibodies inhibit TIGIT by consuming regulatory T cells (e.g., when binding to FcγR).

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.

In some embodiments, the anti-TIGIT antibody is selected from the group consisting of: tirelinumab (MTIG7192A, RG6058 or RO7092284), vimbrolizumab (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: tirelinumab (MTIG7192A, RG6058, or RO7092284), vimbrolizumab (MK-7684), ASP8374 (PTZ-201), EOS-448, and SEA-TGT (SGN-TGT). The anti-TIGIT antibody may be tirelinumab (MTIG7192A, RG6058, or RO7092284).

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

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 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 tirelenumab, ASP8374 (PTZ-201), BGB-A1217, BMS-986207 (ONO-4686), COM902 (CGEN-15137), M6223, IBI939, EOS884448 (EOS-448), domvanalimab (AB154), vimbrolizumab (MK-7684), and SEA-TGT (SGN-TGT).

In some embodiments, the anti-TIGIT antibody comprises the heavy and light chains of any anti-TIGIT antibody 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 tirerelimumab, ASP8374 (PTZ-201), BGB-A1217, BMS-986207 (ONO-4686), COM902 (CGEN-15137), M6223, IBI939, EOS884448 (EOS-448), domvanalimab (AB154), vimbrolizumab (MK-7684), and SEA-TGT (SGN-TGT).

VIII. Bispecific antibodies targeting PD-1 and LAG3

A. An exemplary bispecific antibody combining PD-1 and LAG3

In one aspect, the present invention provides a bispecific antibody comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to LAG3, wherein the first antigen-binding domain specifically binding to PD-1 comprises a VH domain, which includes:

(i) HVR-H1, which contains the amino acid sequence GFSFSSY (SEQ ID NO:25),

(ii) HVR-H2, which contains the amino acid sequence GGR, and

(iii) HVR-H3, comprising the amino acid sequence of TGRVYFALD (SEQ ID NO:26); and a VL domain, comprising

(i)HVR-L1, which contains the amino acid sequence SESVDTSDNSF (SEQ ID NO:27);

(ii) HVR-L2, which contains the amino acid sequence RSS, and

(iii) HVR-L3, which contains the amino acid sequence of NYDVPW (SEQ ID NO:28).

In one aspect, the bispecific antibody comprises an Fc domain, said Fc domain being IgG, specifically an IgG1 Fc domain or an IgG4 Fc domain, and wherein the Fc domain has a reduced or even eliminated effector function. Specifically, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, specifically binding to an Fcγ receptor.

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 bispecific antibody comprises an Fc domain that is IgG, specifically an IgG1 Fc domain or an IgG4 Fc domain, and wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, specifically binding to an Fcγ receptor.

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, which contains...

(i) HVR-H1, which contains the amino acid sequence DYTMN (SEQ ID NO:31),

(ii) HVR-H2, which contains the amino acid sequence VISWDGGGTYYTDSVKG (SEQ ID NO:32), and

(iii) HVR-H3, which contains the amino acid sequence GLTDTTLYGSDY (SEQ ID NO:33); and

VL structuring domain, which contains

(i) HVR-L1, which contains the amino acid sequence RASQSISSYLN (SEQ ID NO:34),

(ii) HVR-L2, which contains the amino acid sequence AASTLQS (SEQ ID NO:35), and

(iii) HVR-L3, which contains the amino acid sequence QQTYSSPLT (SEQ ID NO:36).

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:

The VH structuring domain contains

The amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYTMSWVRQAPGKGLEWVATISGGGRDIYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVLLTGRVYFALDSWGQGTLVTVSS (SEQ ID NO:29); and the VL domain, which contains

The amino acid sequence of DIVMTQSPDSLAVSLGERATINCKASESVDTSDNSFIHWYQQKPGQSPKLLIYRSSTLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNYDVPWTFGQGTKVEIK (SEQ ID NO: 30).

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:

The VH struct contains EVQLLESGGGLVQPGGSLRL

SCAASGFIFDDYTMNWVRQAPGKGLEWVAVISWDGGGTYYTDSVKGRFTISRDDFKNTLY

The amino acid sequence of LQMNSLRAEDTAVYYCAKGLTDTTLYGSDYWGQGTLVTVSS (SEQ ID NO:37), and the VL domain, which contains

The amino acid sequence of DIQMTQSPSSSLSASVGDRVTITCRASQSISLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSSPLTFGGGTKVEIK (SEQ ID NO: 38).

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

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

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

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 containing the amino acid sequence of SEQ ID NO:29; and a VL domain containing the amino acid sequence of SEQ ID NO:30.

Furthermore, the second antigen-binding domain that specifically binds to LAG3 comprises: 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.

In another aspect, the 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, humanized, or chimeric antibody. Specifically, the antibody is a humanized or chimeric antibody.

In one respect, 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. This means 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 (in a 1+1 form).

In one aspect, a bispecific antibody is provided comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to LAG3, wherein the bispecific antibody comprises an Fc domain, a first Fab fragment, and a second Fab fragment, the first Fab fragment comprising the antigen-binding domain specifically binding to PD-1, and the second Fab fragment comprising the antigen-binding domain specifically binding to LAG3. In a particular aspect, in one of the Fab fragments, variable domains VL and VH are interchanged, 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 the antigen-binding domain specifically binding to PD-1, variable domains VL and VH are interchanged.

In one particular aspect, a bispecific antibody is provided comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to LAG3, wherein the bispecific antibody comprises a first heavy chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:39; a first light chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:40; a second heavy chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:41; and a second light chain comprising an amino acid sequence having at least 95% sequence identity with 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).

In another aspect, a bispecific antibody is provided comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to LAG3. The bispecific antibody comprises an Fc domain, a first Fab fragment, and a second Fab fragment. The first Fab fragment contains the antigen-binding domain specifically binding to PD-1, and the second Fab fragment contains the antigen-binding domain specifically binding to LAG3. The second Fab fragment is fused to the C-terminus of the Fc domain. Specifically, the Fab fragment containing the antigen-binding domain specifically binding to LAG3 is fused to the C-terminus of the Fc domain via its VH domain (in a trans 1+1 configuration).

In one aspect, the bispecific antibody comprises a first heavy chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:39; a first light chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:40; a second heavy chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:61; and a second light chain containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:42. More particularly, the bispecific antibody may comprise 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:61; and a second light chain containing the amino acid sequence of SEQ ID NO:42.

i. Fc domain modifications that reduce Fc receptor binding and/or effector function

In some respects, 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 containing one or more amino acid modifications that reduce binding to Fc receptors, specifically Fcγ receptors, and reduce or eliminate effector function.

In some respects, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., the human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

The following sections describe preferred aspects of the bispecific antigen-binding molecules of the present invention comprising Fc domain modifications that reduce Fc receptor binding and/or effector function. In one 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 one or more amino acid substitutions that reduce binding to the Fc receptor, specifically to the Fcγ receptor. Specifically, the Fc domain belongs to the human IgG1 subclass and has amino acid mutations L234A, L235A, and P329G (according to Kabat EU index number).

The Fc domain endows the bispecific antibodies of the present invention with favorable pharmacokinetic properties, including a long serum half-life that facilitates good accumulation in target tissues and a favorable tissue-blood partition ratio. However, at the same time, it may cause the bispecific antibodies of the present invention to undesirably target cells expressing Fc receptors, rather than preferred antigen-carrying cells. Therefore, in certain embodiments, the Fc domain of the bispecific antibodies of the present invention exhibits reduced binding affinity to the Fc receptor and/or reduced effector function compared to the native IgG Fc domain, specifically the IgG1 Fc domain or the IgG4 Fc domain. More specifically, the Fc domain is the IgG1 FC domain.

In one such aspect, compared to a natural IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing a natural IgG1 Fc domain), the Fc domain (or the bispecific antigen-binding molecule of the present invention containing the Fc domain) exhibits a binding affinity to the Fc receptor of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5%; and/or compared to a natural IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing a natural IgG1 Fc domain), the Fc domain (or the bispecific antigen-binding molecule of the present invention containing the Fc domain) exhibits effector function of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5%. In one aspect, the Fc domain (or the bispecific antigen-binding molecule of the present invention containing the Fc domain) does not significantly bind to the Fc receptor and/or induce effector function. In a particular aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In one specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the Fc receptor is an inhibitory Fc receptor. In one specific aspect, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In one aspect, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a particular aspect, the effector function is ADCC. In one aspect, the Fc domain exhibits substantially similar binding affinity to the nascent Fc receptor (FcRn) compared to the native IgG1 Fc domain. When the Fc domain (or the bispecific antigen-binding molecule of the present invention containing the Fc domain) exhibits a binding affinity for FcRn greater than about 70%, specifically greater than about 80%, more specifically greater than about 90% of that of the natural IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing the natural IgG1 Fc domain), substantially similar binding to FcRn is achieved.

In a particular aspect, the Fc domain is engineered to have reduced binding affinity for the Fc receptor and/or reduced effector function compared to an unengineered Fc domain. In a particular aspect, the Fc domain of the bispecific antigen-binding molecule of the present invention comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptor and/or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In another aspect, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In one aspect, the bispecific antigen-binding molecule of the present invention comprising an engineered Fc domain exhibits less than 20%, specifically less than 10%, and more specifically less than 5% binding affinity to the Fc receptor compared to a bispecific antibody of the present invention comprising an unengineered Fc domain. In a particular aspect, the Fc receptor is an Fcγ receptor. In other respects, the Fc receptor is a human Fc receptor. In one respect, the Fc receptor is an inhibitory Fc receptor. In a specific respect, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In some respects, the Fc receptor is an activated Fc receptor. In a specific respect, the Fc receptor is an activated 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 respects, the binding affinity to the complementary component, especially the specific binding affinity to C1q, is also reduced. In one respect, the binding affinity to the nascent Fc receptor (FcRn) is not reduced. When the Fc domain (or the bispecific antigen-binding molecule of the present invention comprising the Fc domain) exhibits a binding affinity for FcRn greater than about 70% for the unengineered form of the Fc domain (or the bispecific antigen-binding molecule of the present invention comprising the unengineered form of the Fc domain), substantially similar binding to FcRn is achieved, i.e., the binding affinity of the Fc domain to the receptor is preserved. The Fc domain or the bispecific antigen-binding molecule of the present invention comprising the Fc domain may exhibit such an affinity greater than about 80% or even greater than about 90%. In some embodiments, the Fc domain of the bispecific antigen-binding molecule of the present invention is engineered to have reduced effector function compared to the unengineered Fc domain. Reduced effector functions may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immune complex-mediated antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signal transduction-induced apoptosis, reduced dendritic cell maturation, or reduced T cell initiation.

Antibodies with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acids at positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581). Certain antibody variants with improved or reduced binding to the FcR have been described (e.g., US Patent No. 6,737,056; WO 2004/056312, and Shields, R.L. et al., J. Biol. Chem. 276(2001) 6591-6604).

In one aspect of the invention, the Fc domain contains amino acid substitutions at positions E233, L234, L235, N297, P331, and P329. In some aspects, the Fc domain contains 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 contains 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 contains an amino acid substitution at position P329 and further contains additional amino acid substitutions selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A, and P329G (“P329G LALA”). The amino acid-substituted “P329G LALA” combination almost completely eliminates Fcγ receptor binding of the human IgG1 Fc domain, as described in PCT patent application number WO2012/130831A1. The document also describes methods for preparing such mutant Fc domains and methods for determining their properties, such as Fc receptor binding or effector function. Such antibodies are IgG1 with mutants L234A and L235A or with mutants L234A, L235A, and P329G (numbered according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

In one aspect, the bispecific antibody of the present invention comprises (all positions according to Kabat's EU index) (i) optionally having a homodimeric Fc region of a human IgG1 subclass with mutant P329G, L234A, and L235A, or (ii) optionally having a homodimeric Fc region of a human IgG4 subclass with mutant P329G, S228P, and L235E, or (iii) optionally having a homodimeric Fc region of a human IgG4 subclass with mutant P329G, L234A, L235A, and I253A, H310A and H435A, or optionally, homodimeric Fc regions of human IgG1 subclasses having mutant P329G, L234A, L235A, H310A, H433A, and Y436A, or (iv) heterodimeric Fc regions, wherein one Fc region polypeptide contains mutant T366W and the other Fc region polypeptide contains mutant T366S, L368A, and Y407V, or one Fc region polypeptide contains mutant T366W and Y349C and the other Fc region polypeptide contains mutant T329G, L234A, L235A, H310A, H433A, and Y436A. One Fc region polypeptide contains the mutants T366S, L368A, Y407V, and S354C; or one Fc region polypeptide contains the mutants T366W and S354C and the other Fc region polypeptide contains the mutants T366S, L368A, Y407V, and Y349C; or (v) a heterodimeric Fc region of a human IgG1 subclass, wherein both Fc region polypeptides contain the mutants P329G, L234A, and L235A, and one Fc region polypeptide contains the mutant... T366W, while another Fc region polypeptide contains mutants T366S, L368A, and Y407V; or one Fc region polypeptide contains mutants T366W and Y349C, while another Fc region polypeptide contains mutants T366S, L368A, Y407V, and S354C; or one Fc region polypeptide contains mutants T366W and S354C, and another Fc region polypeptide contains mutants T366S, L368A, Y407V, and Y349C.

In one aspect, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain contains an amino acid substitution at position S228 (Kabat number), specifically an IgG4 Fc domain with amino acid substitution at S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing amino acid substitutions at L235E and S228P, as well as P329G. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). Therefore, in one aspect, a bispecific antibody is provided comprising (all positions according to Kabat's EU index) heterodimeric Fc regions of human IgG4 subclasses, wherein both Fc region polypeptides contain mutants P329G, S228P, and L235E and one Fc region polypeptide contains mutant T366W while the other Fc region polypeptide contains mutants T366S, L368A, and Y407V, or one Fc region polypeptide contains mutants T366W and Y349C while the other Fc region polypeptide contains mutants T366S, L368A, Y407V, and S354C, or one Fc region polypeptide contains mutants T366W and S354C while the other Fc region polypeptide contains mutants T366S, L368A, Y407V, and Y349C.

Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), responsible for transferring maternal IgG to the fetus (Guyer, R.L. et al., J. Immunol. 117 (1976) 587-593 and Kim, J.K. et al., J. Immunol. 24 (1994) 2429-2434), are described in US 2005/0014934. These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants with substitutions 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, such as the substitution of Fc region residue 434 (US 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; US 5,648,260; US 5,624,821; and WO 94/29351.

Binding to the Fc receptor can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare), and the Fc receptor can be obtained, for example, through recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of the Fc domain or cell-activating bispecific antigen-binding molecules containing the Fc domain to the Fc receptor can be evaluated using cell lines known to express a specific Fc receptor, such as human NK cells expressing the FcγIIIa receptor. The effector function of the Fc domain, or the bispecific antibody containing the Fc domain of the present invention, can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Patent No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, non-radioactive assays may be used (see, for example, the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or otherwise, the ADCC activity of the molecule of interest can be assessed in vivo in animal models such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95,652-656 (1998).

The following sections describe preferred aspects of the bispecific antibodies of the present invention comprising Fc domain modifications that reduce Fc receptor binding and/or effector function. In one 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 one or more amino acid substitutions that reduce the binding affinity of the antibody to the Fc receptor, particularly to the Fcγ receptor. In another 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 one or more amino acid substitutions that reduce effector function. In a particular aspect, the Fc domain belongs to the human IgG1 subclass and has amino acid mutations L234A, L235A, and P329G (according to Kabat EU index number).

ii. Modification of Fc domains to promote heterodimerization

The bispecific antigen-binding molecule of the present invention comprises a different antigen-binding domain fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain can be contained in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides result in several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antibody of the present invention in recombinant production, it is advantageous to introduce modifications that promote the association of the desired polypeptide into the Fc domain of the bispecific antigen-binding molecule of the present invention.

Therefore, 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 modifications that promote association between the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one aspect, the modification is in the CH3 domain of the Fc domain.

In a specific aspect, the modification is a so-called "mortar and pestle" modification, which includes a "mortar" modification in one of the two subunits of the Fc domain and a "mortar" modification in the other of the two subunits of the Fc domain. Therefore, 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, according to the mortar and pestle method, the first subunit of the Fc domain contains a mortar and the second subunit of the Fc domain contains a mortar. In a particular aspect, the first subunit of the Fc domain contains amino acid substitutions of S354C and T366W (EU number) and the second subunit of the Fc domain contains amino acid substitutions of Y349C, T366S, and Y407V (according to Kabat EU index number).

The pestle-and-mortar structure technique is described, for example, in US 5,731,168; US 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 protrusion (“pestle”) at the interface of a first polypeptide and a corresponding cavity (“mortar”) at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote the formation of a heterodimer and inhibit the formation of a homodimer. 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 compensating cavity having 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).

Therefore, in one aspect, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen-binding molecule of the present invention, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit, which can be localized in a cavity within the CH3 domain of the second subunit. Conversely, in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit, in which the protrusion within the CH3 domain of the first subunit can be localized. The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide (e.g., through site-specific mutagenesis or through peptide synthesis). Specifically, 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), while 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 aspect, in the second subunit of the Fc domain, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A).

In a further aspect, in the first subunit of the Fc domain, the serine residue at position 354 is replaced by a cysteine residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C). The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a specific aspect, the first subunit of the Fc domain contains amino acid substitutions of S354C and T366W (EU number), and the second subunit of the Fc domain contains amino acid substitutions of Y349C, T366S, and Y407V (according to Kabat EU index number).

However, other pestle-and-mortar structure techniques, such as those described in EP 1 870 459, may also be used alternatively or additionally. In one embodiment, the multispecific antibody comprises mutations R409D and K370E in the CH3 domain of the "pepper chain" and mutations D399K and E357K (according to Kabat EU index numbers) in the CH3 domain of the "mortar chain".

In one aspect, the bispecific antibody contains the T366W mutation in the CH3 domain of the "pestle chain" and the mutations T366S, L368A, and Y407V in the CH3 domain of the "mortar chain," as well as the additional mutations R409D and K370E in the CH3 domain of the "pestle chain" and the mutations D399K and E357K in the CH3 domain of the "mortar chain" (according to Kabat EU index numbers).

In one aspect, the bispecific antibody comprises mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A, and Y407V in the other of the two CH3 domains; or the multispecific antibody comprises mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A, and Y407V in the other of the two CH3 domains, as well as additional mutations R409D and K370E in the CH3 domain of the "mortar chain" and mutations D399K and E357K in the CH3 domain of the "mortar chain" (according to Kabat EU index numbers).

In an alternative aspect, modifications that promote association between the first and second subunits of the Fc domain include modifications that mediate electrostatic reorientation effects, such as those described in PCT Publication WO 2009/089004. Typically, this method involves replacing one or more amino acid residues at the interface between the two Fc domain subunits with charged amino acid residues, making homodimer formation electrostatically unfavorable but heterodimerization electrostatically favorable.

Besides the "mortar and pestle structure technique," other techniques for modifying the CH3 domain of the heavy chain of multispecific antibodies to implement heterodimerization are also known in the art. These techniques, especially 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 considered herein as alternatives to the combination of the "mortar and pestle structure technique" with bispecific antibodies.

In one aspect, in bispecific antibodies, the method described in EP 1870459 is used to support the heterodimerization of the first and second heavy chains of a multispecific antibody. This method is based on introducing charged amino acids with opposite charges at specific amino acid sites in the CH3/CH3-domain interface between the first and second heavy chains.

Therefore, in this aspect of the tertiary structure of a 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, wherein the corresponding amino acid sequence of the CH3 domain of the first heavy chain and the amino acid sequence of the CH3 domain of the second heavy chain each contain a set of amino acids located within the interface in the tertiary structure of the antibody, wherein from the set of amino acids located in the interface of the CH3 domain of one heavy chain, the first amino acid is replaced by a positively charged amino acid, and from the set of amino acids located in the interface of the CH3 domain of the other heavy chain, the second amino acid is replaced by a negatively charged amino acid. The bispecific antibody according to this aspect is also referred to herein as a "CH3(+/-) engineered bispecific antibody" (where the abbreviation "+/-" represents the introduction of oppositely charged amino acids in the respective CH3 domains).

In one aspect, in a CH3(+/-) engineered bispecific antibody, the positively charged amino acid is selected from K, R, and H, while the negatively charged amino acid is selected from E or D.

In one aspect, in a CH3(+/-) engineered bispecific antibody, the positively charged amino acid is selected from K and R, while the negatively charged amino acid is selected from E or D.

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

In one aspect, in a bispecific antibody engineered with CH3(+/-) in the CH3 domain of one heavy chain, amino acid R at position 409 is replaced by D and amino acid K at position 309 is replaced by E, while in the CH3 domain of the other heavy chain, amino acid D at position 399 is replaced by K and amino acid E at position 357 is replaced by K (according to Kabat EU index number).

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

In another aspect, in the CH3 domain of one heavy chain, amino acid T at position 366 is substituted with K and amino acid L at position 351 is substituted with K, while in the CH3 domain of the other heavy chain, amino acid L at position 351 is substituted with D (according to Kabat EU index number). Additionally, at least one of the following substitutions is included in the CH3 domain of the other heavy chain: amino acid Y at position 349 is substituted with E, amino acid Y at position 349 is substituted with D, and amino acid L at position 368 is substituted with E (according to Kabat EU index number). In one embodiment, amino acid L at position 368 is substituted with E (according to Kabat EU index number).

In one aspect, the method described in WO 2012/058768 is used to support the heterodimerization of the first and second heavy chains of a multispecific antibody. In one aspect, in the CH3 domain of one heavy chain, amino acid L at position 351 is substituted with Y and amino acid Y at position 407 is substituted with A, while in the CH3 domain of the other heavy chain, amino acid T at position 366 is substituted with A and amino acid K at position 409 is substituted with F (according to Kabat EU index number). In another embodiment, in addition to the above substitutions, in the CH3 domain of the other heavy chain, at least one amino acid selected from the following positions is substituted (according to Kabat EU index number): amino acid at position 411 (originally T), amino acid at position 399 (originally D), amino acid at position 400 (originally S), amino acid at position 405 (originally F), amino acid at position 390 (originally N), and amino acid at position 392 (originally K). Preferred substitutions are:

- Replace amino acid T at position 411 with an amino acid selected from N, R, Q, K, D, E, and W (according to the Kabat EU index number).

- Replace amino acid D at position 399 with an amino acid selected from R, W, Y, and K (according to the Kabat EU index number).

- Replace amino acid S at position 400 with an amino acid selected from E, D, R, and K (according to the Kabat EU index number).

- Replace amino acid F at position 405 with an amino acid selected from I, M, T, S, V and W (according to the Kabat EU index number);

- Replace amino acid N at position 390 with an amino acid selected from R, K, and D (according to the Kabat EU index number); and

- Replace amino acid K at position 392 with an amino acid selected from V, M, R, L, F, and E (according to the Kabat EU index number).

In another aspect, a bispecific antibody is engineered according to WO 2012/058768, wherein in the CH3 domain of one heavy chain, amino acid L at position 351 is replaced by Y and amino acid Y at position 407 is replaced by A, while in the CH3 domain of the other heavy chain, amino acid T at position 366 is replaced by V and amino acid K at position 409 is replaced by F (according to Kabat EU index number). In another embodiment of the multispecific antibody, in the CH3 domain of one heavy chain, amino acid Y at position 407 is replaced by A, while in the CH3 domain of the other heavy chain, amino acid T at position 366 is replaced by A and amino acid K at position 409 is replaced by F (according to Kabat EU index number). In a final embodiment of the above, in the CH3 domain of the other heavy chain, amino acid K at position 392 is replaced by E, amino acid T at position 411 is replaced by E, amino acid D at position 399 is replaced by R, and amino acid S at position 400 is replaced by R (according to Kabat EU index number).

In one aspect, the method described in WO 2011/143545 is used to support the heterodimerization of the first and second heavy chains of a multispecific antibody. In another aspect, amino acid modifications (according to Kabat EU index numbers) are introduced at positions 368 and/or 409 in the CH3 domain of both heavy chains.

In one aspect, the method described in WO 2011/090762 is used to support the heterodimerization of the first and second heavy chains of a bispecific antibody. WO 2011/090762 relates to amino acid modifications according to the "mortar and pestle" (KiH) technique. In one embodiment, in the CH3 domain of one heavy chain, amino acid T at position 366 is substituted with W, while in the CH3 domain of the other heavy chain, amino acid Y at position 407 is substituted with A (according to Kabat EU index number). In another embodiment, in the CH3 domain of one heavy chain, amino acid T at position 366 is substituted with Y, while in the CH3 domain of the other heavy chain, amino acid Y at position 407 is substituted with T (according to Kabat EU index number).

In one aspect, the method described in WO 2009/089004 is used to support the heterodimerization of the first and second heavy chains of a bispecific antibody. In one embodiment, in the CH3 domain of one heavy chain, amino acid K or N at position 392 is substituted with a negatively charged amino acid (substituted with E or D in one embodiment, and D in a preferred embodiment), while in the CH3 domain of the other heavy chain, amino acid D at position 399, amino acid E or D at position 356, or amino acid E at position 357 is substituted with a positively charged amino acid (substituted with K or R in one embodiment, and K in a preferred embodiment; in a preferred embodiment, amino acid at position 399 or 356 is substituted with K) (according to Kabat EU index number). In another embodiment, in addition to the above substitutions, in the CH3 domain of one heavy chain, amino acid K or R at position 409 is substituted with a negatively charged amino acid (substituted with E or D in one embodiment, and D in a preferred embodiment) (according to Kabat EU index number). In another aspect, as a supplement to or alternative to the above substitutions, in the CH3 domain of a heavy chain, amino acid K at position 439 and/or amino acid K at position 370 are independently substituted by negatively charged amino acids (substituted by E or D in one embodiment, and by D in a preferred embodiment) (according to Kabat EU index number).

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

The C-terminus of the heavy chain of the bispecific antibody reported herein can be a full C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain can also be a shortened C-terminus in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG.

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

iii. Modifications in the Fab domain

In one aspect, the present 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, variable domains VH and VL or constant domains CH1 and CL are exchanged. The bispecific antibody is prepared according to the Crossmab technique.

Multispecific antibodies (CrossMabVH-VL or CrossMabCH-CL) with domain substitution/exchange in a single binding arm are described in detail in WO2009/080252, WO2009/080253 and Schaefer, W. et al., PNAS, 108(2011)11187-1191. They significantly reduce byproducts resulting from mismatches between the light chain targeting the first antigen and the incorrect heavy chain targeting the second antigen (compared to methods without such domain exchange).

In one specific aspect, the present 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, variable domains VL and VH are interchanged such that the VH domain is part of a light chain and the VL domain is part of a heavy chain. In another specific aspect, the bispecific antibody is one in which the variable domains VL and VH are interchanged in a first Fab fragment comprising an antigen-binding domain that specifically binds to PD-1.

On the other hand, and to further improve correct pairing, bispecific antibodies containing a first Fab fragment that specifically binds to PD-1 and a second Fab fragment that specifically binds to LAG3 may contain different charged amino acid substitutions (so-called “charged residues”). These modifications are introduced into the cross- or non-cross-connected CH1 and CL domains. Such modifications are described, for example, in WO2015/150447, WO2016/020309, and PCT/EP2016/073408.

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

In one particular aspect, the present invention relates to a bispecific antibody comprising a first Fab fragment specifically binding to PD-1 and a second Fab fragment specifically binding to LAG3, wherein in one of the CL domains, the amino acid at position 123 (EU number) has been replaced by arginine (R) and the amino acid at position 124 (EU number) has been replaced by lysine (K), and wherein in one of the CH1 domains, the amino acids at positions 147 (EU number) and 213 (EU number) have been replaced by glutamate (E). In another particular aspect, the bispecific antibody is a bispecific antibody in which, in the second Fab fragment comprising an antigen-binding domain specifically binding to LAG3, the amino acid at position 123 (EU number) has been replaced by arginine (R) and the amino acid at position 124 (EU number) has been replaced by lysine (K), and wherein in one of the CH1 domains, the amino acids at positions 147 (EU number) and 213 (EU number) have been replaced by glutamate (E).

On the other hand, bispecific antibodies are divalent antibodies, which contain...

a) The first light chain and the first heavy chain of the antibody that specifically binds to the first antigen, and

b) The second light chain and the second heavy chain of an antibody that specifically binds to the second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are interchanged.

The antibody under a) does not contain the modifications reported under b), and the heavy and light chains under a) are separate chains.

In the antibody under b), the variable light chain domain VL within the light chain is replaced by the variable heavy chain domain VH of the antibody, while the variable heavy chain domain VH within the heavy chain is replaced by the variable light chain domain VL of the antibody.

In one aspect, (i) in the constant domain CL of the first light chain under a), the amino acid at position 124 (according to the Kabat number) is replaced by a positively charged amino acid, while in the constant domain CH1 of the first heavy chain under a), the amino acid at position 147 or the amino acid at position 213 (according to the Kabat EU index number) is replaced by a negatively charged amino acid; or (ii) in the constant domain CL of the second light chain under b), the amino acid at position 124 (according to the Kabat number) is replaced by a positively charged amino acid, while in the constant domain CH1 of the second heavy chain under b), the amino acid at position 147 or the amino acid at position 213 (according to the Kabat EU index number) is replaced by a negatively charged amino acid.

In another aspect, (i) in the constant domain CL of the first light chain under a), the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in a preferred embodiment, it is independently substituted with lysine (K) or arginine (R), while in the constant domain CH1 of the first heavy chain under a), the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing). (ii) In the constant domain CL of the second light chain under b), the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat number) (in a preferred embodiment, it is substituted with lysine (K) or arginine (R) independently), while in the constant domain CH1 of the second heavy chain under b), the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

In one aspect, in the constant structural domain CL of the second heavy chain, the amino acids at positions 124 and 123 are substituted with K (according to the Kabat EU index number).

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

In one aspect, in the constant structural domain CH1 of the second light chain, the amino acids at positions 147 and 213 are substituted with E (according to the Kabat EU index number).

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

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

In one aspect, in the constant domain CL of the second heavy chain, amino acids at positions 124 and 123 are substituted with K, and in the constant domain CH1 of the second light chain, amino acids at positions 147 and 213 are substituted with E; in the variable domain VL of the first light chain, amino acid at position 38 is substituted with K; in the variable domain VH of the first heavy chain, amino acid at position 39 is substituted with E; in the variable domain VL of the second heavy chain, amino acid at position 38 is substituted with K; and in the variable domain VH of the second light chain, amino acid at position 39 is substituted with E (according to Kabat EU index number).

In one respect, bispecific antibodies are divalent antibodies, which contain

a) The first light chain and the first heavy chain of the antibody that specifically binds to the first antigen, and

b) The second light chain and the second heavy chain of an antibody that specifically binds to the second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are interchanged with each other, while the constant domains CL and CH1 of the second light chain and the second heavy chain are interchanged with each other.

The antibody in a) does not contain the modifications reported in b), and the heavy and light chains in a) are separate chains. In the antibody in b), within the light chain, the variable light chain domain VL is replaced by the variable heavy chain domain VH of the antibody, while the constant light chain domain CL is replaced by the constant heavy chain domain CH1 of the antibody; within the heavy chain, the variable heavy chain domain VH is replaced by the variable light chain domain VL of the antibody, while the constant heavy chain domain CH1 is replaced by the constant light chain domain CL of the antibody.

In one respect, bispecific antibodies are divalent antibodies, which contain

a) The first light chain and the first heavy chain of the antibody that specifically binds to the first antigen, and

b) The second light chain and the second heavy chain of an antibody that specifically binds to the second antigen, wherein the constant structural domains CL and CH1 of the second light chain and the second heavy chain are interchanged.

The antibody in a) does not contain the modifications reported in b), and the heavy and light chains in a) are separate chains. In the antibody in b), within the light chain, the constant light chain domain CL is replaced by the constant heavy chain domain CH1 of the antibody; while within the heavy chain, the constant heavy chain domain CH1 is replaced by the constant light chain domain CL of the antibody.

In one respect, a bispecific antibody is a bispecific antibody that includes the following:

a) A full-length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains; and

b) Specifically binds to one, two, three, or four single-chain Fab fragments of the second antigen.

The single-chain Fab fragment in b) is fused to the full-length antibody in a) via a peptide linker at the C-terminus or N-terminus of the heavy or light chain of the full-length antibody.

In one aspect, one or two identical single-chain Fab fragments binding 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.

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

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

In one aspect, two identical single-chain Fab (scFab) fragments binding to a second antigen are fused to the full-length antibody via peptide linkers at the C-terminus of each heavy or light chain of the full-length antibody.

In one aspect, two identical single-chain Fab (scFab) fragments binding to a second antigen are fused to the full-length antibody via peptide linkers at the C-terminus of each heavy chain of the full-length antibody.

In one aspect, two identical single-chain Fab (scFab) fragments binding to a second antigen are fused to the full-length antibody via peptide linkers at the C-terminus of each light chain of the full-length antibody.

In one respect, bispecific antibodies are trivalent antibodies, which contain...

a) A full-length antibody, wherein the full-length antibody specifically binds to the first antigen and is composed of two antibody heavy chains and two antibody light chains.

b) The first polypeptide, which consists of the following:

ba) Antibody heavy chain variable domain (VH), or

bb) Antibody heavy chain variable domain (VH) and antibody constant domain 1 (CH1),

The first polypeptide is fused with 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) The second polypeptide, which consists of the following:

ca) Antibody light chain variable domain (VL), or

cb) Antibody light chain variable domain (VL) and antibody light chain constant domain (CL),

The second polypeptide is fused to the C-terminus of the other of the two heavy chains of the full-length antibody via a peptide linker at the N-terminus of the VL domain, and

The antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen-binding domain that specifically binds to the second antigen.

In one aspect, the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via interchain disulfide bridges by introducing disulfide bonds between the following positions:

(i) The variable structure domain of the heavy chain is 44 bits to the variable structure domain of the light chain is 100 bits, or

(ii) The variable structure domain of the heavy chain is from bit 105 to bit 43 of the variable structure domain of the light chain, or

(iii) Variable structural domains from position 101 of the heavy chain to position 100 of the light chain (always according to the Kabat EU index number).

Techniques for stabilizing by introducing non-natural disulfide bridges 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 the polypeptide under b) and c) is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In another embodiment, the optional disulfide bond between the variable domains of the polypeptide under b) and c) is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (always according to Kabat numbering). In one embodiment, a trivalent bispecific antibody stabilized without the optional disulfide bond between the variable domains VH and VL of the single-chain Fab fragment is preferred.

In one aspect, a bispecific antibody is a trispecific or tetraspecific antibody that includes the following:

a) The first light chain and the first heavy chain of the full-length antibody that specifically binds to the first antigen, and

b) A full-length antibody that specifically binds to a second antigen, wherein the second (modified) light chain and the second (modified) heavy chain, wherein the variable domains VL and VH are interchanged with each other, and/or wherein the constant domains CL and CH1 are interchanged with each other, and

c) One to four antigen-binding domains that specifically bind to one or two other antigens (i.e., a third and/or a fourth antigen) are fused to the C-terminus or N-terminus of the light or heavy chain of a) and/or b) via a peptide linker.

The antibody under a) does not contain the modifications reported under b), and the heavy and light chains under a) are separate chains.

In one aspect, under c), a trispecific or tetraspecific antibody contains one or two antigen-binding domains that specifically bind to one or two other antigens.

In one aspect, the antigen-binding domain is selected from the group consisting of scFv fragments and scFab fragments.

In one aspect, the antigen-binding domain is the scFv fragment.

In one aspect, the antigen-binding domain is the scFab fragment.

In one aspect, the antigen-binding domain is fused to the C-terminus of the heavy chain under a) and/or b).

In one aspect, under c), a trispecific or tetraspecific antibody contains one or two antigen-binding domains that specifically bind to another antigen.

In one aspect, the trispecific or tetraspecific antibody under c) comprises two identical antigen-binding domains that specifically bind to a third antigen. In a preferred embodiment, both such identical antigen-binding domains are fused to the C-terminus of the heavy chain under a) and b) via the same peptide linker. In a preferred embodiment, the two identical antigen-binding domains are scFv or scFab fragments.

In one aspect, the trispecific or tetraspecific antibody under c) comprises two antigen-binding domains that specifically bind to the third and fourth antigens. In one embodiment, the two antigen-binding domains are fused to the C-terminus of the heavy chain under a) and b) via the same peptide linker. In a preferred embodiment, the two antigen-binding domains are scFv fragments or scFab fragments.

In one aspect, a bispecific antibody is a bispecific tetravalent antibody, which contains

a) The two light chains and two heavy chains of an antibody that specifically binds to the first antigen (and contains two Fab fragments).

b) Two additional Fab fragments of an antibody that specifically binds to the second antigen, wherein each of the additional Fab fragments is fused to the C-terminus or N-terminus of the heavy chain of a) via a peptide linker.

The following modifications are performed in the Fab fragment.

(i) In the two Fab segments of a), or in the two Fab segments of b), the variable domains VL and VH are interchanged with each other, and/or the constant domains CL and CH1 are interchanged with each other, or

(ii) In the two Fab segments of a), the variable domains VL and VH are interchanged with each other, and the constant domains CL and CH1 are interchanged with each other; while in the two Fab segments of b), the variable domains VL and VH are interchanged with each other, or the constant domains CL and CH1 are interchanged with each other, or

(iii) In the two Fab segments of a), the variable domains VL and VH are interchanged with each other, or the constant domains CL and CH1 are interchanged with each other; while in the two Fab segments of b), the variable domains VL and VH are interchanged with each other, and the constant domains CL and CH1 are interchanged with each other, or

(iv) In the two Fab segments of a), the variable domains VL and VH are interchanged, while in the two Fab segments of b), the constant domains CL and CH1 are interchanged, or

(v) In the two Fab segments of a), the constant domains CL and CH1 are interchanged with each other, while in the two Fab segments of b), the variable domains VL and VH are interchanged with each other.

In one aspect, the additional Fab fragments are all fused to the C-terminus of the heavy chain of a) or the N-terminus of the heavy chain of a) via a peptide linker.

In one aspect, the additional Fab fragment is fused to the C-terminus of the heavy chain of a) via a peptide linker.

In one aspect, the additional Fab fragment is fused to the N-terminus of the heavy chain of a) via a peptide linker.

In one aspect, within a Fab segment, the following modifications are performed: in the two Fab segments of a), or in the two Fab segments of b), the variable domains VL and VH are substituted for each other, and/or the constant domains CL and CH1 are substituted for each other.

In one aspect, bispecific antibodies are tetravalent antibodies, which comprise:

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 the second VH-CH1 domain pair of the first antibody is fused to the C-terminus of the heavy chain via a peptide linker.

b)a) The two light chains of the first antibody,

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 the 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)c) The second antibody has two (modified) light chains, each containing a pair of CL-CH1 domains.

In one aspect, bispecific antibodies include

a) The heavy and light chains of the first full-length antibody that specifically binds to the 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 connected to the C-terminus of the light chain via a peptide linker.

The antibody in a) does not contain the modifications reported in b), and the heavy and light chains are separate chains.

In one aspect, bispecific antibodies include

a) A full-length antibody that specifically binds to the first antigen and consists of two antibody heavy chains and two antibody light chains; and

b) An Fv fragment that specifically binds to a second antigen, the Fv fragment comprising a VH2 domain and a VL2 domain, wherein the two domains are interconnected via disulfide bridges.

Only one of the VH2 or VL2 domains is fused to the heavy or light chain of a full-length antibody that specifically binds to the first antigen via a peptide linker.

In bispecific antibodies, the heavy and light chains in a) are separate chains.

In one aspect, another heavy or light chain of a full-length antibody that specifically binds to the first antigen is fused to the VH2 or VL2 domain without via a peptide linker.

In all aspects reported in this paper, the first light chain comprises the VL and CL domains, and the first heavy chain comprises the VH, CH1, hinge region, CH2, and CH3 domains.

In one respect, bispecific antibodies are trivalent antibodies, which contain

a) Specifically binds to two Fab fragments of the first antigen.

b) Specifically binds to a CrossFab fragment of the second antigen, in which the CH1 and CL domains are exchanged.

c) An Fc region containing a first Fc region heavy chain and a second Fc region heavy chain.

The C-terminus of the CH1 domain of two Fab fragments is attached to the N-terminus of the Fc region of the heavy chain polypeptide, while the C-terminus of the CL domain of the CrossFab fragment is attached to the N-terminus of the VH domain of one of the Fab fragments.

In one respect, bispecific antibodies are trivalent antibodies, which contain

a) Specifically binds to two Fab fragments of the first antigen.

b) Specifically binds to a CrossFab fragment of the second antigen, in which the CH1 and CL domains are exchanged.

c) An Fc region containing a first Fc region heavy chain and a second Fc region heavy chain.

The C-terminus of the CH1 domain of the first Fab fragment is attached to the N-terminus of one of the heavy chain Fc region peptides, and the C-terminus of the CL domain of the CrossFab fragment is attached to the N-terminus of another heavy chain Fc region peptide, while the C-terminus of the CH1 domain of the second Fab fragment is attached to the N-terminus of the VH domain of the first Fab fragment or to the N-terminus of the VH domain of the CrossFab fragment.

In one aspect, bispecific antibodies include

a) A full-length antibody that specifically binds to the 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, the Fab fragment comprising a VH2 domain and a VL2 domain constituting a heavy chain fragment and a light chain fragment, wherein within the light chain fragment, the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of the antibody, and within the heavy chain fragment, the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of the antibody.

The heavy chain Fab fragment is inserted between the CH1 domain of one of the heavy chains of the full-length antibody and the corresponding 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, wherein the light chain of the full-length antibody is paired with the heavy chain of the full-length antibody into which the heavy chain Fab fragment has been inserted.

In one aspect, bispecific antibodies include

a) A full-length antibody that specifically binds to the 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, the Fab fragment comprising a VH2 domain and a VL2 domain containing a heavy chain fragment and a light chain fragment, wherein within the light chain fragment, the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of the antibody, and within the heavy chain fragment, the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of the 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 the 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 the full-length antibody, the light chain of the full-length antibody pairing with the heavy chain of the full-length antibody to which the heavy chain fragment of the Fab fragment is conjugated.

B. Administration of bispecific antibodies binding to PD-1 and LAG3

For the prevention or treatment of disease, the appropriate dosage of the bispecific antibody of the present invention, comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to LAG3 (when used alone or in combination with one or more additional therapeutic agents), will depend on the type of disease to be treated, the route of administration, the subject's weight, the type of fusion protein, the severity and progression of the disease, whether the bispecific antibody is administered for prophylactic or therapeutic purposes, prior or concurrent therapeutic interventions, the subject's clinical history and response to the fusion protein, and the judgment of the attending physician. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for the individual subject. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

Bispecific antibodies, as defined herein, comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, are appropriately administered to a subject at one time or in a series of treatments. Depending on the type and severity of the disease, a bispecific antibody at a dose of about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg–10 mg/kg) may be an initial candidate dose administered to the subject, for example, by a single or multiple administrations alone or by continuous infusion. Depending on the factors described above, a typical daily dose range may be about 1 μg/kg to 100 mg/kg or more. For repeated administrations over several days or longer, depending on the condition, treatment typically continues until the desired suppression of disease symptoms occurs. An exemplary dose range for bispecific antibodies is about 0.005 mg/kg to about 10 mg/kg. In other examples, the dosage may also include administration of 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 more, and any range derived therefrom. In examples of ranges derived from the figures listed herein, the dosage may be based on the figures above, ranging from 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. Therefore, subjects may be administered 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). Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., so that the subject receives about two to about twenty, or for example, about six, doses of the fusion protein). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. Progression of the therapy can be easily monitored using routine techniques and assays.

In one particular aspect, a bispecific antibody targeting PD-1 and LAG3 is administered to subjects every three weeks (Q3W) at a fixed dose of approximately 600 mg, for example, at a fixed dose of 600 mg Q3W.

In another aspect, subjects were administered a bispecific antibody targeting PD-1 and LAG3 every three weeks at a fixed dose of approximately 1200 mg, for example, at a fixed dose of 1200 mg Q3W.

In another aspect, subjects are given a bispecific antibody targeting PD-1 and LAG3 every two weeks (Q2W) at a fixed dose of approximately 2100 mg, for example, at a fixed dose of 2100 mg Q2W.

IX. VEGF antagonists

VEGF antagonists include any molecule capable of binding to VEGF, reducing or neutralizing VEGF expression levels, blocking, inhibiting, eliminating, reducing or interfering with VEGF biological activity. An example of human VEGF is shown in UniProtKB/Swiss-Prot accession number P15692, gene ID (NCBI): 7422.

In some cases, VEGF antagonists are anti-VEGF antibodies. In some embodiments, the anti-VEGF antibody is bevacizumab, also known as "rhuMab VEGF" or bevacizumab as a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al. (Cancer Res. 57:4593-4599, 1997). It comprises a mutated human IgG1 framework region and an antigen-binding complementarity-determining region derived from mouse anti-hVEGF monoclonal antibody A.4.6.1 that blocks the binding of human VEGF to its receptor. Approximately 93% of the amino acid sequence of bevacizumab, including the majority of the framework region, is derived from human IgG1, and approximately 7% of the sequence is derived from mouse 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 U.S. Patent No. 6,884,879, issued February 26, 2005, the entire disclosure of which is expressly incorporated herein by reference.

Other preferred antibodies include G6 or B20 series antibodies (e.g., G6-31, B20-4.1), as described in PCT application publication number WO 2005/012359. For other preferred antibodies, see U.S. Patent Nos. 7,060,269, 6,582,959, 6,703,020; 6,054,297; WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1; U.S. Patent Application Publication Nos. 2006009360, 20050186208, 20030206899, 20030190317, 20030203409 and 20050112126; and Popkov et al. (Journal of Immunological Methods 288:149-164, 2004). Other preferred antibodies include antibodies that bind to functional epitopes on human VEGF containing residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104, or alternatively, containing residues F17, Y21, Q22, Y25, D63, 183, and Q89.

In other cases, 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), or ziv-aflibercept (VEGFTrap); a bispecific VEGF antibody (e.g., MP-0250, vanucizumab (VEGF-ANG2), or the bispecific antibody disclosed in US2001/0236388); a bispecific antibody comprising a combination of two of the anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms; an anti-VEGFA antibody (e.g., bevacizumab, sevacizumab); an anti-VEGFB antibody; an anti-VEGFR2 ... VEGFC antibodies (e.g., VGX-100); anti-VEGFD antibodies; or non-peptide small molecule VEGF antagonists (e.g., pazopanib, axitinib, vandetanib, stivarga, cabozantinib, lenvatinib, nintedanib, orantinib, telatinib, dovitinig, cediranib, motesanib, sulfatinib, apatinib, foretinib, famitinib, or tivozanib). In some examples, VEGF antagonists can be tyrosine kinase inhibitors, including receptor tyrosine kinase inhibitors (e.g., multi-target receptor tyrosine kinase inhibitors, such as sunitinib or axitinib).

X.PD-1 axis-binding antagonist

PD-1 axis binding antagonists may include PD-L1 binding antagonists, PD-1 binding antagonists, and PD-L2 binding antagonists. Any suitable PD-1 axis binding antagonist may be used to treat subjects with cancer.

A. PD-L1 binding antagonists

In some cases, PD-L1 binding antagonists inhibit the binding of PD-L1 to one or more of its ligand-binding partners. In other cases, PD-L1 binding antagonists inhibit the binding of PD-L1 to PD-1. In still other cases, PD-L1 binding antagonists inhibit the binding of PD-L1 to B7-1. In some cases, PD-L1 binding antagonists inhibit the binding of PD-L1 to both PD-1 and B7-1. PD-L1 binding antagonists can be, but are not limited to, antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, or small molecules. In some cases, PD-L1 binding antagonists are small molecules that inhibit PD-L1 (e.g., GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK041). In some cases, PD-L1 binding antagonists are small molecules that inhibit both PD-L1 and VISTA. In some cases, the PD-L1 binding antagonist is CA-170 (also known as AUPM-170). In some cases, the PD-L1 binding antagonist is a small molecule that inhibits both PD-L1 and TIM3. In some cases, this small molecule is a compound described in WO 2015/033301 and WO 2015/033299.

In some cases, PD-L1 binding antagonists are anti-PD-L1 antibodies. Various anti-PD-L1 antibodies are considered and described herein. In any case herein, isolated anti-PD-L1 antibodies can bind to human PD-L1 (e.g., human PD-L1 as shown in UniProtKB/Swiss-Prot accession number Q9NZQ7-1, or a variant thereof). In some cases, anti-PD-L1 antibodies are capable of inhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In some cases, anti-PD-L1 antibodies are monoclonal antibodies. In some cases, anti-PD-L1 antibodies are antibody fragments selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab') ₂ fragments. In some cases, anti-PD-L1 antibodies are humanized antibodies. In some cases, anti-PD-L1 antibodies are human antibodies. Exemplary anti-PD-L1 antibodies include atezolizumab, MDX-1105, MEDI4736 (dvorumab), MSB0010718C (avimab), SHR-1316, CS1001, envorimab, TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, cochilimumab, lodalimumab, 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 that can be used in the methods of the present invention and methods for their preparation are described in International Patent Application Publication No. WO2010/077634 and U.S. Patent No. 8,217,149, each of which is incorporated herein by reference in its entirety.

In some cases, anti-PD-L1 antibodies contain:

(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) The 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).

In one embodiment, the anti-PD-L1 antibody comprises:

(a) Heavy chain variable region (VH) containing the following amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS(SEQ ID NO:70), and

(b) Light chain variable region (VL) containing the following amino acid sequence:

DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:71).

In some cases, the anti-PD-L1 antibody comprises (a) a VH containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:9 (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) or the sequence of SEQ ID NO:9; (b) a VL containing an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:10 (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) or the sequence of SEQ ID NO:10; or (c) a VH as in (a) and a VL as in (b).

In one embodiment, the anti-PD-L1 antibody comprises atezolizumab, which includes:

(a) The following heavy chain amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:62), and

(b) The following light chain amino acid sequence:

DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:63).

In some cases, the anti-PD-L1 antibody is avermab (CAS Registry No.: 1537032-82-8). Avermab, also known as MSB0010718C, is a human monoclonal IgG1 anti-PD-L1 antibody (Merck KGaA, Pfizer).

In some cases, the anti-PD-L1 antibody is durvalumab (CAS Registry No.: 1428935-60-7). Durvalumab, also known as MEDI4736, is the Fc-optimized human monoclonal IgG1κ anti-PD-L1 antibody described in WO 2011/066389 and US 2013/034559 (MedImmune, AstraZeneca).

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

In some cases, the anti-PD-L1 antibody is LY3300054 (Eli Lilly).

In some cases, the anti-PD-L1 antibody is STI-A1014 (Sorrento Biosciences). STI-A1014 is a human anti-PD-L1 antibody.

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

In some cases, anti-PD-L1 antibodies contain a cleavable portion or linker that, when cleaved (e.g., by proteases in the tumor microenvironment), activates the antibody antigen-binding domain to bind its antigen, for example, by removing the non-binding spatial portion. In some cases, the anti-PD-L1 antibody is CX-072 (CytomX Therapeutics).

In some cases, anti-PD-L1 antibodies comprise six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and/or heavy chain variable domains and light chain variable domains from the anti-PD-L1 antibodies described in the following patents: US20160108123, WO 2016/000619, WO 2012/145493, US Patent No. 9,205,148, WO 2013/181634, or WO 2016/061142.

In a more specific context, anti-PD-L1 antibodies exhibit reduced or minimal effector function. More specifically, minimal effector function arises from "effectless Fc mutations" or glycosylation-free mutations. In a further specific case, the effectless Fc mutation is an N297A or D265A/N297A substitution in the constant region. In a further specific case, the effectless Fc mutation is an N297A substitution in the constant region. In some cases, isolated anti-PD-L1 antibodies are glycosylated. Antibody glycosylation is typically N-linked or O-linked. N-linking refers to the carbohydrate moiety being linked to the asparagine residue side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic linking of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-glycosylation refers to the linkage of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used). Glycosylation sites can be conveniently removed from antibodies by altering the amino acid sequence to remove one of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Mutation can be achieved by substituting an asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conserved substitution).

B. PD-1 binding antagonists

In some cases, PD-1 axis binding antagonists are PD-1 binding antagonists. For example, in some cases, PD-1 binding antagonists inhibit the binding of PD-1 to one or more of its ligand-binding partners. In some cases, PD-1 binding antagonists inhibit the binding of PD-1 to PD-L1. In other cases, PD-1 binding antagonists inhibit the binding of PD-1 to PD-L2. In still other cases, PD-1 binding antagonists inhibit the binding of PD-1 to both PD-L1 and PD-L2. PD-1 binding antagonists can be, but are not limited to, antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, or small molecules. In some cases, PD-1 binding antagonists are immunoadhesins (e.g., immunoadhesins containing an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). For example, in some cases, PD-1 binding antagonists are Fc fusion proteins. In some cases, the PD-1 binding antagonist is AMP-224. AMP-224, also known as B7-DCIg, is the PD-L2-Fc fusion soluble receptor described in WO 2010/027827 and WO 2011/066342. In some cases, the PD-1 binding antagonist is a peptide or a small molecule compound. In some cases, the PD-1 binding antagonist is AUNP-12 (Pierre Fabre/Aurigene). See, for example, WO 2012/168944, WO 2015/036927, WO 2015/044900, WO 2015/033303, WO 2013/144704, WO 2013/132317 and WO 2011/161699. In some cases, PD-1 binding antagonists are small molecules that inhibit PD-1.

In some cases, the PD-1 binding antagonist is an anti-PD-1 antibody. A variety of anti-PD-1 antibodies can be used in the methods and uses disclosed herein. In any of the cases herein, the PD-1 antibody can bind to human PD-1 or a variant thereof. In some cases, the anti-PD-1 antibody is a monoclonal antibody. In some cases, 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 cases, the anti-PD-1 antibody is a humanized antibody. In other cases, the anti-PD-1 antibody is a human antibody. Exemplary anti-PD-1 antagonist antibodies include nivolumab, pembrolizumab, MEDI-0680, PDR001 (spartazumab), REGN2810 (cimiprizumab), BGB-108, palolizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dotalimab, rivanlimab, sazasanlimab, penaplimab, CS1003, HLX10, SCT-I10A, cepalimumab, baritelimab, genolimab, BI 754091, cilimab, YBL-006, BAT1306, HX008, bouglimab, and AMG. 404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM 388D4, STI-1110, AK-103 and hAb21.

In some cases, the anti-PD-1 antibody is nivolumab (CAS Registry No.: 946414-94-4). Nivolumab (Bristol-Myers Squibb/Ono Pharmaceutical (Ono)) is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and is the anti-PD-1 antibody described in WO 2006/121168.

In some cases, the anti-PD-1 antibody is pembrolizumab (CAS Registry No.: 1374853-91-4). Pembrolizumab (Merck) is also known as MK-3475, Merck 3475, pembrolizumab, SCH-900475, and is the anti-PD-1 antibody described in WO 2009/114335.

In some cases, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is PDR001 (CAS registration 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.

In some cases, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is BGB-108 (BeiGene).

In some cases, the anti-PD-1 antibody is BGB-A317 (BeiGene).

In some cases, the anti-PD-1 antibody is JS-001 (Shanghai Junshi Biosciences). JS-001 is a humanized anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is STI-A1110 (Sorrento Biosciences). STI-A1110 is a human anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD-1 antibody.

In some cases, the anti-PD-1 antibody is PF-06801591 (Pfizer).

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

In some cases, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).

In some cases, 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 preventing PD-L1 from binding to PD-1.

In some cases, 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.

In some cases, anti-PD-1 antibodies comprise six HVR sequences (e.g., three heavy chain HVRs and three light chain HVRs) and/or heavy chain variable domains and light chain variable domains from the anti-PD-1 antibodies described in the following patents: WO 2015/112800, WO 2015/112805, WO 2015/112900, US20150210769, WO2016/089873, WO 20 15/035606、WO 2015/085847、WO 2014/206107、WO 2012/145493、US 9,205,148、WO 2015/119 930, WO 2015/119923, WO 2016/032927, WO 2014/179664, WO 2016/106160 and WO 2014/194302.

In a more specific context, anti-PD-1 antibodies exhibit reduced or minimal effector function. More specifically, minimal effector function arises from "effectless Fc mutations" or glycosylation-free mutations. In a further specific case, the effectless Fc mutation is an N297A or D265A/N297A substitution in the constant region. In some cases, the isolated anti-PD-1 antibody is glycosylated.

C. PD-L2 binding antagonists

In some cases, PD-1 axis binding antagonists are PD-L2 binding antagonists. In other cases, PD-L2 binding antagonists are molecules that inhibit the binding of PD-L2 to its ligand-binding partner. In specific instances, the PD-L2 binding ligand partner is PD-1. PD-L2 binding antagonists can be, but are not limited to, antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, or small molecules.

In some cases, the PD-L2 binding antagonist is an anti-PD-L2 antibody. In any of the cases described herein, the anti-PD-L2 antibody may bind to human PD-L2 or a variant thereof. In some cases, the anti-PD-L2 antibody is a monoclonal antibody. In some cases, 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 cases, the anti-PD-L2 antibody is a humanized antibody. In other cases, the anti-PD-L2 antibody is a human antibody. In a more specific aspect, the anti-PD-L2 antibody has reduced or minimal effector function. In a more specific aspect, minimal effector function arises from an "effectless Fc mutation" or a glycosylation-free mutation. In a more specific case, the effectless Fc mutation is an N297A or D265A/N297A substitution in the constant region. In some cases, the isolated anti-PD-L2 antibody is glycosylated.

XI. Pharmaceutical compositions and formulations

This disclosure also provides pharmaceutical compositions and formulations comprising a bispecific antibody targeting PD-1 and LAG3 and an optional pharmaceutical carrier. This 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 an optional pharmaceutical carrier; and (ii) pharmaceutical compositions and formulations comprising an anti-TIGIT antagonist antibody and a bispecific antibody targeting PD-1 and LAG3 and an optional pharmaceutical carrier. Pharmaceutical compositions and formulations of bispecific antibodies targeting PD-1 and LAG3 and/or other agents described herein (e.g., dexamethasone) can be prepared as lyophilized formulations or aqueous solutions by mixing one or more agents having the desired purity with one or more optional pharmaceutical carriers (Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). In some embodiments, motuzumab is formulated for subcutaneous administration. In some embodiments, motuzumab is formulated for intravenous administration.

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

An exemplary tislelizumab formulation comprises a histidine solution containing polysorbate 20, sucrose, L-methionine, and WFI. tislelizumab can be provided in 15 mL vials containing 10 mL of the tislelizumab drug product, with an approximate antibody concentration of 60 mg/mL.

Pharmaceutical carriers are generally non-toxic to the treated individual at the doses and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); and low molecular weight (less than about 10 residues). Polypeptides; 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers described herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006/044908, the latter of which comprises histidine-acetate buffer.

The formulations described herein may also contain more than one active ingredient essential for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. For example, it may be desirable to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitors, and/or anti-hormonal agents, such as those described above). Such active ingredients are suitably present in a combination of amounts effective for the intended purpose.

The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules) prepared by, for example, cohesive techniques or interfacial polymerization; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980).

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being in the form of a molded article, such as a membrane or microcapsule.

Formulations intended for internal administration are typically sterile. For example, sterility can be easily achieved through filtration using a sterile filter membrane.

XII. Products or reagent kits

A. Kit including bispecific antibodies targeting PD-1 and LAG3 and anti-TIGIT antagonist antibodies.

In another aspect, this document provides products or kits comprising bispecific antibodies targeting PD-1 and LAG3 and anti-TIGIT antagonist antibodies (e.g., tislelizumab). In some cases, the products or kits further include a packaging insert containing instructions on using the combination of an anti-TIGIT antagonist antibody and a bispecific antibody targeting PD-1 and LAG3 to treat a subject's cancer or delay cancer progression in a subject. The products or kits may include any of the bispecific antibodies targeting PD-1 and LAG3 and/or any of the anti-TIGIT antagonist antibodies described herein.

In another embodiment of the invention, a kit is provided comprising a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody being used in combination with an anti-TIGIT antagonist antibody according to any of the methods described herein to treat a subject with cancer. In some cases, the kit further comprises an anti-TIGIT antagonist antibody. In some cases, the article or kit further comprises a packaging insert containing instructions on using a combination of a bispecific antibody targeting PD-1 and LAG3 with an anti-TIGIT antagonist antibody (e.g., tislelizumab) to treat a subject's cancer or delay cancer progression in a subject.

In some cases, bispecific antibodies targeting PD-1 and LAG3 and anti-TIGIT antagonist antibodies are contained in the same container or in separate containers. Suitable containers include, for example, vials, bottles, bags, and syringes. Containers can be formed from a variety of materials, such as glass, plastics (e.g., polyvinyl chloride or polyolefins), or metal alloys (e.g., stainless steel or Hastelloy). In some cases, the container holds the formulation, and a label on or associated with the container may indicate instructions for use. The product or kit may also include other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. In some cases, the product further includes one or more other pharmaceutical agents (e.g., additional chemotherapeutic agents and antitumor agents). Suitable containers for one or more reagents include, for example, vials, bottles, bags, and syringes.

The product or kit may include any of the bispecific antibodies targeting PD-1 and LAG3 described herein and/or an anti-TIGIT antagonist antibody. Either the product or kit may include instructions for administering the bispecific antibody targeting PD-1 and LAG3 and/or the anti-TIGIT antagonist antibody to a subject according to any method described herein, such as any method detailed in Section III above.

B. Kits including bispecific antibodies targeting PD-1 and LAG3 and anti-VEGF antibodies.

In another aspect, this document provides products or kits comprising bispecific antibodies targeting PD-1 and LAG3 and anti-VEGF antibodies (e.g., bevacizumab). In some cases, the products or kits further include a packaging insert containing instructions on using the combination of an anti-VEGF antibody and a bispecific antibody targeting PD-1 and LAG3 to treat a subject's cancer or delay cancer progression in a subject. The products or kits may include any of the bispecific antibodies targeting PD-1 and LAG3 and/or any of the anti-VEGF antibodies described herein.

In another embodiment of the invention, a kit is provided comprising a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody being used in combination with an anti-VEGF antibody according to any of the methods described herein to treat a subject with cancer. In some cases, the kit further comprises an anti-VEGF antibody. In some cases, the article or kit further comprises a packaging insert containing instructions on using the combination of the bispecific antibody targeting PD-1 and LAG3 with an anti-VEGF antibody to treat a subject's cancer or to delay the progression of the subject's cancer.

In some cases, bispecific antibodies targeting PD-1 and LAG3 and anti-VEGF antibodies are contained in the same container or in separate containers. Suitable containers include, for example, vials, bottles, bags, and syringes. Containers can be formed from a variety of materials, such as glass, plastics (e.g., polyvinyl chloride or polyolefins), or metal alloys (e.g., stainless steel or Hastelloy). In some cases, the container holds the formulation, and a label on or associated with the container may indicate instructions for use. The product or kit may also include other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. In some cases, the product further includes one or more other pharmaceutical agents (e.g., additional chemotherapeutic agents and antitumor agents). Suitable containers for one or more reagents include, for example, vials, bottles, bags, and syringes.

The product or kit may include any of the bispecific antibodies targeting PD-1 and LAG3 and/or any of the anti-VEGF antibodies described herein. Either the product or kit may include instructions for administering the bispecific antibody targeting PD-1 and LAG3 and/or the anti-VEGF antibody to a subject according to any method described herein, such as any method detailed in Section III above.

Example

Example 1: A phase Ib/II, open-label, multicenter, randomized umbrella study evaluating the efficacy and safety of multiple treatment combinations in patients with melanoma.

Melanoma is a potentially fatal form of 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 are diagnosed with melanoma globally each year, and 57,000 die from the disease. Clinical outcomes for patients with melanoma are highly dependent on the stage at presentation. Most people presenting with more advanced melanoma have a poorer prognosis (Finn et al., BMC Med. 10:23, 2012). Patients with lymph node metastasis (stage III) have a high risk of local and distant recurrence after surgery, and the 5-year survival rate in this patient group is 32%–93% (Gershenwald et al., CA Cancer J Clin. 67:472-492, 2017). A very small number of patients present with metastatic disease (stage IV), but some develop metastases after their initial definitive treatment. Immunotherapy and targeted therapy have improved outcomes for these patients, with a 5-year survival rate 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(Supplement 15):10013, 2020). Despite recent advances in treatment, melanoma remains a serious health problem with high medical demand, and its incidence has steadily increased over the past 30 years (Bataille. Expert Rev Dermatol. 4:533-539, 2009).

BO43328 is a phase Ib/II, open-label, multicenter, randomized, umbrella study in patients with resectable stage III (cohort 1) or stage IV (cohort 2) melanoma. The study is designed to be flexible enough to open new treatment arms when new therapies become available, close existing treatment arms that demonstrate minimal clinical activity or unacceptable toxicity, adjust patient cohorts (e.g., based on prior anticancer treatment or biomarker status), or introduce additional cohorts of patients with other types of melanoma.

A. Overview of Research Design

This study evaluated the efficacy, safety, and pharmacokinetics of the treatment combination in patients with resectable stage III melanoma who had not received cancer immunotherapy (CIT) (cohort 1) and in patients with stage IV melanoma (cohort 2). The specific study objectives and corresponding endpoints for cohort 1 (see Table 5) and cohort 2 (see Table 6) are summarized below.

Table 5. Goals and corresponding endpoints of queue 1

ADA = Anti-drug antibody; ASTCT = American Society for Transplantation and Cell Therapy; 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 Standard 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 = Standards for Evaluation of Treatment in Solid Tumors, version 1.1; RFS = Recurrence-free survival.

Table 6. Goals and corresponding endpoints of queue 2

ADA = Anti-drug antibody; ASTCT = American Society for Transplantation and Cell Therapy; CR = Complete response; CRS = Cytokine release syndrome; DOR = Duration of response; iRECIST = RECIST v1.1 revised for immunotherapy; NCI CTCAE v5.0 = National Cancer Institute Common Terminology Standard 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 = Standards for evaluating efficacy in solid tumors, version 1.1.

Note: Researchers used RECIST v1.1 to assess the overall response at a single time point.

This study included two cohorts. Cohort 1 included patients with resectable stage III melanoma who had measurable lymph node metastases that could be biopsied (according to RECIST v1.1 criteria for efficacy evaluation of solid tumors), who had no history of transitional metastasis in the past 6 months, and who had not received systemic CIT for their disease, such as PD-1/PD-L1 and/or CTLA-4 blockers or other agents.

Cohort 2 included patients with stage IV melanoma who had experienced disease progression during or after at least one, but no more than two, lines of treatment for metastatic disease. A maximum of two lines of checkpoint inhibitor therapy (monotherapy or combination therapy) were permitted. Patients with BRAF-mutant disease may have already received an additional line of targeted therapy (before, between, or after checkpoint inhibitor therapy), or may have simultaneously received targeted therapy and checkpoint inhibitor therapy as a combination therapy.

Treatment allocation

In cohort 1, patients were randomly assigned to either the control arm (nivolumab plus ipilimumab (Nivo+Ipi)) or the experimental arm consisting of RO7247669 (a bispecific antibody binding to PD-1 and LAG3), a combination of atezolizumab and tirelinumab (Atezo+Tira), or a combination of RO7247669 and tirelinumab (RO7247669+Tira). Patients were stratified by geographic region (Australia vs. the rest of the world) and baseline LDH (≤Upper Limit of Normal (ULN) vs. >ULN). Detailed information on the treatment regimens is provided in Table 7 and Figure 1.

In cohort 2, patients were enrolled in the experimental arm consisting of the combination of RO7247669 and tislelizumab (RO7247669+Tira). Enrollment began with a safety introductory period of 6 patients.

Approximately 61–191 patients were enrolled during the study, including about 6 patients enrolled in the safety introductory phase of cohort 2. Enrollment for each experimental arm was conducted in two phases: the initial phase and the expansion phase. Approximately 15–20 patients were enrolled in each treatment arm during the initial phase. If clinical activity (pathological remission in cohort 1) was observed in the experimental arm during the initial phase, approximately 20 additional patients could be enrolled in that arm during the expansion phase.

The organizer may decide to delay or suspend enrollment for a given treatment arm. Experimental arms with insufficient clinical activity or unacceptable toxicity will not be expanded. Additional patients may be included to ensure a balance between treatment arms in terms of demographics and baseline characteristics, including potential predictive biomarkers, for further subgroup analyses.

The randomization rate depends on the number of available experimental arms (e.g., if an arm is added or enrollment in an arm is paused pending preliminary phase results analysis), where the probability of being assigned to the control arm is specified to be no more than 35%. Randomization takes into account arm-specific exclusion criteria. If a patient meets any exclusion criteria outlined for that arm, they are not eligible for inclusion in that particular arm.

Table 7 provides details of the treatment plan.

Table 7. Treatment Plan

^The organizer may decide to delay or suspend enrollment for a given treatment arm. Therefore, all experimental arms may not be open for enrollment simultaneously.

During the ^safety introduction phase, patients are assigned to available treatment arms. The treatment allocation ratio depends on the number of open-enrollment experimental arms.

^The randomization ratio depends on the number of experimental arms in the open randomization (e.g., if an arm is added or randomization of an arm is paused to await preliminary results analysis), where the probability of being allocated to the control arm is specified to be no more than 35%.

^d. If clinical activity is observed in the experimental arm during the initial 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 be expanded.

Enrollment was paused in the ^e- cohort 1 RO7247669, Atezo+Tira and RO7247669+Tira arms to allow for safety assessment of at least 6 patients.

^After safety assessment of the treatment combination in cohort 2, open enrollment was conducted for RO7247669+Tira arm in cohort 1.

In cohort 1, patients in both the control and experimental arms received neoadjuvant therapy over a 6-week period. Upon completion of neoadjuvant therapy, or if discontinued due to toxicity and without disease progression, patients underwent surgery (complete lymph node dissection (CLND)) at week 7. Outside of this study, patients were subsequently either initiated adjuvant therapy or observed at week 13, based on the researchers' judgment (Figure 2).

Because immune cell infiltration can lead to an initial increase in metastatic lymph node size in the context of a T-cell response to CIT (known as pseudoprogression), suspected clinical or radiographic progression according to RECIST v1.1 may not indicate true disease progression. Patients who meet the RECIST v1.1 criteria for disease progression during CIT treatment are permitted to continue study treatment until surgery, provided there are no unacceptable toxicities. Progression is confirmed by additional expert reviewers via biopsy or repeat radiographic evaluation before discontinuation of study treatment and/or cancellation of surgery. All patients are expected to proceed with surgery if there are no distant metastases and the surgeon believes the disease can be completely removed.

In cohort 2, patients continued treatment until unacceptable toxicity or loss of clinical benefit was determined by investigators based on a comprehensive assessment of radiographic and biochemical data, local biopsy results (if any), and clinical status (e.g., worsening symptoms, such as disease-related pain). Because immune cell infiltration can lead to an initial increase in tumor burden in the context of a T-cell response to atezolizumab and other CITs (known as pseudoprogression), radiographic progression according to RECIST v1.1 may not indicate true disease progression. In the absence of unacceptable toxicity, patients who met the RECIST v1.1 criteria for disease progression during treatment with combination CITs were permitted to continue treatment if all of the following criteria were met:

• Evidence of clinical benefit, such as that determined by researchers after reviewing all available data.

• There are no symptoms or signs indicating clear disease progression (including laboratory values such as new or worsening hypercalcemia).

• There were no Eastern Cooperative Oncology Group (ECOG) performance status declines attributable to disease progression.

• No tumor progression in key anatomical sites (e.g., leptomeningeal diseases) where medical interventions permitted by the protocol cannot resolve the issue.

Researchers inform patients who meet the post-progression treatment criteria 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. Furthermore, researchers have the right to withdraw patients from the study if researchers or the sponsor determine that the patient's continued participation in the study may jeopardize the patient's safety.

If subsequent tumor assessments rule out pseudoprogression and confirm disease progression, the patient's study treatment will be discontinued.

Security assessment period (cohort 1)

To assess the toxicity of the experimental treatment in the neoadjuvant setting, enrollment was paused after approximately six patients had been enrolled to allow for a safety assessment. The safety assessment was based on safety data from a minimum of six patients who had received at least one dose of treatment (i.e., one dose of each agent in a given combination) and completed a safety follow-up assessment up to surgery. Notably, timely surgery (CLND) is an indicator of treatment tolerability. Enrollment for that combination was paused if ≥30% of patients experienced one or more of the following events deemed at least likely to be related to the study treatment during the safety assessment of the six patients, or at any time after the safety assessment, while the sponsor assessed the benefit-risk profile of the treatment:

• Treatment-related ≥ grade 3 adverse events that do not improve to grade 2 or better within 2 weeks (regardless of whether treatment was received).

• Treatment-related adverse events that cause surgery to be delayed by more than 2 weeks.

• Treatment-related serious adverse events.

• Treatment-related adverse events requiring permanent discontinuation of the investigational drug.

• Death, except for death that is indisputably related to the progression of disease or external causes such as accidents.

If no new safety signals are detected, enrollment in that arm will resume.

Safety introduction phase (Queue 2)

To assess the safety and tolerability of the novel combination in its first clinical trial, an initial safety lead-in period was implemented in cohort 2. Approximately six patients with metastatic disease were treated with the novel combination (i.e., RO7247669 + Tira) and safety and tolerability were assessed for at least 28 days.

At least six patients in Cohort 2 must complete the initial safety introductory period. If the RO7247669+Tira combination is determined to be tolerable, enrollment for the initial phase can be opened, and enrollment for the RO7247669+Tira arm in Cohort 1 can also be opened. Patients in the safety introductory period are enrolled and treated sequentially, with at least a one-week interval between the first patient and the remaining patients.

The assessment was based on safety data from a minimum of six patients who had received at least one dose of treatment (i.e., one dose of each drug) and completed a safety follow-up assessment within at least 28 days. Enrollment for the combination was suspended if ≥30% of patients experienced one or more of the following events deemed at least potentially related to the study treatment, while the sponsor assessed the benefit-risk profile of the treatment:

• Treatment-related ≥ grade 3 adverse events that do not improve to grade 2 or better within 2 weeks (regardless of whether treatment was received).

• Treatment-related serious adverse events.

• Treatment-related adverse events requiring permanent discontinuation of the investigational drug.

• Death, except for death that is indisputably related to the progression of disease or external causes such as accidents.

If no new security signal is detected, the combination is also initiated in queue 1.

B. Study completion and duration

The study was defined as ending on the date the last patient completed their final visit, including survival follow-ups conducted by telephone or at the clinic. The total length of the study (from the screening of the first patient to the study endpoint) was expected to be approximately 5 years.

C. Basic Principles of Research Design

The basic principles of patient groups

Cohort 1 included patients with resectable stage III melanoma who had measurable lymph node metastases that could be biopsied (according to RECIST v1.1) and no history of migrating metastases in the past 6 months. Enrolled patients had not previously received immunotherapy for their disease.

The same patient population was included in the OpACIN and OpACIN-neo studies, including the PRADO extended cohort. These studies evaluated neoadjuvant (and adjuvant) combinations of nivolumab and ipilimumab in patients with resectable melanoma. Neoadjuvant therapy was found to have a statistically significant and clinically meaningful benefit compared to adjuvant therapy (Rozeman et al. Lancet Oncol. 20:948-960, 2019; Blank et al. J Clin Oncol. 38:15S, 2020; Rozeman et al. NatMed. 27:256-263, 2021). Furthermore, the safety profile of the treatment was found tolerable with an optimized treatment schedule.

Despite recent demonstrations of the benefits of checkpoint inhibition therapy, there remains a need for more effective (i.e., broader and deeper pathological responses in surgical samples) and better-tolerated treatment options for patients with resectable melanoma. The multiple treatment options in this study are expected to stimulate the immune system through various mechanisms. The aim is to extend the benefits of CIT to a larger population with resectable melanoma beyond current checkpoint inhibition methods.

Cohort 2 included patients with stage IV melanoma who had experienced disease progression during or after at least one, but no more than two, lines of treatment for metastatic disease. A maximum of two lines of checkpoint inhibitor therapy (monotherapy or combination therapy) were permitted. Patients with BRAF-mutant disease may have already received an additional line of targeted therapy (before, between, or after checkpoint inhibitor therapy), or may have simultaneously received targeted therapy and checkpoint inhibitor therapy as a combination therapy.

Novel combinations of compounds with clinical and/or biological principles of anti-melanoma activity that have not yet been clinically tested were investigated in Cohort 2. Importantly, for the individual compounds considered for use in the novel combinations, safety and tolerability had been established in other studies, and safe dosages and schedules were available. The Cohort 2 safety introductory phase assessed the safety of the novel combinations in terms of potential overlapping toxicities.

D. Inclusion criteria

Common inclusion criteria for queue 1 and queue 2

Patients must meet all of the following criteria to be eligible for entry into both Quota 1 and Quota 2:

• The person must be at least 18 years old when signing the informed consent form.

• Availability of representative tumor specimens suitable for determining PD-L1 and/or other biomarker status via central testing.

- Baseline tumor tissue samples were collected from all patients (except those in the safety introduction period of Cohort 2) by performing biopsies on metastatic lymph nodes (Cohort 1) or other metastatic lesions (Cohort 2) at screening.

- Additionally, if available, submit archived primary tumor tissue from all patients. Enrollment is permitted if no archived primary tissue is available (e.g., for patients with unknown primary tumors). For archived tissue, formalin-fixed, paraffin-embedded (FFPE) tumor samples (preferably) in paraffin blocks of sufficient size and representative of tumor contents, preferably including invasive margins, or at least 16 slides containing unstained, freshly cut, serial sections must be submitted with the relevant pathology report. If only 10 to 15 slides are available, the patient still qualifies for the study.

• Adequate blood and end-organ function, defined by the following laboratory test results obtained within 14 days prior to the initiation of study treatment: absolute neutrophil count (ANC) ≥1.5 × ^10⁹ /L (1500/μL); lymphocyte count ≥0.5 × ^10⁹ cells/L (500/μL) (marginalized lymphocyte counts can be confirmed by manual counting); platelet count ≥100 × 10⁹ ^/L. /L (100,000/μL); Hemoglobin ≥90 g/L (9 g/dL); AST, ALT, and ALP ≤2.5×ULN (AST and ALT can be ≤5×ULN for participants with a history of liver metastases; ALP can be ≤5×ULN for participants with a history of liver or bone metastases); Total bilirubin ≤1.5×ULN (bilirubin levels can be ≤3×ULN for patients with known Gilbert's disease); Creatinine ≤1.5×ULN or creatinine clearance ≥30 mL/min (calculated using the Cockcroft-Gault formula); Serum albumin ≥25 g/L (2.5 g/dL). INR and aPTT can be ≤1.5×ULN in patients not receiving therapeutic anticoagulation.

• For patients receiving therapeutic anticoagulation: a stable anticoagulation regimen (i.e., no new thrombosis, thromboembolic events, or bleeding events within 3 months prior to the start of study treatment).

• The HIV test was negative at the time of screening. Unless local regulations prohibit it, no patient who previously tested positive for HIV is tested for HIV at the time of screening.

• At screening, both the hepatitis B surface antibody (HBsAb) and total hepatitis B core antibody (HBcAb) tests must be negative. If the patient tests negative for hepatitis B surface antigen (HBsAg) and positive for total HBcAb at screening, a hepatitis B virus (HBV) DNA test must also be performed to rule out active HBV.

• The patient is negative for hepatitis C virus (HCV) antibodies at screening; or positive for HCV antibodies at screening but negative for HCV RNA afterwards. HCV RNA testing is performed only on patients who are positive for HCV antibodies.

• For women of childbearing age: Agree to abstain from sex (avoid heterosexual intercourse) or use contraception.

For men: consent to abstinence (avoiding heterosexual intercourse) or use of contraception, and consent not to donate sperm, as outlined in each specific treatment arm.

Selection criteria for queue 1

Patients must meet all of the following criteria to be eligible for entry into queue 1:

• ECOG Performance Status (PS) is 0 or 1.

• Histologically confirmed resectable stage III melanoma (T: T0, Tx, or T1-4; N: cN1-3, pN1b/2b/3b; M: M0, according to AJCC-8 (Gershenwald et al. CA Cancer J Clin. 67:472-492, 2017)) and no history of transition or metastasis in the past 6 months.

- Patients may present with primary melanoma with regional lymph node metastasis, or with a history of primary melanoma or an unknown primary melanoma and clinically detected regional lymph node recurrence, and may belong to any of the following groups: primary cutaneous melanoma with clinically/radiologically significant regional lymph node metastasis; recurrent melanoma with clinically/radiologically detected proximal regional lymph node depression; or clinically/radiologically detected lymph node melanoma originating from an unknown primary site (if at a single site).

• Suitable for and planned for CLND (assessed by a surgeon according to local guidelines prior to randomization).

• Measurable disease (at least one target lesion) according to RECIST v1.1. • At least one grossly visible lymph node metastasis to be biopsied (measurable according to RECIST v1.1).

Selection criteria for queue 2

Patients must meet all of the following criteria to be eligible for entry into Cohort 2:

• ECOGPS is 0, 1, or 2.

• Life expectancy ≥ 3 months, if determined by the researchers.

• Histologically confirmed stage IV (metastatic) melanoma, according to AJCC-8 (Gershenwald et al. CA Cancer J Clin. 67:472-492, 2017).

• Disease progression during or after at least one, but no more than two, lines of treatment for metastatic disease. A maximum of two lines of checkpoint inhibitor therapy (monotherapy or combination therapy) are permitted. Patients with BRAF-mutant disease may have already received an additional line of targeted therapy (before, between, or after checkpoint inhibitor therapy), or may have simultaneously received targeted therapy and checkpoint inhibitor therapy as a combination therapy.

Patients who relapse or experience systemic progression during or within 6 months of completing adjuvant therapy for local melanoma may be eligible.

• Measurable disease (at least one target lesion) according to RECIST v1.1.

At least one transfer (measurable according to RECIST v1.1).

E. Exclusion Criteria

If a patient meets any of the applicable criteria outlined in subsequent sections (as specified for the treatment arm below), the patient will be denied enrollment in that specific arm:

Exclusion criteria for queue 1 and queue 2

Patients meeting any of the following criteria will be excluded from the study:

• Mucosal and uveal melanoma.

- Cohort 1 excludes acral lentigines-like melanoma.

- For cohort 2, acral lentigines are permitted; however, the proportion of patients with acral lentigines should not exceed 20% of those evaluable for response.

• Treat with the investigational therapy within 28 days prior to the start of the investigational treatment.

• Treat with systemic immunostimulants (including, but not limited to, IFN and IL-2) within 4 weeks prior to the start of study treatment or within 5 drug elimination half-lives (whichever is longer).

• Previous allogeneic stem cell or solid organ transplantation.

• Patients with known immunodeficiency or conditions requiring systemic immunosuppressive agents (including but not limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-tumor necrosis factor-alpha agents), or those expected to require systemic immunosuppressive agents during the study treatment, with the following exceptions: Patients receiving alternative doses of corticosteroids for pituitary hypofunction or adrenal insufficiency are eligible for this study; patients receiving acute, low-dose, systemic immunosuppressive agents or a single pulse dose of systemic immunosuppressive agents (e.g., 48-hour corticosteroids for contrast agent allergy) are eligible after discussion with a medical monitor; patients receiving mineralocorticoids (e.g., fludrocortisone), corticosteroids for chronic obstructive pulmonary disease or asthma, or low-dose corticosteroids for orthostatic hypotension or adrenal insufficiency are eligible for this study.

• Treatment with a live attenuated vaccine within 4 weeks prior to the start of study treatment, or if such a vaccine is expected to be needed during study treatment or within 5 months after the last dose of study treatment.

• Active or history of autoimmune diseases or immunodeficiency, including but not limited to myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, antiphospholipid syndrome, Wegener's granulomatosis, Sjögren's syndrome, Guillain-Barré syndrome, or multiple sclerosis, except in the following cases:

Patients with a history of autoimmune-related hypothyroidism who are receiving a stable dose of thyroid replacement hormone are eligible for this study.

Patients with type 1 diabetes who are on a stable insulin regimen are eligible for this study.

Patients with eczema, psoriasis, chronic simple lichen simplex, or vitiligo with only dermatological manifestations (e.g., excluding patients with psoriatic arthritis) are eligible for this study if they meet all of the following criteria: the rash must cover <10% of the body surface area; the disease is well controlled at baseline and requires only low-potency topical corticosteroids; and there have been no acute exacerbations of the underlying condition requiring psoralen plus UVA radiation, methotrexate, retinoids, biologics, oral calcineurin inhibitors, or high-potency or oral corticosteroids within the past 12 months.

• A history of idiopathic pulmonary fibrosis, organizing pneumonia (e.g., bronchiolitis obliterans), drug-induced lung inflammation, or idiopathic lung inflammation, or evidence of active lung inflammation on a chest computed tomography (CT) scan. Patients with a history of CIT-related pneumonia <2 may qualify after discussion with a medical monitor.

• Patients with a history of malignant tumors other than malignant melanoma within 2 years prior to screening are excluded, provided that the risk of metastasis or death is negligible (e.g., 5-year OS rate >90%), such as well-treated cervical carcinoma in situ, non-melanoma skin cancer, localized prostate cancer, ductal carcinoma in situ, or stage I uterine cancer.

• Active pulmonary tuberculosis (TB).

• A serious infection occurred within 4 weeks prior to the initiation of the study treatment, including but not limited to hospitalization due to complications of infection, bacteremia, or severe pneumonia, or any active infection that the researchers believe may affect the patient's safety.

• Treat with therapeutic or prophylactic oral or IV antibiotics within 2 weeks prior to the start of the study treatment.

• Significant cardiovascular disease, such as New York Heart Association heart disease (Class II or higher), myocardial infarction or stroke within 3 months prior to the start of study treatment, unstable arrhythmia or unstable angina.

• Uncontrolled hypertension (defined as resting systolic blood pressure >150 mmHg and/or diastolic blood pressure >100 mmHg in two or more consecutive measurements).

• Had major surgery other than for diagnosis within 4 weeks prior to the start of the study treatment, or is expected to need major surgery other than CLND during the study period.

- Placing a central venous access catheter (e.g., port or similar catheter) is not considered a major surgery and is therefore permissible.

• Any other disease, metabolic disorder, physical examination findings, or clinical laboratory findings that preclude the use of the investigational drug, may affect the interpretation of the results, impair the patient's ability to participate in the study, or may place the patient at high risk of treatment complications.

• History of severe allergic reactions to chimeric antibodies, humanized antibodies, or fusion proteins.

• It is known to be hypersensitive to Chinese hamster ovary cell products or recombinant human antibodies.

• Known allergic or hypersensitive reactions to any investigational drug or its excipients.

• Known intolerance to any of the drugs required for prophylactic use (acetaminophen, ranitidine, diphenhydramine, and methylprednisolone).

• Pregnant or breastfeeding, or planning to become pregnant during the study.

-Women of childbearing potential must have a negative serum pregnancy test result within 14 days prior to the initiation of the study treatment.

• Meets the criteria for the control arm only.

Exclusion criteria for queue 1

Patients meeting any of the following criteria will be excluded from Cohort 1:

• Metastatic melanoma

• History of migration or relocation within the past 6 months

Previous radiation therapy

• Previous immunotherapies, including anti-CTLA-4, anti-PD-1, and anti-PD-L1 therapeutic antibodies, as well as other systemic therapies for melanoma.

Exclusion criteria for queue 2

Patients meeting any of the following criteria will be excluded from Cohort 2:

• Symptomatic, untreated, or progressive CNS metastases.

- Asymptomatic patients with treated CNS lesions are eligible if they meet all of the following criteria: measurable disease according to RECIST v1.1 must be outside the CNS; the patient has no history of intracranial or spinal hemorrhage; CNS metastases have been stable for ≥4 weeks prior to study initiation, or neurosurgical resection has occurred ≥28 days prior to study initiation; the patient does not require corticosteroids as therapy for CNS disease for at least 14 days prior to study initiation; and stable doses of anticonvulsant therapy are permitted.

• History of active carcinomatous meningitis/leptomeningeal disease or carcinomatous meningitis/leptomeningeal disease.

• Uncontrolled tumor-related pain. Patients requiring analgesia must be on a stable regimen at screening. Symptomatic lesions suitable for palliative radiotherapy (e.g., bone metastases or metastases causing nerve contact) should be treated prior to enrollment. Patients should have recovered from the effects of radiotherapy. A minimum recovery period is not required. Asymptomatic metastatic lesions that may lead to functional impairment or intractable pain if they continue to grow (e.g., epidural metastases currently unrelated to spinal cord compression) should be considered for localized treatment (if appropriate) prior to enrollment.

• Uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage (once a month or more frequently). Patients with indwelling catheters (e.g., [insert catheter here]) are permitted.

• Uncontrolled or symptomatic hypercalcemia (ionized calcium >1.5 mmol/L, calcium >12 mg/dL, or corrected calcium >ULN).

• Any history of an immune-mediated grade 4 adverse event attributable to a previous CIT (excluding endocrine disorders controlled by alternative therapy or asymptomatic elevation of serum amylase or lipase) that led to permanent discontinuation of the previous immunotherapy.

• All immune-mediated adverse events associated with previous immunomodulatory therapy that have not completely resolved to baseline (excluding endocrine disorders or stable vitiligo controlled by replacement therapy). Patients treated with corticosteroids for immune-mediated adverse events (excluding corticosteroid replacement therapy for adrenal insufficiency (provided the patient is receiving ≤10 mg prednisone/day or equivalent)) must not develop related symptoms or signs within ≥4 weeks after discontinuation of corticosteroids.

• Adverse events associated with any prior radiotherapy, chemotherapy, targeted therapy, CPI therapy, or surgery must have resolved to Grade 1 or better, except for alopecia (any grade) at stable doses of hormone replacement therapy (e.g., thyroxine, hydrocortisone, prednisolone, etc.), Grade 2 peripheral neuropathy, and hypothyroidism and/or hypopituitarism.

Exclusion criteria for arms containing RO7247669 (Queues 1 and 2)

Patients meeting any of the following criteria will be excluded from the arm containing RO7247669:

• Previously treated with anti-LAG-3 medications.

• History of myocarditis (regardless of the cause).

• Within 6 months prior to the start of the study treatment, the left ventricular ejection fraction (LVEF) was <50% as assessed by transthoracic echocardiography (TTE) or multi-gated acquisition (MUGA) scan (TTE's preferred test).

• Troponin T (TnT) or Troponin I (TnI) > institutional ULN. Patients with TnT or TnI levels between >1 and <2 × ULN are eligible if the repeat level over 24 hours is ≤1 × ULN. If the repeat level over 24 hours is between >1 and <2 × ULN, the patient requires cardiac evaluation, and treatment may be considered if no clinically significant findings are found.

Exclusion criteria for arms containing tislelizumab (cohort 1 and cohort 2)

Patients meeting any of the following criteria will be excluded from the arm containing tislelizumab:

• Previously treated with anti-TIGIT medications.

• Active Epstein-Barr virus (EBV) infection or known or suspected chronic active EBV infection at the time of screening. Patients who test positive for EBV viral capsid antigen (VCA) IgM at the time of screening should be excluded from the arm containing tisrelimumab. EBV PCR testing should be performed according to clinical indications to screen for active infection or suspected chronic active infection. Patients who test positive for EBV PCR should be excluded from the arm containing tisrelimumab.

F. Research on Treatment

The drugs studied in this study were atezolizumab, tirelinumab, RO7247669, nivolumab, and ipilimumab.

Control arm (nivolumab + ipilimumab)

Patients in the nivolumab plus ipilimumab (Nivo+Ipi) control arm received two cycles (6 weeks) of treatment as outlined in Table 8, until surgery, or until unacceptable toxicity or loss of clinical benefit occurs, whichever comes first. Treatment is recommended to begin no later than 7 days after randomization.

Table 8. Treatment regimens for nivolumab + ipilimumab arm

Nivolumab was administered via IV infusion at a dose of 3 mg/kg on day 1 of each 21-day cycle (Q3W). Ipilimumab was administered via IV infusion at a dose of 1 mg/kg on day 1 of each 21-day cycle (Q3W).

RO7247669 arm

Patients with RO7247669 arms should receive two cycles (6 weeks) of treatment as outlined in Table 9, until surgery, or until unacceptable toxicity or loss of clinical benefit occurs, whichever comes first. Treatment should be initiated no later than 7 days after randomization.

Table 9. Treatment plan for RO7247669 arm

RO7247669 was administered at a fixed dose of 2100 mg every 3 weeks (2100 mg on day 1 of each 21-day cycle). RO7247669 administration was carried out in a monitored environment with immediate access to trained personnel and adequate equipment and medication to manage potential critical reactions. RO7247669 infusions were administered according to the instructions outlined in Table 10.

Table 10. Administration of the first and second RO7247669 infusions

IRR = Infusion-related response.

For patients experiencing a Grade 2 infusion-related reaction (IRR), prophylactic administration of acetaminophen 500–1000 mg orally (PO) or intravenously and diphenhydramine 25–50 mg PO or intravenously (or an adequate dose of an alternative histamine H _1/2 antagonist) is required prior to subsequent infusions. If a Grade 3 or 4 IRR related to study treatment occurs, the patient should permanently discontinue study treatment.

Dosage adjustments for RO7247669 are not permitted.

Atezolizumab + Tirelimumab

Patients on the atezolizumab plus tireliumab (Atezo+Tira) arm will receive two cycles (6 weeks) of treatment as outlined in Table 11, until surgery, or until unacceptable toxicity or loss of clinical benefit occurs, whichever comes first. Treatment is recommended to begin no later than 7 days after randomization.

Table 11. Treatment regimen of atezolizumab + tirelinumab arm

Atezolizumab is administered at a fixed dose of 1200 mg every 3 weeks (Q3W) (1200 mg on day 1 of each 21-day cycle). Atezolizumab administration is performed in a monitored environment with immediate access to trained personnel and adequate equipment and medication to manage potential critical reactions. Administer atezolizumab infusions according to the instructions outlined in Table 12.

Dosage adjustments for atezolizumab are not permitted.

Table 12. Administration of the first and second atezolizumab infusions

IRR = Infusion-related response.

Tirelinumab is administered at a fixed dose of 600 mg IV Q3W (600 mg on day 1 of each 21-day cycle). Tirelinumab administration is performed in a monitored environment with immediate access to trained personnel and adequate equipment and medication to manage potential critical reactions. Administer tisrelinumab infusions according to the instructions outlined in Table 13.

Table 13. Administration of the first and subsequent tisrelimumab infusions

IRR = Infusion-related response.

In patients experiencing toxicities deemed relevant to the study treatment, atezolizumab and tislelizumab treatment may be temporarily suspended. If corticosteroid treatment toxicities are initiated, they must be gradually tapered down to an equivalent dose of ≤10 mg/day of oral prednisone over ≥1 month before study treatment can be resumed (if necessary) before medication can be restarted. In neoadjuvant settings, study treatment is limited to a 6-week preoperative window. Treatment should not be interrupted during this period unless the patient experiences toxicities. Atezolizumab and/or tislelizumab should be interrupted/discontinued if toxicities meet the criteria for interruption/discontinuation of atezolizumab and/or tislelizumab. After toxicities resolve, subsequent treatment cycles should only be considered if the benefit/risk profile is acceptable and surgery can be performed within 2 weeks of the planned date. Otherwise, subsequent treatment cycles should be omitted so that the patient can proceed directly to surgery without further delay.

Based on its existing mechanism of action, tisrelimumab may cause adverse events similar to, but independent of, atezolizumab. Tirelimumab may also exacerbate the frequency or severity of atezolizumab-related adverse events, or may have toxicities that do not overlap with atezolizumab. Because these conditions may be indistinguishable from each other in a clinical setting, adverse events should generally be attributed to both agents, and dose interruption or treatment discontinuation should be applied to both tisrelimumab and atezolizumab. If atezolizumab is discontinued or discontinued, tisrelimumab should also be discontinued or discontinued.

RO7247669 + tislelizumab (cohort 1 and cohort 2)

Patients receiving RO7247669 plus tireliumab (RO7247669+Tira) received the treatments outlined in Table 14.

Patients in Cohort 1 received two cycles (6 weeks) of treatment until surgery, or until unacceptable toxicity or loss of clinical benefit occurred, whichever came first.

Patients in Cohort 2 received treatment until unacceptable toxicity or loss of clinical benefit was determined by the investigators after a comprehensive assessment of imaging and biochemical data, local biopsy results (if any), and clinical status (e.g., worsening of symptoms, such as disease-related pain).

Treatment is recommended to begin no later than 7 days after randomization (cohort 1) or enrollment (cohort 2).

Table 14. Treatment regimen for RO7247669 + tislelizumab arm

Once ^a safety profile is established, the organizer may decide to explore lower doses (e.g., 1200 mg and 600 mg).

RO7247669 was administered via IV infusion at a fixed dose of 2100 mg on day 1 of each 21-day cycle. Tirelimumab was administered via IV infusion at a fixed dose of 600 mg on day 1 of each 21-day cycle, with a post-infusion observation period as described in Table 13.

RO7247669 administration is performed in a monitored environment with immediate access to trained personnel and adequate equipment and medication to manage potential critical reactions. RO7247669 infusion is administered according to the instructions outlined in Table 15.

Table 15. Administration of the first, second, and subsequent RO7247669 infusions

IRR = Infusion-related response.

For patients experiencing a grade ≥2 infusion-related reaction (IRR), prophylactic administration of acetaminophen 500–1000 mg orally (PO) or intravenously and diphenhydramine 25–50 mg PO or intravenously (or an adequate dose of an alternative histamine H _1/2 antagonist) is required before subsequent infusions. If a grade 3 or 4 IRR related to study treatment occurs, the patient should permanently discontinue study treatment.

Dosage adjustments to RO7247669 are not permitted. However, based on emerging safety and efficacy data, the sponsor may explore lower doses (e.g., 1200 mg and 600 mg). RO7247669 treatment may be discontinued for reasons other than toxicity (e.g., surgery).

Researchers and medical monitors will determine the acceptable length of treatment interruption.

In patients experiencing toxicities deemed relevant to the study treatment, treatment with RO7247669 and tislelizumab may be temporarily suspended. If corticosteroid treatment toxicities are initiated, the dose must be gradually tapered down to an equivalent dose of ≤10 mg/day of oral prednisone over ≥1 month before treatment can be resumed (if necessary).

For Cohort 1, in the neoadjuvant setting, study treatment was limited to a 6-week preoperative window. Treatment should not be interrupted during this period unless the patient experiences toxicity. If toxicity meets the criteria for discontinuing/stopping RO7247669 and tislelizumab, then RO7247669 and tislelizumab should be discontinued/stopped. After toxicity subsides, subsequent treatment cycles should only be considered if the benefit/risk profile is acceptable and surgery can be performed within 2 weeks of the planned date. Otherwise, subsequent treatment cycles should be omitted so that the patient can proceed directly to surgery without further delay.

For Cohort 2, if RO7247669 and tislelizumab are discontinued for 12 weeks or longer due to toxicity, patients should discontinue both RO7247669 and tislelizumab. However, RO7247669 and tislelizumab may be discontinued for more than 12 weeks to allow for gradual reduction of corticosteroids before resuming treatment. RO7247669 and tislelizumab treatment may be resumed after more than 12 weeks if the medical monitor agrees that the patient may receive clinical benefit. RO7247669 and tislelizumab treatment may be suspended for reasons other than toxicity (e.g., surgery). The acceptable length of any extension must be agreed upon by the investigator and the medical monitor.

Based on its existing mechanism of action, tisrelimumab may cause adverse events similar to, but independent of, RO7247669. Tirelimumab may also exacerbate the frequency or severity of adverse events associated with RO7247669, or may have toxicities that do not overlap with RO7247669. Because these situations may not be distinguishable from each other in a clinical setting, adverse events should generally be attributed to both agents, and dose interruption or treatment discontinuation should be applied to both tisrelimumab and RO7247669. If RO7247669 is discontinued or discontinued, tisrelimumab should also be discontinued or discontinued.

G. Companion Therapy

Concomitant therapy consists of any medications used by the patient other than the study treatments specified in the protocol (e.g., prescription drugs, over-the-counter drugs, vaccines, herbal or homeopathic medicines, nutritional supplements) from 7 days before the start of the study treatment until the end of the treatment visit.

Generally, in accordance with local nursing practice, researchers should manage patient care (including pre-existing conditions) using supportive therapy rather than therapies defined as cautious or contraindicated based on clinical indications. Patients experiencing infusion-related symptoms may be treated symptomatically with acetaminophen, ibuprofen, diphenhydramine, and/or H2 receptor antagonists (e.g., famotidine, cimetidine) or equivalents as per local standard practice. Serious infusion-related events presenting with dyspnea, hypotension, wheezing, bronchospasm, tachycardia, decreased oxygen saturation, or respiratory distress should be treated with supportive therapy as clinically indicated (e.g., oxygen supplementation and β2-adrenergic agonists).

Permissible therapies for RO7247669, Atezo+Tira, and RO7247669+Tira arms

Patients are permitted to use the following therapies during the study:

• Oral contraceptives with an annual failure rate of <1%.

Hormone replacement therapy.

• Prophylactic or therapeutic anticoagulation therapy (such as stable doses of warfarin or low molecular weight heparin).

• Inactivated vaccines (e.g., influenza vaccines).

• Administer medroxyprogesterone acetate as an appetite stimulant

• Mineralocorticoids (e.g., fludrocortisone)

• Administer corticosteroids for chronic obstructive pulmonary disease (COPD) or asthma.

• Administer low-dose corticosteroids for orthostatic hypotension or adrenal insufficiency.

• Local treatment (e.g., surgery (excluding complete lymph node dissection (CLND) and non-melanoma-specific procedures).

For the RO7247669 arm, at the discretion of the researchers, prophylactic administration of antihistamines, antipyretics, and/or analgesics may only be administered for the second RO7247669 infusion.

For the atezolizumab plus tirelinumab arm, at the discretion of the investigator, prophylactic administration of antihistamines, antipyretics, and/or analgesics may only be administered for the second atezolizumab and tirelinumab infusion.

Additional permissible therapy in cohort 2: RO7247669 + Tira arm

Patients are permitted to use the following therapies during the study:

• Palliative radiotherapy as outlined below (e.g., treatment of known bone metastases or relief of pain symptoms): Palliative radiotherapy is permitted as long as it does not interfere with the assessment of the tumor target lesion (e.g., the lesion to be irradiated is not the only site of measurable disease). Tirelimumab treatment may continue during palliative radiotherapy. RO7247669 treatment may continue during the following palliative periods, with one exception: palliative radiotherapy is not permitted on days when RO7247669 is administered.

• Local therapies outlined below (e.g., surgery, stereotactic radiosurgery, radiotherapy, radiofrequency ablation): Patients experiencing a mixed response requiring local therapy to control three or fewer lesions remain eligible to continue study treatment after obtaining approval from the medical monitor. Patients receiving local therapy targeting the lesion will no longer be evaluable for radiographic response, but progression can still be evaluated.

At the discretion of the researchers, prophylactic use of antihistamines, antipyretics, and/or analgesics may only be administered for the second and subsequent RO7247669 and tirelinumab infusions.

H. Assessment

Adverse events in all patients were closely monitored throughout the study. Adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0 (NCI CTCAE v5.0). The severity of cytokine release syndrome (CRS) was also graded according to the American Society for Transplantation and Cell Therapy (ASTCT) CRS Consensus Grading Scale.

Patients in Cohort 1 received two cycles (6 weeks) of neoadjuvant therapy and underwent surgery in week 7 (CLND). All patients are expected to proceed with surgery if there are no distant metastases and the surgeon deems the disease completely resectable. Pathological response was assessed through local, independent pathology review.

Patients who discontinued treatment due to unacceptable toxicity and continued treatment without evidence of metastatic disease were still eligible for surgery and would continue CLND after the adverse events had subsided and stage III disease was confirmed by re-staging. If disease progression was confirmed, patient management and treatment options were determined by the attending physician. These patients remained in the study for follow-up.

Patients underwent radiographic oncology assessment 6 weeks prior to surgery (starting on day 1 of cycle 1). Response was assessed and determined by investigators according to RECIST v1.1, but confirmation by subsequent imaging studies was not required.

Patients in Cohort 2 underwent tumor assessment every 9 weeks (starting on day 1 of Cycle 1) for the first 54 weeks, followed by assessment every 12 weeks. Response was assessed by investigators using RECIST v1.1. Response to RECIST v1.1 according to the modified immunotherapy-based treatment (iRECIST) was determined programmatically by the sponsor based on individual lesion data assessed by investigators.

For Cohort 1 and Cohort 2, if clinical activity is demonstrated in the experimental arm, the sponsor may require submission of a tumor assessment scan of that arm for evaluation by an independent review body.

Tumor and response assessment

All measurable and evaluable lesions should be assessed and documented at screening. Tumor assessments performed as standard of care before obtaining informed consent and within 14 days prior to randomization/enrollment do not need to be repeated at screening.

For both Cohort 1 and Cohort 2, all measurable and/or evaluable lesions identified at baseline should be reassessed in subsequent tumor evaluations. Subsequent tumor evaluations should use the same radiological procedures (e.g., the same contrast agent protocol as used for CT scans) used to assess the disease site at screening.

Cohort 1 Tumor and Response Assessment

The pathological and radiological responses of patients in Cohort 1 to treatment were assessed. Patients underwent pathological tumor evaluation at baseline and 6 weeks after surgery (CLND). Complete resection of stage III lymph nodes (CLND) at week 7 must be performed according to the appropriate surgical procedure standards for therapeutic lymph node dissection. If patients are receiving corticosteroids or other anti-inflammatory drugs to treat immune-mediated adverse events, CLND should be performed as planned, provided these drugs are administered at a stable or gradually decreasing dose and the severity of the adverse event is grade 2 or better. CLND may be delayed by up to 2 weeks if the treatment-related adverse events investigated at the time of surgery planning have not adequately improved. Pathological response was determined by local and independent pathological review according to the INMC guidelines (Tetzlaff et al., Ann Oncol. 29:1861-1868, 2018).

Surgical complications were scored according to the Clavien-Dindo classification. The complication rate for each level was reported and scored for patients undergoing CLND.

Patients underwent radiographic assessment according to RECIST v1.1 at baseline, 6 weeks after treatment prior to surgery (CLND), and at the completion/discontinuation of treatment at week 13.

Researchers used RECIST v1.1 to assess the overall response at individual time points.

Disease follow-up and confirmation of disease progression or recurrence

During neoadjuvant therapy, the diagnosis of disease progression should be confirmed by clinical, laboratory, radiological, and/or histological examination results. Following surgery, tumor assessment should be performed at week 13 before initiating adjuvant therapy or observation to mark the end of the neoadjuvant-surgical intervention window.

Subsequently, during the adjuvant therapy/observation phase outside the study (i.e., starting at week 13), all patients must be followed up to assess disease relapse and survival for each arm.

Patients who complete treatment will have their first survival follow-up 3 months after surgery. Patients who prematurely discontinue the study drug will have their first survival follow-up 3 months after the last study dose. Disease recurrence, whether local, regional, or distant, can only be specified when the diagnosis is confirmed by clinical, laboratory, radiological, and/or histological findings.

During the postoperative period, the disease status should be clinically assessed and documented according to institutional guidelines (e.g., every 3 months for the first 2 years; every 6 months for the third year; and annually from the fourth year onwards). In addition, when clinical evidence suggests the exclusion of metastatic disease, liver function tests, bone scans, chest X-rays/diagnostic CT scans, liver imaging, and/or other radiological imaging modalities may be considered.

Whenever clinically possible, the diagnosis of progression or recurrence should be confirmed histologically. The earliest date of diagnosis of disease progression or recurrence should be used and recorded. This date should be based on objective clinical, radiological, histological, or cytological evidence. Recurrence includes local, regional, or distant recurrence.

The following provides the definition and confirmation procedures for disease relapse, death, and other notable events during follow-up. Recording of relapses requires specifying all sites involved to establish a relapse pattern. The following criteria for treatment failure constitute the only acceptable evidence of disease relapse:

• Lungs: Positive cytology or biopsy, with a single new lesion or multiple lesions consistent with metastatic disease.

• Liver: Positive cytology or biopsy, with a single new lesion or multiple lesions consistent with metastatic disease.

Central nervous system: Positive brain CT or MRI scan or CSF cytology.

• Recurrence in the skin, subcutaneous tissue, and lymph nodes: Positive cytology or biopsy, presence of a single new lesion or multiple lesions consistent with metastatic disease.

• Bone and other organs: Positive cytology or biopsy, presence of a single new lesion or multiple lesions consistent with metastatic disease identified in two different radiological studies (i.e., positive radionuclide bone scan or PET scan and contrast-enhanced GI series or abdominal ultrasound, X-ray or CT for abdominal disease).

Cohort 2 Tumor and Response Assessment

Patients in Cohort 2 underwent tumor assessment every 9 (±1) weeks (starting on day 1 of Cycle 1) for the first 54 weeks, and then every 12 (±2) weeks, regardless of dose delay. An exception was patients who continued treatment after radiographic progression of the disease. These patients underwent tumor assessment every 9 weeks until investigators determined a loss of clinical benefit. Therefore, for patients who discontinued treatment for reasons other than loss of clinical benefit, tumor assessment should continue as planned, even if they begin a new non-regimen-specified anticancer therapy. Tumor assessment may be repeated at the investigator's discretion if disease progression is suspected.

Brain metastases treated with radiation therapy or surgery are not considered measurable or evaluable, but are recorded as sites of metastatic disease during screening. Brain metastases identified at baseline that have been treated with radiation therapy or surgery are not considered measurable or evaluable unless there is suspected disease progression in the brain (i.e., the patient develops symptoms). Therefore, subsequent head scans are not required unless there is a clinical indication.

To facilitate assessment of responses according to iRECIST in Cohort 2, for patients continuing treatment after progression, tumor assessment must continue following disease progression according to RECIST v1.1. This includes continuous measurement of target lesions, assessment of non-target lesions (including monitoring for further deterioration of any non-target lesions that have shown clear progression), and assessment of any newly identified lesions (including measurement, if the lesion is measurable) in all subsequent assessments.

Researchers used RECIST v1.1 to assess the overall response at individual time points.

Biomarker assessment

Baseline tumor tissue samples were collected from all patients (except those in the safety introductory period of cohort 2) by biopsy of metastatic lymph nodes (cohort 1) or other metastatic lesions (cohort 2) at screening. For patients in cohort 1, in-treatment tissue samples were collected by biopsy on day 1 of cycle 2 and at surgery (CLND). For patients included in cohort 2, in-treatment tissue samples were collected by biopsy on day 8 of cycle 2.

Exploratory biomarker analyses are conducted to understand the association between biomarkers and responses to investigational drugs, considering both efficacy and safety endpoints. Exploratory biomarker studies may include, but are not limited to, analysis of genes or gene signatures associated with tumor immunobiology, PD-L1, lymphocyte subsets, T cell receptor lineages, or cytokines associated with T cell activation. Studies may involve DNA or RNA extraction, somatic mutation analysis, and the use of next-generation sequencing (NGS) (including whole-exome sequencing (WES)). Studies may involve extracting DNA, cell-free DNA, or RNA; analyzing mutations, single nucleotide polymorphisms, and other genomic variations; and performing genomic analysis using NGS with a comprehensive genome. DNA extracted from blood can be compared with DNA extracted from tissues to identify somatic variants by distinguishing between germline and somatic variants.

NGS methods may include whole-genome sequencing (WGS) or whole-electrode sequencing (WES) of tissue and blood samples. At participating sites, blood samples are collected for DNA extraction to enable WGS or WES, thereby identifying variants that can predict responses to investigational drugs, are associated with progression to more severe disease states, are associated with acquired resistance to investigational drugs, or are associated with susceptibility to developing adverse events. This can improve adverse event surveillance or investigation, or increase knowledge and understanding of disease biology and drug safety. DNA extracted from blood can be compared with DNA extracted from tissue to identify somatic variants by distinguishing between germline variants and somatic variants.

I. Analysis

Final study analyses are based on patient data collected upon study termination. Unless otherwise specified, efficacy analyses are based on the efficacy-evaluable population, defined as all patients receiving at least one dose of each drug in their assigned treatment regimen, and safety analyses are based on the safety-evaluable population, defined as all patients receiving any amount of study treatment.

Enrollment was summarized by region, country, and treatment arm. Patient profiles were summarized by treatment arm. Major protocol biases, including major biases regarding inclusion and exclusion criteria, were summarized by treatment arm.

For patients for whom safety can be assessed, study drug administration data are tabulated or listed by treatment arm, with any dose modifications marked. Mean and standard deviation are used to summarize the total dose and dose intensity for each study drug. Reasons for discontinuation of study drugs are tabulated.

Demographic and baseline characteristics (including age, sex, race/ethnicity, weight, duration of malignancy, site of metastatic disease (if applicable), and baseline ECOGPS) were summarized overall and by treatment arm.

Determining the sample size

This study was not intended to provide definitive efficacy and Type I error considerations for hypothesis testing. Rather, it aimed to obtain preliminary efficacy, safety, and pharmacokinetic (PK) data when a treatment or combination of treatments was administered to patients with melanoma. Cohort 1 consisted of patients with resectable stage III melanoma who had not received prior systemic therapy for their disease. Cohort 2 consisted of patients with stage IV melanoma who had experienced disease progression during or after at least one, but no more than two, lines of therapy for metastatic disease.

In cohort 1, approximately 55 to 145 patients were randomly assigned to the control arm and the experimental arm during the study. In cohort 2, approximately 6 to 46 patients were assigned to the experimental arm.

Therapeutic effect analysis

The primary efficacy endpoint of cohort 1

The primary efficacy endpoint for Cohort 1 was pRR at surgery. pRR was assessed at week 7 after neoadjuvant therapy at CLND. pRR was defined as the proportion of patients achieving pCR (complete absence of viable tumor cells in the treated tumor bed) as determined by independent pathological review, near-complete pathological response (pnCR; <10% viable tumor cells in the treated tumor bed), or partial pathological response (pPR; <50% of the treated tumor bed occupied by viable tumor cells). pRR and 90% CI were calculated for each arm. The difference in pRR between the experimental and control arms, and its 90% CI, was also calculated. Confidence intervals were estimated using either the exact method or the Wald method, depending on the sample size.

Secondary therapeutic endpoints in cohort 1

Secondary efficacy endpoints for cohort 1 were pRR, event-free survival (EFS), RFS, OS, and preoperative ORR, as determined by local pathological assessment at the time of surgery. The definition of pRR is shown in Table 5.

EFS is defined as the time from randomization to any of the following events (whichever occurs first): disease progression excluding surgery as assessed by the investigator according to RECIST v1.1; local, regional, or distant disease recurrence; or death from any cause. Patients who have not experienced such events are censored at the last tumor assessment.

RFS is defined as the time from surgery to the first recorded disease recurrence or death from any cause. For patients with no recorded disease recurrence or death, RFS is censored on the day of the last oncology assessment.

OS is defined as the time from randomization to death from any cause. Patients still alive at the time of OS analysis are censored if their last known date of survival is unknown.

The Kaplan-Meier method was used to estimate the median of RFS, EFS, and OS, with the 90% CI constructed using the Brookmeyer and Crowley methods. The Kaplan-Meier method was also used to estimate the rates of RFS, EFS, and OS at specific time points, with the 90% CI calculated based on Greenwood estimates of variance.

ORR was assessed according to RECIST v1.1 at week 7 after neoadjuvant therapy and defined as the proportion of patients achieving CR or PR as determined by investigators according to RECIST v1.1. Patients with missing or no response assessments were classified as nonresponders. Note that ORR was determined using unconfirmed preoperative radiological responses. Although RECIST v1.1 requires confirmatory imaging assessments to be completed at least 4 weeks after the initial response, these responses could not be confirmed by subsequent imaging due to the CLND timeline.

ORR and 90% CI were calculated for each arm using the Clopper-Pearson method. The ORR difference between the experimental and control arms, and its 90% CI, were also calculated. CI was estimated using either the exact method or the Wald method, depending on the sample size.

Exploratory efficacy endpoints in cohort 1

The exploratory efficacy endpoints for cohort 1 were the endpoints of EFS, RFS, and OS at specific time points (1 year, 2 years, 3 years, and 5 years).

The Kaplan-Meier method was used to estimate the landmark EFS rate, landmark RFS rate, and landmark OS rate for each study arm, and the 90% CI was calculated using the Greenwood formula.

The primary efficacy endpoint of cohort 2

The primary efficacy endpoint for cohort 2 was ORR, as defined in Table 6. ORR was determined by the investigators according to RECIST v1.1. Patients with missing or no response assessments were classified as nonresponders.

Calculate the ORR (observable response rate) for each arm, i.e., the proportion of patients achieving complete or partial remission, and the 90% CI (Clopper-Pearson method). CI is estimated using either the exact method or the Wald method, depending on the sample size.

Secondary therapeutic endpoints in cohort 2

Secondary efficacy endpoints for cohort 2 were PFS, OS, OS at specific time points (e.g., 6 months), duration of response (DOR), and disease control, as defined in Table 6. PFS, DOR, and disease control were determined by the investigators according to RECIST v1.1.

DOR originates from patients with evaluable efficacy of CR or PR.

For patients whose disease progression or death was never recorded, PFS and DOR were censored on the day of the last oncology assessment.

Patients still alive at the time of OS analysis were censored at the last known date of their survival.

The Kaplan-Meier method was used to estimate the median of PFS, OS, and DOR, and a 90% CI was constructed using the Brookmeyer and Crowley methods. The Kaplan-Meier method was also used to estimate the OS rate at specific time points, and a 90% CI was calculated based on the Greenwood estimate of variance.

Calculate the disease control rate (proportion of patients with SD ≥12 weeks), PR or CR for each treatment arm, and estimate the 90% CI using the Clopper-Pearson precision method.

Exploratory therapeutic endpoints in cohort 2

The exploratory efficacy endpoints were ORR, PFS, DOR, and disease control as determined by researchers according to iRECIST.

ORR, PFS, DOR, and disease control were analyzed using the same methods described in the sections “Primary Efficacy Endpoints in Cohort 2” and “Secondary Efficacy Endpoints in Cohort 2” above. DOR was derived for patients with evaluable efficacy of complete or partial remission.

Security Analysis

Adverse event terminology is mapped word-for-word to the thesaurus of terms in the regulatory activity medical dictionary, and the severity of adverse events is graded according to NCI CTCAE v5.0 and the ASTCT CRS consensus grading scale based on CRS.

Safety was assessed by summarizing adverse events, changes in laboratory test results, changes in vital signs and ECG, and exposure to the investigational drug. The duration of follow-up for exposure and safety of combination therapy was summarized per treatment arm.

All adverse event terms were mapped verbatim to the thesaurus of terms in the regulatory activity medical dictionary. Adverse event severity was graded according to NCI CTCAE v5.0, and CRS severity was also graded by the researchers according to the ASTCT consensus grading (Lee et al., Biol Blood Marrow Transplant. 25:625-638, 2019). All adverse events occurring during or after the first administration of study treatment, critical adverse events, adverse events leading to death, adverse events of particular concern, and adverse events leading to termination of study treatment (i.e., adverse events occurring during treatment) were summarized using the mapped terms, appropriate thesaurus levels, and severity grades. For events of different severity levels, the highest grade was used in the summary. Deaths and causes of death were summarized.

Relevant laboratory data, vital signs (pulse rate, respiratory rate, blood pressure, pulse oxygen saturation, and body temperature), and ECG data will be displayed over time, with grades indicated where appropriate. Additionally, a transposed table of selected laboratory test results will be used to summarize the severity grades at baseline and after maximum baseline. Changes in vital signs and ECG will be summarized.

In addition, in Cohort 1, the incidence and nature of grade ≥3 immune-related adverse events during the first 12 weeks, as well as the rate and duration of delayed surgery due to treatment-related adverse events, will be summarized by treatment arm. CLND may be delayed by up to 2 weeks if treatment-related adverse events are not adequately mitigated when surgery is planned.

In addition, surgical complications were scored according to the Clavien-Dindo classification. The complication rate for each level was reported and scored for patients undergoing CLND.

Immunogenicity analysis

If appropriate, the immunogenicity of atezolizumab and other investigational treatments can be assessed. Immunogenicity analysis includes all patients who underwent assessment for at least one antidrug antibody (ADA). Patients are grouped according to the treatment received, or, if they were not treated prior to study discontinuation, according to their assigned treatment.

For atezolizumab, the number and proportion of ADA-positive and ADA-negative patients were summarized by treatment group at baseline (baseline incidence) and after drug administration (post-baseline incidence). In determining the post-baseline incidence, a patient was considered ADA-positive if they were ADA-negative or had missing baseline data but developed an ADA response after investigational drug exposure (treatment-induced ADA response), or if they were ADA-positive at baseline and one or more post-baseline samples had a titer at least 0.60 titers higher than the baseline sample titer (treatment-enhanced ADA response). A patient was considered ADA-negative if they were ADA-negative or had missing baseline data and all post-baseline samples were negative, or if they were ADA-positive at baseline but no post-baseline sample titer was at least 0.60 titers higher than the baseline sample titer (treatment unaffected).

For other research treatments testing ADA, a positive result was determined according to standard methods established in previous studies of the drug.

The relationships between ADA status and safety, efficacy, pharmacokinetic (PK) and biomarker endpoints can be analyzed and reported using descriptive statistics.

Mid-term analysis

Given the exploratory nature of this study, interim analyses are expected during the study period. The earliest interim analysis will be conducted when at least one experimental arm has completed initial enrollment and patients have completed their pathological response assessment (cohort 1), or when at least one experimental arm has completed initial enrollment and patients have been followed up for at least 9 weeks for the primary endpoint analysis (ORR) (cohort 2). Further interim analyses may be conducted if deemed appropriate by the sponsor. In cohort 1, the posterior probability, based on the interim analysis of clinical activity in the experimental arm compared to the control arm, can be used to guide further enrollment. If the interim analysis indicates that the experimental arm has higher activity than the control arm, then 20 additional patients may be enrolled in the experimental arm (extension phase).

In cohort 2, based on an interim analysis of clinical activity in the experimental arm compared to a predetermined ORR threshold (defined as an improvement compared to the standard of care), posterior probabilities can be used to guide further enrollment in the treatment arm. For example, if available data indicate that the standard of care ORR is 10%, and a 10% improvement in ORR is considered a clinically meaningful change, this would result in an ORR threshold of 20% when calculating the posterior probability.

For the Cohort 2 patient population that had received at least two prior lines of treatment at the time of analysis, the ORR of standard care was based on internal and external data from similar immunomodulatory studies and other emerging compounds.

The organizer may decide on enrollment for the extended arm based on the total amount of available data, including but not limited to observed duration of remission, PFS, and potential early OS data. Safety and biomarker data (which are available when making this decision) are also considered from a full benefit-risk assessment perspective.

Example 2: Basic principles of 600mg Q3W dosage and schedule

A. Preface

RO7247669 (PD1-LAG3) is a novel bispecific antibody under investigation for the treatment of inflammatory solid tumor types. It is designed to revitalize T cells by blocking two co-inhibitory checkpoint receptors, PD-1 and LAG-3. RO7247669 binds with both monovalent high affinity for PD-1 and monovalent high affinity for LAG-3 (20-fold lower than PD-1), achieving an affinity-mediated increase in selectivity. LAG-3 is expressed at higher levels on regulatory T cells than on other T cells, and it has been reported that blocking it with a monospecific anti-LAG3 antibody adversely enhances its inhibitory activity. However, Treg cells express lower levels of PD-1 compared to dysfunctional T cells, making them less likely to be targeted and “activated” in response to treatment with RO7247669, where binding to PD-1 also acts as a pathway. Recently, LAG3 has been validated as a clinical target for first-line (1L) treatment of melanoma, and the combination of the anti-LAG3 antibody renalalimab and the anti-PD-1 antibody nivolumab has recently been approved.

In recent years, the shift from cytotoxic agents to cancer immunotherapy and molecularly targeted drugs has necessitated a paradigm shift in dosing optimization and dosing selection in oncology. Despite the need for thorough characterization of doses and timelines prior to registration trials, the notion that higher doses provide greater efficacy persists.

The pharmacological action of agents that block inhibitory checkpoint receptors is driven by binding to receptors on immune cells. Therefore, target binding (TE) saturation can be used as a substitute for pharmacological saturation (i.e., maximum effect on downstream signaling). Thus, once the target receptor is saturated, additional drug action is not expected to produce additional pharmacological effects. Therefore, the dose required for tumor target saturation can be recommended as the recommended phase II dose.

B. Method

i. Clinical research

RO7247669 is currently being evaluated as a single agent in the Phase I study NP41300. Study NP41300 is an open-label, multicenter, dose-escalation Phase I study designed to evaluate the safety/tolerability, pharmacokinetics (PK), pharmacodynamics, and preliminary antitumor activity of RO7247669 in patients with locally advanced and/or metastatic solid tumors. The study consists of a dose-escalation arm (Part A) with two timelines and a tumor-specific extension arm (Part B). Part A comprises a dose-escalation design of Part A1 (dosing every two weeks (Q2W)) and Part A2 (dosing every three weeks (Q3W)) to determine the maximum tolerated dose (MTD) and/or extended recommended dose (RDE) of RO7247669 in patients with solid tumors. Part B comprises a tumor-specific extension of RO7247669 administered at the MTD and/or RDE, as well as other target doses, for future development in a subset of patients with selected solid tumor indications. The protocol includes adult patients with advanced and/or metastatic solid tumors.

ii. Clinical pharmacokinetics

Serum concentrations of RO7247669 were determined using a validated target binding capacity (PK) assay. To assess RO7247669 PK, serum samples were collected from patients at regularly pre-specified time points during the NP41300 study. These included peak (within 30 minutes after infusion) and trough (within 24 hours before the next dose) samples, as well as abundant samples collected during cycles 1 and 5.

Population PK analysis was performed using a nonlinear mixed-effects modeling approach. One-compartment and two-compartment models were evaluated, and different residual error structures were tested. The models were then refined by testing the inter-individual variability of each PK parameter, and the correlations between random effect values were examined to guide the development of the reduced ω structure. Model selection was based on log-likelihood criteria, goodness-of-fit plots, and scientific rationality.

iii. Tumor receptor occupancy modeling

The pharmacological effects of PD1-LAG3 are driven by the binding of PD1 and LAG3 to CD8+ T cells. Therefore, saturation of these receptors on CD8 cells can serve as a substitute for the maximum effect on downstream signaling; additional PD1-LAG3 is not expected to elicit additional pharmacological effects.

Tumor receptor occupancy modeling was performed. The goal was to characterize intratumoral PD1 and LAG3 binding as a function of RO7247669 concentration and dose. To estimate the dose required to saturate the target using PK data, a minimal physiology-based pharmacokinetic (mPBPK) model was used. This model simplifies different tissues/organs to two different tissue types sufficient to describe systemic concentrations of RO7247669 and includes tumor compartments to describe intratumoral RO7247669 concentration and PD1 and LAG3 binding, thus providing a combined approach. Two tumor subcompartments represent vascular and stromal spaces. PD1-LAG3 enters and leaves the tumor vascular space based on tumor blood flow rates. Once within the tumor, PD1-LAG3 can bind to PD-1 and/or LAG3 on tumor-associated immune cells and be eliminated via target-mediated drug disposal (TMDD) or by convection. Tumor compartments were also added to the model to describe tumor uptake of RO7247669. Li et al., Clinical Pharmacology and Therapeutics, 110(1):200-209, 2021, previously described a simpler model (with only one target) to predict the recommended phase 2 dose of pembrolizumab (Figure 3). Subsequent randomized dose comparison studies confirmed RP2D proposed by Li et al. as a key dose across tumor types. The additional LAG3 receptor added to the PD1-LAG3 model is shown in Figure 4. Population PK model parameters are shown in Table 16. Model parameters beyond the pembrolizumab model reported by Li et al. are provided in Table 17.

Table 16. Parameters of the Group PK Model

parameter estimate relative standard error 95% confidence interval Shrinkage CL(L/h) 0.0086 4.70 0.00785-0.00944 - V1(L) 3.11 2.08 2.98-3.24 - Q(L/h) 0.0229 7.77 0.0194-0.0264 - V2(L) 1.98 7.64 1.68-2.27 - HT on V1 1.99 43.1 0.31-3.67 - WT on CL 2.35 12.7 1.76-2.93 - HT on Q 0.572 52.3 -0.0146 -1.16 - IIV-CL 0.114 17.7 0.0748-0.154 11.2 IIV-V1 0.0217 20.1 0.0131-0.0303 12.8 IIV-Q 0.214 31.6 0.0814-0.346 32.1 IIV-V2 0.209 22.8 0.116-0.303 23.6 proportional error 0.148 4.61 0.134-0.161 - Addition error 0.712 35.6 0.214-1.21 -

CL: clearance rate; V1: central volume; Q: inter-particulate clearance rate; V2: peripheral volume; IIV: inter-individual variability.

Table 17. Model parameters other than the pembrolizumab model

C. Result

i. Clinical efficacy and safety

At the clinical data cutoff (March 1, 2022), a total of 35 patients were treated with RO7247669 at six dose levels in Part A1 (Q2W). In Part B, a total of 83 patients were treated. Table 18 provides a summary of patients who received RO7247669 in NP41300.

During the dose escalation period from 50 mg to 2100 mg Q2W, the maximum tolerated dose (MTD) was not reached, and no dose-limiting toxicities (DLTs) were observed. Following the initial administration of 50 mg RO7247669, occupancy of PD-1 and LAG3 receptors on peripheral CD8+ cells was observed to exceed 90%, and saturation was observed at all doses up to 2100 mg. Persistent ADA formation against RO7247669 was observed in less than 20% of patients. Reduced exposure occurred in some ADA-positive patients who received 300 mg or less. Clinical responses were observed at 600 mg and higher doses during the dose escalation period.

Throughout the clinical dose range (50 to 2100 mg Q2W), mean exposure increased in a dose-proportional manner with increasing dose.

Table 18. Summary of patients who received RO7247669 in NP41300

As of March 1, 2022, the disease control rate (DCR) of the NP41300 A portion of the study was 51.4% (18/35 patients), and the objective response rate (ORR) was 17.1% (6/35 patients). Among patients receiving the RDE of 2100 mg Q2W, 7 out of 13 patients (53.8%) achieved a best response of stable disease or better (DCR 53.8%), with an ORR of 30.8% (4/13 patients).

In addition to the confirmed partial remission (cPR) observed in the RDE, there were 2 other cPRs (ORR 50.0%) at the 600 mg dose level.

In Part B of this study, as of March 1, 2022, in patients treated with RDE at 2100 mg Q2W (Parts B1, B2, and B3), the DCR was 48.3% (28/58) and the ORR was 5.2% (3/58). In patients treated with 600 mg Q2W (Part B5), the DCR was 40% (4/10) and the ORR was 10% (1/10). In patients treated with 600 mg Q3W, the DCR was 28.6% (2/7) and the ORR was 14.3% (1/7).

As of March 1, 2022, the disease control rate (DCR) for melanoma patients in Part B1 who experienced checkpoint inhibitor (CPI) treatment was 43.8% (14/32), and the objective response rate (ORR) was 6.3% (2/32). In Part B2, among CPI-treated NSCLC patients, 50% experienced clinical benefit (9/10 DCR), but none achieved a response. In Part B3, among CPI-untreated esophageal squamous cell carcinoma (ESCC), the DCR was 62.5%, and the ORR was 12.5% (1/8 patients). In the biomarker cohort, the DCRs for Parts B5 and B6 were 40% (44/10) and 28.6% (2/7), respectively, while the ORRs were 10% (1/10) and 14.3% (1 partial response (PR)/7), respectively.

In Part A1 (Q2W dosing), the majority of patients (94.3%) reported at least one adverse event (AE). The most frequently reported AEs (occurring in at least 20% of patients) were anemia (37.1%), constipation (31.4%), dyspnea (28.6%), fatigue (25.7%), asthenia and decreased appetite (22.9% each), diarrhea and fever (20.0% each). Overall, 62.9% of patients reported treatment-related AEs. The incidence of serious adverse events (SAEs) was 25.7%, of which 6 (17.1%) were treatment-related SAEs. A total of 6 patients experienced SAEs that the investigators considered to be related to the study treatment. Treatment-related adverse events (SAEs) included one case of elevated bilirubin in the 150 mg cohort, one case of myositis in the 600 mg cohort, two cases of adverse events (one case of dyspnea and one case of hyperthyroidism) in the 1200 mg cohort, and two cases of adverse events (one case of pleural effusion and one case of anemia) in the 2100 mg cohort.

As of the clinical cutoff date of March 1, 2022, preliminary safety data were available for 118 patients with solid tumors who received RO7247669 monotherapy at Q2W or Q3W in Parts A1 and B. A total of 748 adverse events (AEs) were reported by 112 of the 118 patients (94.9%). Overall, 19 patients (54.3%) reported Grade 1 or 2 AEs as the maximum severity, and 14 patients (40.0%) reported 21 Grade 3 AEs. No Grade 4–5 AEs were observed. Of the 21 Grade 3 AEs, 6 patients (17.1%) reported 6 Grade 3 AEs that were considered related to the study treatment. Of these 6 related Grade 3 AEs, 4 were considered serious. Figures 5 and 6 provide an overview of adverse events in patients with evaluable safety in the dose-escalation (Part A1, Q2W) portion of the study.

ii. Clinical pharmacokinetics

The PopPK model was used to simulate the C- _{valence at} 600 mg and 1200 mg doses under two dosing regimens, Q2W and Q3W. As shown in Figure 7, the valence concentrations after Q3W and Q2W for 600 mg and 1200 mg doses are expected to overlap after the first and third administrations.

Due to limited datasets, covariate analysis including tumor type has not yet been performed on the PD1-LAG3 PopPK model; however, analyses of nivolumab and pembrolizumab have reported minimal impact of tumor type on pharmacokinetics. In the nivolumab analysis, clearance (CL) was similar across all tumor types (NSCLC, melanoma, and RCC), indicating that PK was independent of tumor type (Bajaj et al., CPT Pharmacometrics Syst Pharmacol, 6(1):49-57, 2017). In the pembrolizumab analysis, the PopPK model included patient data from advanced melanoma, non-small cell lung cancer (NSCLC), and other solid tumor types. Clearance was 13.9% higher in NSCLC patients than in other tumor types; however, this was clinically irrelevant (Ahamadi et al., CPT Pharmacometrics Syst Pharmacol, 6(1):58-66, 2017).

iii. Tumor receptor binding modeling

Multiple doses of RO7247669 simulated PD1 and LAG3 conjugation, ranging from 0.015 to 1500 mg administered over Q3 weeks (Figure 8). Simulations showed that PD1 and LAG3 were expected to saturate in tumors at 60 mg Q3 weeks, with a LAG3 receptor occupancy (RO) of 90% at 60 mg, and a PD1 RO of 90% at 2.37 mg Q3 weeks. Population pharmacokinetic (PK) models showed linear clearance at these doses, indicating target-mediated drug disposal (TMDD) saturation, consistent with predictions from tumor receptor conjugation models.

To account for the spatial heterogeneity of tumors and the predicted range of tumor penetration associated with RO7247669, the model simulated a range of vascularizations, where poorly vascularized areas were 25-fold lower than well-vascularized areas. Furthermore, some inter-subject variability is expected with RO7247669 exposure: therefore, 600 mg Q3W is expected to ensure at least 90% LAG3 RO in most patients, regardless of tumor uptake levels.

D. Discussion

The recommended RO7247669 dose and 600 mg Q3W schedule were estimated using clinical and pharmacokinetic data from the NP41003 dose-escalation study in solid tumors. Quantitative models based on published models combined with the RO7247669 PopPK model and PD1 and LAG3 target characterization data were used to model target binding across a series of clinical doses in the Q3W regimen. Considering inter- and intra-subject variability in tumor exposure and vascularization, 600 mg Q3W was expected to saturate PD1 and LAG3 receptors on CD8 cells in tumors. The pharmacological effects of blocking inhibitory checkpoint receptors are driven by receptor binding to immune cells; therefore, target binding saturation can be used as a substitute for pharmacological saturation (i.e., maximum effect on downstream signaling). Thus, once target receptor saturation is achieved, additional drug administration is not expected to produce additional pharmacological effects.

This conclusion is supported by NP41300 clinical data, in which responses were observed in the 600 mg Q2W and higher dose cohorts. Furthermore, the 600 mg Q2W dose was well-tolerated in the dose escalation cohort. The predicted C- _valence overlaps between 600 mg Q2W and Q3W, and therefore no clinically relevant differences are expected between the two schedules. Therefore, the more patient-centered 600 mg Q3W schedule was chosen for further investigation.

Example 3: A randomized, open-label, multicenter, phase II study of multiple doses of RO7247669 in participants with previously untreated unresectable or metastatic melanoma.

A. Research Design

Despite the emergence of several new agents that offer survival benefits to patients, cancer remains a leading cause of death worldwide. Many cancer indications have poor prognoses, and the management of most advanced solid tumors remains challenging due to high rates of tumor recurrence or distant metastasis. While currently available checkpoint inhibitor (CPI) therapies are effective against a variety of tumor types, including melanoma, other treatment options targeting immune checkpoints are still needed because patients may eventually progress after an initial response or fail to respond to PD-1/L1 or cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) checkpoint blockade.

Current standards of care for metastatic melanoma include treatment with immune checkpoint inhibitors (CPIs) alone or in combination, as well as therapy targeting the v-Raf murine sarcoma virus oncogene homolog B1 (BRAF) and mitogen-activated protein kinase (MEK) inhibitors. Interleukin-2 (IL-2), oncolytic viruses, and interferon (IFN) therapy remain options for a subset of patients. Overall, despite advances in treatment options, patients with metastatic melanoma continue to experience poor long-term clinical outcomes, reflecting the aggressive nature of the disease and its complex etiology, and highlighting ongoing unmet medical needs.

RO7247669 (PD1-LAG3) is a bispecific antibody (BsAb) against programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG3) designed to target dysfunctional tumor antigen-specific T lymphocytes to establish or restore an effective anti-tumor immune response in cancer patients with highly unmet medical needs. By targeting both PD-1 and LAG3 to dysfunctional tumor-specific T lymphocytes, RO7247669 aims to restore an effective anti-tumor immune response and provide cancer patients with greater survival benefits than existing agents. Compared to blocking PD-1 alone, the combination of blocking LAG3 and PD-1 may have the potential to improve efficacy without increasing significant toxicity and become a treatment option for melanoma patients.

Recently, the combination of renalalimab and nivolumab has provided a clinical proof-of-concept for dual inhibition of PD-1 and LAG3 in patients with previously untreated metastatic or unresectable melanoma (Tawbi et al., N Engl J Med, 386(1):24-34, 2022). Inhibition of both immune checkpoints provided a greater benefit in progression-free survival (PFS) compared to inhibition of PD-1 alone.

The purpose of the study BP43963 described in this example is to evaluate the efficacy, safety, pharmacokinetics (PK), and pharmacodynamics of two dose levels of RO7247669 in participants with unresectable or metastatic melanoma in order to select a recommended dose for further development. The study objectives and endpoints are summarized in Table 19.

Table 19. Objectives and endpoints of the BP43963 study

The main objectives are expressed in the estimation framework through the five attributes described in Table 20.

Table 20. Estimates

ORR: Objective response rate; PFS: Progression-free survival; Q3W: Every 3 weeks; TA: Tumor assessment.

i. Overall Design

BP43963 is a phase II, randomized, open-label, global, multicenter study designed to evaluate the safety and clinical activity of two different dose levels of RO7247669 in participants with unresectable or metastatic melanoma who have not previously received systemic therapy for their metastatic or unresectable disease. An overview of the study design is provided in Figure 9.

This study enrolled approximately 80 participants aged ≥18 years with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Participants were randomized 1:1 to receive RO7247669 at 600 mg every 3 weeks (Q3W) or 1200 mg Q3W. Prior adjuvant or neoadjuvant CPI therapy (yes or no) and PD-L1 expression (≥1% vs. <1% expression, based on immunohistochemistry (IHC) using antibody clones 22C3, SP263, or 28-8) were used as stratification factors.

Participants may continue treatment as long as they are experiencing clinical benefits as assessed by the researchers, and without unacceptable toxicity or symptom worsening attributable to disease progression after a comprehensive assessment of up to 24 months of radiological data, biopsy results (if available), and clinical status. Participants meeting the disease progression criteria according to RECIST v1.1 are permitted to continue study treatment if they meet all post-progression treatment criteria.

Participants underwent their first tumor assessment 12 weeks (±1 week) from the date of Day 1 of Cycle 1 (C1D1). Subsequent tumor assessments were performed every 9 weeks (±1 week) until week 48, and then every 12 weeks (±1 week) from the date of C1D1, regardless of any delay in treatment.

Response was assessed according to RECIST v1.1. Researchers determined objective responses at individual time points according to RECIST v1.1. Survival follow-up information was collected approximately every 3 months via telephone, participant medical records, and/or outpatient visits after study treatment discontinuation and disease progression according to RECIST v1.1, up to a maximum of 24 months after death, loss to follow-up, study termination, or randomization.

The first occurrence shall prevail.

During the study, serum samples were collected to assess RO7247669 PK and to detect the presence of antibodies against RO7247669. Samples were also collected from participants, including archived and fresh tumor tissue, serum, plasma, and blood samples, for biomarker assessment.

Safety assessment includes the incidence, nature, and severity of adverse events (AEs), as well as other protocol-specified tests, such as laboratory abnormalities, which are considered to be related to the safety profile of an individual.

Safety assessments are crucial for research.

Study duration and end of study

The study duration for each participant, from screening to safety follow-up, is a maximum of approximately 25 months (excluding long-term follow-up (LTFU)).

The duration of each study period for each participant is as follows:

• Screening: Maximum 28 days before randomization

• Treatment period: Day 1 of cycle 1 (C1D1) up to a maximum of 24 months.

• Survival follow-up: 90 (±1 week) days after the last dose of study treatment; then every 3 months (±2 weeks) until death, loss to follow-up, study termination or randomization for a maximum of 24 months.

The study endpoint was defined as the date of the last participant's last observation (LPLO). The LPLO was expected to occur no later than 24 months after the last participant was randomized.

Participant Group

The participant population consisted of female and male participants with unresectable or metastatic melanoma who had no prior systemic anticancer therapy for the unresectable or metastatic disease.

Number of participants

The planned number of participants randomized to the study treatment was 80, which means that approximately 80 participants will be available for assessment in the primary endpoint analysis.

Concomitant medication

Generally, medications used to treat melanoma are not permitted to be used concurrently with medications used to treat adverse events (AEs), unless the underlying principles of the exception are discussed and clearly documented.

Background information on melanoma

Skin cancer is the most common of all cancers. Melanoma, accounting for only 1% of all skin cancers, is the most aggressive and dangerous form of skin cancer, developing from melanocytes. According to the American Cancer Society, approximately 100,000 new cases of aggressive melanoma will be diagnosed in the United States in 2022. Although the outcomes are good for superficial tumors diagnosed in a timely manner, melanoma remains one of the deadliest cancers in metastatic cases, with a 5-year survival rate of 27% (Surveillance, Epidemiology, and Final Outcomes (SEER) 2021, available on the American Cancer Society website). Prior to 2011, approved treatments for metastatic melanoma were limited and included chemotherapy and immunotherapy (IL-2). Since then, new treatment options have been approved, including drugs targeting the tyrosine kinase pathway, CPI therapies targeting CTLA-4 and PD-1 receptors, and vaccines.

The anti-CTLA-4 antibody ipilimumab was the first CPI to demonstrate significant improvement in clinical outcomes and was therefore approved for unresectable or metastatic melanoma (Hodi et al., N Engl J Med, 363:711-723, 2010). Subsequently, monoclonal antibodies binding to the PD-1 receptor (pembrolizumab or nivolumab) produced improved outcomes and reduced toxicity (Robert et al., N Engl J Med, 372:320-330, 2010; Robert et al., N Engl J Med, 372(26):2521-2532, 2010; Weber et al., Lancet Oncol, 16(4):375-384, 2015). The combined CTLA-4/PD-1 blockade produced higher response rates and longer overall survival compared to the individual single-agent activities. However, combination therapy is accompanied by increased toxicity (Wolchok et al., Abstract 9506 ASCO Annual Meeting 2021).

Recent results from the phase 2/3 study (RELATIVITY-047) in previously untreated metastatic or unresectable melanoma showed that combination blocking of PD-1 (via nivolumab) and LAG3 (via renalalimab) improved progression-free survival (PFS) compared to PD-1 inhibition alone (Tawbi et al., N Engl J Med, 386(1):24-34, 2022). The PFS benefit observed in this combination appeared similar to that of the nivolumab and ipilimumab combination, but with a more favorable safety profile (Tawbi et al., N Engl J Med, 386(1):24-34, 2022, Wolchok et al., Abstract 9506 ASCO Annual Meeting 2021). Meanwhile, the combination of renalalimab and nivolumab did not show any new safety signals compared to treatment with nivolumab alone.

Current standards of care for metastatic melanoma include treatment with immune CPIs, alone or in combination, as well as treatment with targeted BRAF and MEK inhibitor therapies. IL-2, oncolytic viruses, and IFN therapy remain options for a subset of patients.

Overall, despite advances in treatment options, patients with metastatic melanoma continue to experience poor long-term clinical outcomes, reflecting the aggressive nature of the disease and its complex etiology, and highlighting ongoing unmet medical needs.

Background information on RO7247669

RO7247669 is a novel Fc-silencing IgG1-based bispecific antibody, employing a 1+1 formulation that incorporates monovalent binding to each of the following two CPIs: PD-1 and LAG3. RO7247669 is engineered to preferentially bind to T cells in the tumor microenvironment that co-express both PD-1 and LAG3, or to a lesser extent, T cells expressing either PD-1 or LAG3 alone. Preferential binding to T cells in the tumor microenvironment avoids targeting other LAG3-expressing cells, such as regulatory T cells in the tumor and periphery (which do not express high levels of PD-1). Monovalent binding to LAG3 reduces antibody (Ab) internalization after binding to the T cell surface. Another feature of PD-1BsAb is its engineered IgG1-based Fc region, which prevents binding to the Fcγ receptor by introducing the LALA P329G mutation. This avoids drug-shaving, thereby avoiding tumor-associated macrophage resistance mechanisms that have been observed in IgG4-based antibodies such as pembrolizumab and nivolumab (Arlauckas et al., Sci Transl Med, 9(389):eaal3604, 2017).

RO7247669 is currently being evaluated in the following four ongoing clinical studies:

1. Phase I human trials in participants with advanced and/or metastatic solid tumors (including the second-line melanoma extension cohort) (Study NP41300);

2. A phase II study in second-line participants with advanced or metastatic esophageal squamous cell carcinoma (study BP42772);

3. A phase Ib/II study (Study GO42216) in combination with bevacizumab in first-line participants with advanced hepatocellular carcinoma; and

4. Phase Ib/II study in participants with neoadjuvant resectable stage III and subsequent line IV melanoma, with or without tislelizumab (Study BO43328).

In all studies, RO7247669 was well tolerated up to the maximum test dose (2100 mg Q2W), and no specific safety issues were identified associated with RO7247669. During the dose escalation portion of the NP41300 study, no dose-limiting toxicities (DLTs) were observed, and no maximum tolerated dose (MTD) was identified.

Study BP42772 was a double-blind study, and studies GO42216 and BO43328 have only recently begun recruiting. Efficacy data are currently available from study NP41300. As of the data cutoff date of January 14, 2022, the disease control rate (DCR) for dose escalation in this study was 51% (18 out of 35 evaluable participants), and the objective response rate (ORR) was 17% (6 out of 35 participants). Responses across tumor types were observed at 600 mg every two weeks (Q2W) and 2100 mg Q2W in both CPI-naïve and CPI-experienced participants. While there were no responders at doses <600 mg (N=12), there were 7 out of 23 responders at doses ≥600 mg Q2W, indicating a dose-response relationship. No significant relationship between exposure and best overall response (BOR) was observed at doses ≥600 mg Q2W. The mean _Cavisss (steady-state mean concentration) in patients with disease progression was similar to that in patients with partial remission in the 600 and 2100 mg cohorts.

As of January 14, 2022, the efficacy of 2100 mg Q2W was evaluated in 28 participants with CPI-experienced uveal (9 of 28 participants) and non-uveal (19 of 28 participants) melanomas with both PDL-1 and LAG3. In the non-uveal melanoma participants, the ORR was 21% (4/19) and the DCR was 58% (11/19).

Responses were observed in two participants who had previously received pembrolizumab, one participant who received nivolumab, and one participant who received a combination of nivolumab and ipilimumab. Enrollment is ongoing.

Benefits-Risk Assessment

Despite limited clinical experience with RO7247669, the clinical efficacy and safety profile of anti-PD-1/PD-L1 monoclonal antibodies has been well characterized and established. In recent years, three CPIs—pembrolizumab, nivolumab, and ipilimumab—have been approved by the U.S. Food and Drug Administration (FDA) and other health authorities for the treatment of unresectable or metastatic melanoma. The reported median overall survival (OS) for patients with advanced melanoma is 72.1 months with the combination of nivolumab and ipilimumab, 36.9 months with nivolumab monotherapy, and 19.9 months with ipilimumab monotherapy (Wolchok et al., Abstract 9506, ASCO Annual Meeting 2021).

Next-generation combination therapies may yield higher response rates, greater depth of response, longer or similar overall survival (OS), and clinically meaningful improvements in safety profiles, as has been seen primarily in anti-PD-1 and anti-CTLA-4 combinations in patients with advanced melanoma. Therefore, better efficacy, primarily from simultaneously targeting PD-1 and LAG3-mediated immune resistance mechanisms, may be expected compared to monospecific PD-1-targeted antibodies.

As discussed above, the combination of renalalimab and nivolumab provides a clinical proof-of-concept for dual inhibition of PD-1 and LAG3 in patients with previously untreated metastatic or unresectable melanoma (Tawbi et al., N Engl J Med, 386(1):24-34, 2022). Inhibition of both immune checkpoints provided a greater benefit in terms of PFS compared to inhibition of PD-1 alone. Meanwhile, the combination of renalalimab and nivolumab did not show any new safety signals and demonstrated a significantly improved safety profile at review compared to the combination of nivolumab and ipilimumab (Tawbi et al., N Engl J Med, 386(1):24-34, 2022, Wolchok et al., Abstract 9506 ASCO Annual Meeting 2021).

As discussed above, RO7247669 has been shown to induce responses in patients with melanoma and other tumor types who have demonstrated resistance to CPI therapies, including combination therapy with nivolumab and ipilimumab. Specifically, the 21% ORR observed in non-uveal melanoma participants in the NP41300 study was a significant improvement compared to the 11.5% over-provision response rate of the nivolumab and rennet combination in a comparable patient population (Ascierto et al., AnnOncol, v611-v612, 2017).

RO7247669 has been tolerated at doses up to 2100 mg Q2W; adverse events (AEs) are manageable, and the safety profile has been consistent across different solid tumor indications and approved PD-1 targeted antibodies. In conclusion, RO7247669 has the potential to provide additional benefit compared to nivolumab monotherapy, with a similar safety profile.

Considering the preliminary efficacy and manageable safety profile, as well as the potential for improved outcomes due to its dual checkpoint inhibitory mechanism of action, RO7247669 treatment shows therapeutic potential for solid tumors such as melanoma. Based on the above considerations, currently available data, and planned safety monitoring and management guidelines, the proposed investigational treatment is considered to have an appropriate benefit/risk profile for the population included in this study.

B. Basic Principles

The basic principles of studying groups

This study included participants with unresectable or metastatic melanoma who had not received prior systemic anticancer therapy for unresectable or metastatic disease. Clinical validation of a combination of monospecific anti-LAG3 and monospecific anti-PD-1 antibodies has recently been conducted in this therapeutic setting (Tawbi et al., N Engl J Med, 386(1):24-34, 2022). Combined with emerging efficacy data from the ongoing NP41300 study in a CPI-experienced melanoma cohort, treatment with RO7247669 is expected to provide clinical benefit in this indication.

Choosing first-line melanoma also allows for testing of two different dose levels of monotherapy RO7247669, as CPI treatment without chemotherapy is the current standard of care for this population. Therefore, dose-dependent effects can be assessed in the absence of potential confounding effects from combination therapy.

The disease-specific eligibility criteria for this study were similar to those used in RELATIVITY-047. A benefit was observed with the combination of nivolumab and pirazumab compared to nivolumab alone, independent of tumor PD-L1 and LAG3 expression and patient BRAF mutation status. Therefore, participants met the criteria for this study regardless of tumor PD-L1 and LAG3 expression.

Expression was not related to BRAF mutation status. Including participants with BRAF-mutant melanoma was also considered reasonable, as current guidelines recommend considering immunotherapy before treatment with BRAF and MEK inhibitors, since it can provide very long-term disease control even after treatment is stopped (Keilholz et al., Ann Oncol, 31(11):1435-1448, 2020).

Basic principles of the primary endpoint

While overall survival (OS) remains the gold standard for demonstrating clinical benefit, measuring this endpoint requires a larger number of patients and longer follow-up periods than other endpoints, and can be confounded by subsequent therapies (Pazdur et al., Oncologist, 13 Supplement 2:19-21, 2008). In contrast, progression-free survival (PFS), including landmark PFS, is an earlier reading that allows for differentiation of the benefits of past first-line treatments for melanoma (Wolchok et al., Abstract 9506, ASCO Annual Meeting 2021). Therefore, PFS has been considered an acceptable moderating endpoint. For example, based on phase 3 clinical trials with PFS as the primary endpoint, the FDA approved dabrafenib and trametinib as monotherapy and in combination for the treatment of unresectable or metastatic melanoma (Flaherty et al., N Engl J Med, 367:107-114, 2012; Hauschild et al., Lancet, 380:358-365, 2012; Long et al., ASCO Annual Meeting Abstract; 32:9011, 2014). PFS has also been the primary endpoint of the RELATIVITY-047 trial, and its benefits have been demonstrated after 6 months of treatment (Tawbi et al., N Engl J Med, 386(1):24-34, 2022).

Compared to OS, PFS is not confounded by post-study treatment. Therefore, we consider PFS to be the preferred option for the selected patient population because clinically active treatment options (e.g., nivolumab, ipilimumab, vemurafenib, dabrafenib, and trametinib) are increasingly available for patients with unresectable or metastatic melanoma, and their use after disease progression in the current study could confound the OS endpoint.

For these reasons, PFS at 6 months was chosen as the primary endpoint of efficacy.

The basic principle of stratification factors

To minimize the possibility of imbalance across treatment arms, the trial used the following two stratification factors: prior adjuvant or neoadjuvant checkpoint inhibitor therapy (yes or no) and PD-L1 expression (based on IHC using antibody clones 22C3, SP263, or 28-8, ≥1% tumor cell surface expression vs. <1% tumor cell surface expression).

The choice of prior adjuvant or neoadjuvant CPI therapy (yes or no) is based on the fact that CPI therapy has improved recurrence-free survival (RFS) following complete resection of high-risk stage II/III melanoma. Adjuvant and neoadjuvant therapy with CPIs has been, or will be, part of the standard of care for melanoma treatment. Therefore, participants who completed adjuvant or neoadjuvant anti-PD-1 or anti-CTLA-4 therapy within at least 6 months between their last dose and the date of recurrence were eligible for this study. Because the effect of prior adjuvant or neoadjuvant anti-PD-1 or anti-CTLA-4 therapy on responsiveness to combination anti-PD-1 and anti-LAG3 therapy in unresectable or metastatic disease is unclear, participants in the current trial were stratified accordingly.

Regarding PD-L1 expression, previous clinical studies using nivolumab monotherapy have shown that patients with PD-L1-positive tumors may have a higher response rate than those with indeterminate expression (Robert et al., N Engl J Med, 372:320-330, 2010). Similarly, it has been reported that in patients with PD-L1 expression of 1% or higher, the median PFS for patients treated with the combination of piracetamab and nivolumab was 12.6 months, while for patients with PD-L1 expression of less than 1%, the median PFS for the combination of piracetamab and nivolumab was 6.4 months (Tawbi et al., N Engl J Med, 386(1):24-34, 2022).

Basic principles of biomarker assessment

The primary hypothesis of this study is that additional checkpoint blockade via PD-1-LAG3 will reactivate depleted T cells and lead to increased CD8 T cell infiltration and proliferation in the tumor microenvironment. Therefore, the exploration of treatment-induced biomarkers will focus on changes from baseline associated with PD-1-LAG3 in both the tumor microenvironment and peripheral blood. Serial blood sampling of peripheral blood was also performed to assess dynamic changes in the immune cell profile, which was then examined for its association with treatment with RO7247669.

To explore predictive biomarkers for response to RO7247669, the initial hypothesis focused on the association between response and baseline PD-L1, CD8, LAG3, and/or PD-1 expression. Other exploratory biomarkers include assessing baseline subsets of immune cells/effect gene signatures or other soluble markers (such as hematologic tumor mutational burden, circulating tumor DNA (ctDNA), etc.) in the peripheral blood and tumor microenvironment. If the initial data provide a strong scientific basis for these measurements, other biomarkers can be measured.

Dosage rationality

RO7247669 is administered at a dose of 600 mg or 1200 mg Q3W (on day 1 of each 21-day cycle).

As discussed in Example 2, RO7247669 was tolerable in the NP41300 study, and no new safety issues related to RO7247669 were identified. No DLT was observed up to the highest dose of 2100 mg Q2W, and no MTD was identified.

• Antitumor activity was observed at 600 mg and 2100 mg Q2W doses as measured by radiographic PR (Study NP41300).

The p-value of RO7247669 was dose-linear within the dose range tested in the NP41300 study.

• A minimal physiology-based pharmacokinetic model was used to further model PD-1 and LAG3 target binding in tumors to characterize intratumoral RO7247669 concentrations and estimate target-mediated drug disposition characterized by RO7247669 binding properties. It was estimated that, taking into account both inter-participant PK variability and spatial heterogeneity of intratumoral RO7247669 distribution, 600 mg Q3W RO7247669 would generate >90% PD-1 and LAG3 target binding at tumor sites in most participants throughout the treatment period. A similar approach was used to confirm the recommended phase II dose as the key dose for pembrolizumab (Li et al., Clinical Pharmacology and Therapeutics, 110(1):200-209, 2021).

• Tumor receptor occupancy simulations were performed for 600 mg Q2W and 600 mg Q3W, and both regimens produced >90% PD-1 and LAG3 receptor occupancy throughout the treatment period. Therefore, the less frequent Q3W regimen was chosen to minimize the treatment burden on participants.

The immunogenicity of RO7247669 and the impact of antidrug antibody (ADA) development on exposure and efficacy are not fully understood. However, there is evidence that ADA-mediated effects on exposure were observed at doses ≤300 mg in the NP41300 study. To date, ADA has not been observed at a dose of 600 mg, which leaves the potential impact of ADA-mediated effects on exposure at 600 mg uncertain.

To date, no effect on exposure has been observed in participants who developed ADA at 1200 mg and 2100 mg: therefore, 1200 mg is expected to saturate cyclic ADA and minimize the effect on exposure.

Therefore, this study selected 600 mg and 1200 mg Q3W dosages and regimens to administer to participants.

C. Inclusion and Exclusion Criteria

The participant cohort consisted of participants diagnosed with unresectable or metastatic melanoma who had no prior systemic anticancer therapy for the unresectable or metastatic disease, who met all given inclusion criteria and did not meet any exclusion criteria.

Inclusion criteria

Participants are eligible to be included in the study only if they meet all of the following criteria:

conventional

1. Able and willing to provide written informed consent and comply with the research protocol of the International Council for Harmonisation (ICH) and local regulations.

2. Age ≥ 18 years old

Participant types and disease characteristics

3. Participants must have histologically confirmed unresectable or metastatic melanoma according to the AJCC staging system (unresectable stage III or IV).

4. Radiometrically measurable disease according to RECIST v1.1. Previously irradiated lesions should not be counted as target lesions unless there is significant progression following radiotherapy.

5. ECOG fitness level is 0 to 1.

6. Participants must be aware of the BRAF V600 mutation status.

7. Participants must be aware of the PD-L1 state.

• PD-L1 expression is defined as the percentage of tumor cells showing any intensity of plasma membrane PD-L1 staining using an approved IHC assay with antibody clone 22C3, SP263, or 28-8.

• PD-L1 expression needs to be determined on tissue samples obtained within 3 months prior to enrollment, and no interventional treatment should have been administered between collection and PD-L1 testing.

8. Tumor tissue from sites of unresectable or metastatic disease must be provided for biomarker analysis.

9. Based on the clinical judgment of the treating physician, participants must have at least one non-target tumor lesion suitable for biopsy and agree to undergo mandatory therapeutic biopsy.

Medical condition

10. Sufficient cardiovascular function.

11. Any adverse events (AEs) arising from any prior radiotherapy, chemotherapy, or surgical procedure must have regressed to ≤ Grade 1, except for alopecia (any grade), vitiligo, endocrine disorders managed with alternative therapy, and Grade 2 peripheral neuropathy.

12. Sufficient hematological function.

13. Sufficient liver function.

14. Sufficient kidney function.

15. Other sufficient laboratory parameters obtained:

• Serum albumin ≥25g/L (2.5g/dL)

• For participants not receiving anticoagulation therapy: prothrombin time (PT) and activated partial thromboplastin time ≤1.5x ULN

• For participants receiving therapeutic anticoagulation: a stable anticoagulation regimen.

contraception

16. Male and/or female participants:

Contraception and abstinence requirements are intended to prevent embryonic exposure to the study treatment. The reliability of sexual abstinence for male and/or female enrollment eligibility should be assessed based on the duration of the clinical study and the participants' preferred and habitual lifestyles. Natural family planning methods (e.g., calendar, ovulation, symptom-basal body temperature, or post-ovulation methods) and withdrawal are unacceptable methods of contraception.

• Female participants: Female participants are eligible if they are not pregnant, not breastfeeding, and meet at least one of the following conditions:

• Women who are infertile (WOCBP).

·WOCBP, them:

• Agree to abstain from sexual intercourse (avoiding heterosexual intercourse) or use a highly effective contraceptive method with an annual failure rate of <1% during the treatment period and for at least 4 months after the last dose of the study drug.

During this same period, women must avoid donating eggs.

• A negative pregnancy test (blood) within 7 days prior to randomization.

• Male participants: During the treatment period and for at least 4 months after the last dose of the study drug, consent was given to:

• Maintain abstinence (avoid heterosexual intercourse) with your WOCBP or pregnant female partner, or use contraception such as condoms to avoid exposing the embryo.

• Avoid donating sperm.

Exclusion criteria

Participants will be excluded from the study if any of the following criteria are met:

conventional

1. Pregnancy, lactation, or breastfeeding.

2. Known hypersensitivity to any component of RO7247669, including but not limited to hypersensitivity to Chinese hamster ovary cell products or other recombinant human or humanized antibodies.

Participant types and disease characteristics

3. Participants must not have ocular melanoma.

Medical condition

4. Symptomatic central nervous system (CNS) metastases. Participants who have previously been treated for brain metastases may participate, provided they:

• Stable (no evidence of progression obtained by computed tomography (CT) or magnetic resonance imaging (MRI) for at least 28 days prior to randomization).

• No evidence of new or expanded brain metastases for at least 28 days prior to randomization.

• No systemic steroid treatment for at least 28 days prior to randomization.

5. Spinal cord compression syndrome that has not been clearly treated with surgery and/or radiation, or for which there is no evidence that the disease was clinically stable for ≥14 days before randomization.

6. History of active carcinomatous meningitis/leptomeningeal disease or carcinomatous meningitis/leptomeningeal disease.

7. Asymptomatic primary or metastatic tumors of the CNS, provided they require steroid or enzyme-induced anticonvulsants within the last 28 days prior to randomization.

8. Uncontrolled tumor-related pain. Participants requiring pain medication must be on a stable protocol upon enrollment in the study.

9. Participants with an active secondary malignancy. Exceptions to concurrent malignancies include: cervical carcinoma in situ that has been cured, ductal carcinoma in situ of the breast with a good prognosis, basal cell or squamous cell skin cancer, or low-grade early-stage localized prostate cancer, and any early-stage non-hematologic malignancy that has been in remission for at least two years after prior treatment.

10. Evidence of significant, uncontrolled comorbidities that may affect protocol adherence or outcome interpretation, including diabetes, a history of related lung disease, a known autoimmune disease or immunodeficiency, or other fibrotic diseases (such as scleroderma, pulmonary fibrosis, emphysema, neurofibromatosis, palmoplantar fibromatosis, etc.).

11. Encephalitis, meningitis, or uncontrolled seizures within one year prior to informed consent.

12. Major cardiovascular/cerebrovascular disease within the 6 months prior to randomization, including any of the following:

• Hypertensive crisis/encephalopathy.

• Unstable angina.

Transient ischemic attack/stroke.

• Congestive heart failure.

• Serious arrhythmias requiring treatment (excluding atrial fibrillation and paroxysmal supraventricular tachycardia).

• History of thromboembolic events (such as myocardial infarction, stroke, or pulmonary embolism)

13. Known active or uncontrolled bacterial, viral, fungal, mycobacterial (including but not limited to tuberculosis and typical mycobacterial diseases), parasitic or other infection (excluding fungal infections of the nail bed) or any major infection outbreak requiring IV antibiotics or hospitalization (related to completion of the course of antibiotics, except for tumor fever) within 28 days prior to randomization.

14. Known clinically significant liver diseases, including alcoholic hepatitis, cirrhosis, and hereditary liver diseases.

15. Major surgical procedure or major trauma (excluding biopsy) within 28 days prior to randomization, or major surgery expected to be performed during the study period.

16. Any other disease, metabolic disorder, physical examination findings, or clinical laboratory findings that reasonably suggest the presence of a disease or condition that contraindicates the use of the study drug or may affect the interpretation of the results or may place the participant at high risk of treatment complications.

17. It will prohibit informed consent for dementia or changes in mental state.

18. Uncontrolled pleural effusion (except in participants with indwelling catheters, for example), pericardial effusion, or ascites requiring repeated drainage procedures (expected to occur monthly or more frequently).

19. Active autoimmune disease or immunodeficiency, or a history of autoimmune disease or immunodeficiency, except in the following cases:

Participants with a history of autoimmune-mediated hypothyroidism or endocrine disorders and who are receiving stable thyroid hormone replacement therapy or appropriate replacement therapy are eligible for this study.

Participants with type 1 diabetes who are on an insulin regimen are eligible for this study.

• Participants with eczema, psoriasis, chronic simple lichen simplex, or vitiligo with only skin manifestations (e.g., excluding participants with psoriatic arthritis) are eligible for this study if they meet all of the following criteria:

• The rash must cover less than 10% of the body surface area.

• The disease is well controlled at baseline and only requires the use of ineffective topical corticosteroids.

• No acute exacerbations of underlying conditions requiring psoralen plus UVA radiation, methotrexate, retinoids, biologics, oral calcineurin inhibitors, or potent or oral corticosteroids have occurred in the past 12 months.

20. The screening test was positive for human immunodeficiency virus (HIV).

21. Participants must be positive for hepatitis B surface antigen (HBsAg) or total hepatitis B core antibody (HBcAb) at the time of screening. Participants who are positive for HBsAg or total HBcAb at the time of screening but subsequently negative for hepatitis B virus (HBV) deoxyribonucleic acid (DNA) are eligible.

22. A positive hepatitis C virus (HCV) antibody test at screening. Participants who tested positive for HCV antibodies at screening but subsequently tested negative for HCV RNA are eligible.

Previous/companion therapy

23. Previous systemic anticancer therapies for unresectable or metastatic melanoma.

24. Prior anticancer therapy using any immunomodulatory agents (including CPIs such as anti-PD-L1/PD-1 and anti-CTLA-4). The following prior adjuvant or neoadjuvant melanoma therapies are permissible if all relevant adverse events have returned to baseline or stabilized:

• Anti-PD-1 and/or anti-CTLA-4 based therapy administered at least 6 months between the last dose and the date of relapse. Participants who relapsed during (neo)adjuvant therapy or within 6 months of its completion were ineligible.

• Administer the last dose of IFN therapy at least 6 weeks prior to randomization.

25. Prior treatment with anti-LAG3 therapy.

26. Any history of life-threatening toxicity associated with prior immunotherapy (e.g., anti-CTLA-4 or anti-PD-1/PD-L1 therapy or any other antibody or drug that specifically targets T-cell co-stimulation or the immune checkpoint pathway), except those unlikely to recur through standard countermeasures (e.g., hormone replacement after adrenal crisis).

27. Receive a live vaccine within 28 days prior to randomization, or if a live attenuated vaccine is expected to be needed during the study.

28. Administer therapeutic oral or IV antibiotics within 14 days prior to randomization.

29. Prior to randomization, the patient was treated concurrently with any other investigational drug (defined as a treatment for an indication that is not currently approved by a regulatory agency) for <28 days or 5 half-lives of the drug (whichever is shorter).

30. Treatment with immunomodulators and immunosuppressants/drugs within <5 half-lives or 28 days (whichever is shorter) prior to randomization, except in the following circumstances:

• Inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone) are permitted.

• Participants who are eligible to receive acute and/or low-dose systemic immunosuppressive drugs (e.g., limited use of dexamethasone for nausea or long-term use of prednisone or equivalent doses of corticosteroids at ≤10 mg/day).

31. Regular immunosuppressive therapy (i.e., organ transplantation, chronic rheumatism).

32. Radiation therapy is not permitted within the last 28 days prior to the initiation of RO7247669 treatment, except for limited palliative radiation therapy.

33. Prior treatment using adoptive cell therapy, such as chimeric antigen receptor T-cell (CAR-T) therapy.

D. Research on treatment

Table 21 summarizes the treatments administered.

Table 21. Summary of the treatments administered

Research Treatment Name RO7247669 IMP and NIMP IMP Dosage formulation liquid Unit dose specification/dose level 300mg/6mL dose 600mg or 1200mg every 3 weeks, for up to 24 months. Application route IV infusion Packaging and Labeling The treatment is provided in vials.

IMP = Investigational Drug Product; NIMP = Non-Investigational Drug Product

RO7247669 is administered intravenously (IV). A 0.2 μm or 0.22 μm tandem filter must be used with the infusion device during administration.

The initial dose of RO7247669 was delivered over 60 ± 10 minutes (the infusion may be slowed or interrupted for participants experiencing infusion-related symptoms), followed by a 60-minute observation period. If the 60-minute infusion was tolerated without infusion-related adverse events (AEs), all subsequent infusions could be delivered over 30 ± 10 minutes, followed by a 30-minute observation period.

Predrugs

No prophylactic medication is expected prior to the first application of RO7247669.

Participants who experienced a Grade 2 infusion-related reaction (IRR) in a previous infusion should receive prophylactic medication for subsequent infusions. Prophylactic medication may be reduced for future cycles, or if the participant does not experience a Grade 2 or higher IRR event in the current infusion.

Permitted therapies

Participants are permitted to use the following therapies during the study:

• Oral contraceptives with an annual failure rate of <1%.

Hormone replacement therapy.

• Prophylactic or therapeutic anticoagulation therapy (such as stable doses of warfarin or low molecular weight heparin).

• Inactivated influenza vaccine.

• Administer medroxyprogesterone acetate as an appetite stimulant.

• Inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone).

• Acute and/or low-dose systemic immunosuppressive medications (e.g., a single dose of dexamethasone for nausea or long-term use of prednisone ≤10 mg/day or equivalent doses of corticosteroids).

Limited on-site palliative radiotherapy is permitted at any time during the study period, except on days when RO7247669 is administered.

Concomitant use of herbal remedies is not recommended because their pharmacokinetics, safety profiles, and potential drug-drug interactions are often unknown. However, herbal remedies not intended to treat cancer may be used during research.

Prohibited Therapy

Generally speaking, no medications are permitted to be used alongside medications used to treat acute exacerbations (AEs).

The following therapies are prohibited during the study period (excluding survival follow-up) and for at least 28 days prior to randomization or for five half-lives of the drug (whichever is shorter) and during study treatment, unless otherwise stated below:

• Research drugs or drugs that are not licensed/approved.

• Therapies intended to treat cancer (including, but not limited to, chemotherapy, hormone therapy, immunotherapy, surgical resection of target lesions and radiotherapy (excluding limited palliative radiotherapy), as well as herbal remedies or traditional Chinese medicines with anticancer activity listed on the label).

• Long-term use of steroids (inhaled and topical steroids permitted) at a baseline of ≤10 mg prednisone/day (or equivalent). Concurrent high-dose systemic corticosteroids.

• Administer a live attenuated vaccine or expect to need such a live attenuated vaccine during the study period or within 4 months after administering the final dose of the study drug.

• Systemic immunostimulants (including but not limited to IFN and IL-2) may increase the risk of autoimmune diseases when combined with CPIs.

• In the use of systemic immunosuppressive drugs (including but not limited to cyclophosphamide, azathioprine, methotrexate, and thalidomide), because these agents may alter the efficacy and safety of the investigational drug.

• Adoptive cell therapy, such as CAR-T therapy.

E. Efficacy evaluation

Participants received their first tumor assessment at week 12 (±1 week) relative to the randomization date. Subsequent tumor assessments were performed every 9 weeks (±1 week) until week 48, and then every 12 weeks (±1 week) relative to the randomization date, regardless of treatment delay. Tumor assessments were performed at the described time intervals, regardless of treatment delay, until the onset of disease progression according to RECIST v1.1 radiographic findings, initiation of a new anticancer therapy, withdrawal of consent, death, study termination, or up to 24 months after randomization, whichever occurred first. Therefore, in participants who discontinued treatment for reasons other than disease progression or loss of clinical benefit, tumor assessments continued according to the schedule. For participants who continued treatment after disease progression according to RECIST v1.1, tumor assessments continued according to the schedule until study treatment was discontinued.

F. Physical condition of the Eastern Oncology Collaboration Group

ECOG performance status was assessed at screening, before each study treatment administration, and at the end of the visit. If possible, it is recommended that the same person assess participants' performance status throughout the study.

G. Infusion-related reactions

Administration of therapeutic antibodies may cause an IRR, characterized by symptoms such as fever, chills, dizziness, hypertension, hypotension, dyspnea, restlessness, sweating, flushing, rash, tachycardia, tachypnea, headache, tumor pain, nausea, and/or vomiting. Respiratory and cardiac symptoms may also occur, such as bronchospasm, laryngeal and pharyngeal irritation, wheezing, laryngeal edema, and atrial fibrillation. Such reactions typically occur during or shortly after infusion, or within 24 hours of a study treatment infusion, primarily at the first infusion. The morbidity and severity generally decrease with subsequent infusions.

Participants may also develop immunoglobulin (Ig)E-mediated hypersensitivity. IRR can be difficult to distinguish from an allergic reaction; however, in cases of IgE-mediated hypersensitivity, symptoms typically appear after prior exposure and rarely occur at the first infusion. If an IgE-mediated hypersensitivity reaction is confirmed, treatment should be permanently discontinued. Table 22 provides recommendations for the prevention and management of infusion-related reactions.

Table 22. Recommendations for the prevention and management of infusion-related reactions

IRR = Infusion-related reaction; NCI CTCAE = National Cancer Institute Criteria for Adverse Events.

^For symptom grading, please refer to the NCI-CTCAE, v5.0 scale.

^b. Supportive care: For supportive care, if the patient has not received acetaminophen/paracetamol and antihistamines such as diphenhydramine within the previous 4 hours, these medications should be administered. Intravenous fluids (e.g., normal saline) may be administered as clinically indicated. For bronchospasm, urticaria, or dyspnea, antihistamines, oxygen, corticosteroids (e.g., 100 mg IV prednisolone or equivalent) and/or bronchodilators may be administered according to institutional practice.

H. Statistical Considerations

Sample quantity determination

The study planned to enroll 80 participants, randomized 1:1 to receive RO7247669Q3W at either 600 mg or 1200 mg. To assess the primary endpoint, participants were followed for at least 6 months after the last participant to ensure no administration censoring was present when calculating 6-month PFS. This study aimed to qualitatively characterize the two dose levels and identify any significant differences between the doses. However, this study was not sufficiently robust to detect all clinically meaningful differences.

Assuming no censoring occurred 6 months prior, some sample size can be considered around the 6-month PFS endpoint. Without censoring, the 6-month PFS would be consistent with the proportion of participants who did not experience any progression or death within the first 6 months, which can be described by a binomial distribution.

Table 23 shows several possible real potential differences in efficacy in 6-month PFS, assuming that the proportion of participants in the least effective arm who did not experience any progression or death at 6 months was approximately 43% (6-month PFS observed in the nivolumab arm in the RELATIVITY-047 trial).

The table shows that the power is 70% for detecting a 20% difference in 6-month PFS (assuming 20% two-sided α), while the power drops to 53% when a 15% difference is present.

Table 23. Efficacy of several possible true 6-month PFS rates (assuming no censoring before 6 months)

6-month PFS rate of the least effective arm 6-month PFS rate of the most effective arm power 43% 53% 36% 43% 58% 53% 43% 63% 70%

PFS = Progression-free survival

Analysis settings

For analytical purposes, the definition of a group is shown in Table 24.

Table 24. Analytical Groups

Demographic and baseline characteristics

Study enrollment, study drug administration, reasons for study drug discontinuation, and reasons for study termination are summarized by treatment arm. Demographics (including age, sex, and self-reported race/ethnicity) and baseline disease characteristics (e.g., ECOG performance status) are summarized overall and by treatment arm. Descriptive statistics (mean, median, standard deviation, and range) are provided for continuous data, and frequencies and percentages are provided for categorical data, as needed.

Baseline measurements are the last available data obtained before participants receive the study treatment.

Therapeutic effect analysis

Primary and secondary efficacy analyses included all participants in the intention-to-treat (ITT) population, who were grouped according to the dose level assigned at random. The statistical methods used for efficacy analysis are shown in Table 25.

Table 25. Statistical Analysis Methods for Treatment Efficacy

CI = Confidence Interval; CR = Complete Response; DCR = Disease Control Rate; DoR = Duration of Response; EORTC = European Organisation for Research and Treatment of Cancer.

HR = hazard ratio; ORR = objective response rate; OS = overall survival; PFS = progression-free survival; PR = partial response; RECIST = criteria for evaluating the efficacy of treatment in solid tumors.

Example 4: A phase Ib/II, open-label, multicenter, randomized umbrella study evaluating the efficacy and safety of multiple immunotherapy-based treatment combinations in patients with advanced hepatocellular carcinoma (MORPHEUS).

A. Research Design

GO42216 is a phase Ib/II, open-label, multicenter, randomized umbrella study evaluating the efficacy, safety, and pharmacokinetics of immunotherapy-based treatment combinations in patients with advanced hepatocellular carcinoma. The study is designed to be flexible enough to open new treatment arms when new therapies become available, close existing treatment arms that demonstrate minimal clinical activity or unacceptable toxicity, adjust the patient cohort (e.g., based on prior anticancer therapy or biomarker status), or introduce additional cohorts of patients with other types of advanced primary hepatocellular carcinoma (e.g., intrahepatic cholangiocarcinoma (iCCA)).

Cohort 1 enrolled patients with locally advanced or metastatic hepatocellular carcinoma (HCC) who had not received prior systemic therapy for their disease (Figures 10 and 11). Eligible patients were initially randomized to one of several treatment arms (Phase 1). Patients experiencing loss of clinical benefit or unacceptable toxicity during Phase 1 may be eligible for treatment using different combinations of therapies (Phase 2).

Phase 1

During Phase 1, patients were randomly assigned to either the control arm (atezolizumab plus bevacizumab (Atezo+bev)) or the experimental arm consisting of a combination of bevacizumab and RO7247669 (RO7247669+bev). The specific objectives and corresponding endpoints of the study are outlined in Table 26 below.

Table 26. Statistical Analysis Methods for Treatment Efficacy

ADA = Anti-drug antibody; DOR = Duration of response; HCC = Hepatocellular carcinoma; HCC mRECIST = HCC-specific modified RECIST; NCI CTCAE v5.0 = National Cancer Institute Common Terminology Standard for Adverse Events version 5.0; ORR = Objective response rate; OS = Overall survival; PFS = Progression-free survival; PK = Pharmacokinetics; RECIST v1.1 = Standards for Efficacy Evaluation in Solid Tumors version 1.1. Note: Researchers used RECIST v1.1 and HCC mRECIST to assess overall response at individual time points.

Approximately 170 to 320 patients were enrolled during Phase 1. Enrollment for each experimental arm was conducted in two phases: a preliminary phase and an extension phase. For most arms, approximately 20 patients were enrolled during the preliminary phase. If clinical activity was observed in the experimental arm during the preliminary phase, approximately 20 additional patients could be enrolled in that arm during the extension phase. In some arms, randomization was paused to allow for safety assessment in a minimum of 6 patients. Experimental arms with minimal clinical activity or unacceptable toxicity were not extended. Additional patients could be enrolled to ensure a balance between treatment arms in terms of demographics and baseline characteristics, including potential predictive biomarkers, for further subgroup analyses.

New experimental arms can be added during the study by revising the protocol. Phase 1 patients are randomized to the treatment arm, and the randomization rate depends on the number of open-enrollment experimental arms (e.g., if an arm is added or enrollment is paused for preliminary phase results analysis), specifying that the probability of being assigned to the control arm will not exceed 35%. Randomization considers arm-specific exclusion criteria. If a patient meets any exclusion criteria outlined for that arm, they are not eligible for inclusion in that arm. Table 27 provides details on treatment allocation and randomization.

Table 27. Treatment Plan for Phase 1

Atezo = Atezolizumab; Bev = Bevacizumab; Q2W = every 2 weeks; Q3W = every 3 weeks.

Enrollment for ^RO7247669 2100mg Q2W+Bev arm was paused to allow for safety assessment of at least 6 patients.

Patients in the control and experimental arms continued treatment until unacceptable toxicity or loss of clinical benefit was determined based on a comprehensive assessment of radiographic and biochemical data, local biopsy results (if any), and clinical status (e.g., worsening symptoms, such as disease-related pain). Because immune cell infiltration can lead to an initial increase in tumor burden in the context of a T-cell response to cancer immunotherapy (CIT) (known as pseudoprogression), radiographic progression according to the RECIST v1.1 criteria for efficacy evaluation in solid tumors may not indicate true disease progression. In the absence of unacceptable toxicity, patients who meet the RECIST v1.1 criteria for disease progression during CIT treatment will be permitted to continue study treatment if they meet all of the following criteria:

• Evidence of clinical benefit, as determined after reviewing all available data.

• There are no symptoms or signs indicating clear disease progression (including laboratory values such as new or worsening hypercalcemia).

• There were no Eastern Cooperative Oncology Group (ECOG) performance status declines attributable to disease progression.

• No tumor progression in key anatomical sites (e.g., leptomeningeal diseases) where medical interventions permitted by the protocol cannot resolve the issue.

Safety assessment period

Given the potential overlapping toxicities of RO7247669 2100 mg every 2 weeks (Q2W) + Bev arm, enrollment was paused after approximately 6 patients had been enrolled to allow for safety assessment. The safety assessment was based on safety data from a minimum of 6 patients who received at least one dose of treatment (i.e., one dose of each agent in a given combination) and completed safety follow-up assessments over at least one complete treatment cycle. Enrollment for that arm was resumed if the combination was determined to be adequately safe. The decision not to resume enrollment was based on ≥ grade 3 events related to the study treatment that were uncontrollable and resulted in at least one-third of patients discontinuing all study drugs.

Phase 2

Patients in the control or experimental arm who experience loss of clinical benefit (as described above) during Phase 1 may be eligible to receive a different combination of treatments during Phase 2, provided they meet the eligibility criteria and the Phase 2 arm is open for enrollment. Patients who experience unacceptable toxicity during Phase 1 may be eligible to receive treatment during Phase 2.

Phase 2 treatment must be initiated within 3 months of the patient experiencing loss of clinical benefit or unacceptable toxicity in Phase 1 and must continue until unacceptable toxicity or loss of clinical benefit is achieved. However, it is recommended that patients begin Phase 2 treatment as soon as possible. There are currently no available Phase 2 treatment options.

Assessment and monitoring

Adverse events in all patients were closely monitored throughout the study and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0 (NCI CTCAE v5.0). The severity of cytokine release syndrome (CRS) was also graded according to the American Society for Transplantation and Cell Therapy CRS Consensus Grading Scale.

Patients underwent tumor assessment every 6 weeks (starting from day 1 of cycle 1) during the first 48 weeks, followed by assessment every 6 or 12 weeks. Responses were assessed using RECIST v1.1 and HCC-specific modified RECIST (HCC mRECIST).

Baseline tumor tissue samples were collected from all patients, preferably via biopsy performed upon entry into the study. These samples were used for biomarker studies.

To characterize the pharmacokinetic (PK) properties and/or immunogenicity of the therapeutic agent, blood samples were extracted at different time points before and during treatment administration.

Based on a review of real-time safety data and available PK data, treatment plans can be modified when deemed appropriate.

target group

Inclusion criteria for Phase 1

Patients must meet all of the following criteria to be eligible to enter Phase 1:

• Age ≥ 18 years old.

• The ECOG fitness level is 0 or 1 within 7 days prior to randomization.

• Locally advanced or metastatic and/or unresectable HCC diagnosed in patients with cirrhosis by histological/cytological or clinical criteria of the American Association for the Study of Liver Diseases (AASLD). For patients with cirrhosis without histological confirmation, clinical confirmation according to AASLD criteria is required.

• Child-Pugh Grade A within 7 days prior to randomization.

• Diseases unsuitable for radical surgery and/or localized therapy. Patients whose disease has progressed after surgery and/or localized therapy are eligible.

• No prior systemic treatment for HCC (including systemic investigational drugs) has been administered. Prior treatment with herbal remedies (including traditional Chinese medicine) that are labeled as having anticancer activity is permitted, provided that these drugs have been discontinued prior to randomization.

• Life expectancy ≥ 3 months.

• Availability of representative tumor specimens suitable for determining PD-L1 and/or other biomarker status via central testing. Baseline tumor tissue samples are collected from all patients, preferably via biopsy performed upon entry into the study.

Inclusion criteria for Phase 1 and Phase 2

Patients must meet all of the following criteria to be eligible to enter Phase 1 and Phase 2:

• The ability to adhere to research protocols.

• Measurable disease (at least one target lesion) according to RECIST v1.1. Patients who have received prior local regional therapy (e.g., radiofrequency ablation, percutaneous ethanol or acetic acid injection, cryoablation, high-intensity focused ultrasound, transarterial chemoembolization, transarterial embolization, etc.) are eligible if the target lesion was not previously treated with local regional therapy or if the target lesion within the local therapy area has subsequently progressed according to RECIST v1.1.

• Appropriate blood and end-organ function, defined by the following laboratory test results obtained within 7 days prior to the initiation of study treatment:

- Without granulocyte colony-stimulating factor support, ANC ≥ ^{1.5 x 10⁹} / L (≥ 1500 / μL).

- Lymphocyte count ≥0.5× ^10⁹ /L (≥500/μL).

- Without blood transfusion, platelet count ≥75× _10⁹ /L (≥75,000/μL).

- Without blood transfusion, hemoglobin ≥90g/L (≥9.0g/dL).

- Patients must not require blood transfusions to meet this criterion during the screening period or within 2 weeks prior to screening.

-AST, ALT, and ALP ≤ 5 × upper limit of normal (ULN).

- Bilirubin ≤3×ULN.

- Creatinine ≤ 1.5 × ULN or creatinine clearance ≥ 50 mL/min (calculated using the Cockcroft-Gault formula).

- In the absence of blood transfusion, albumin ≥28g/L (≥2.8g/dL).

- For patients who have not received anticoagulation therapy: INR or aPTT ≤ 1.5 × ULN.

• Documented virological status of hepatitis, such as confirmed by screening tests for hepatitis B virus (HBV) and hepatitis C virus (HCV). For patients with active HBV: HBV DNA <500 IU/mL during screening, initiation of anti-HBV therapy at least 14 days prior to randomization, and willingness to continue anti-HBV therapy (according to local standards of care; e.g., entecavir) during the study. HCV patients are eligible, whether the infection has regressed (proven by detectable antibodies) or is chronic (proven by detectable HCV RNA).

• The HIV test was negative during screening.

• For women of childbearing age: Agree to abstain from sex (avoid heterosexual intercourse) or use contraception.

For men: Agree to abstain from sex (avoid heterosexual intercourse) or use contraception, and agree not to donate sperm.

Selection criteria for Phase 2

Patients must meet all of the following criteria to be eligible to enter Phase 2:

• ECOG fitness level is 0, 1, or 2.

• Candidates can begin Phase 2 treatment within 3 months of experiencing unacceptable toxicity or loss of clinical benefit unrelated to atezolizumab or RO7247669 during Phase 1 treatment.

• Availability of tumor specimens from biopsies performed at the time of the Phase 1 suspension (if clinically feasible).

Exclusion criteria

Patients who meet any of the following criteria are excluded from enrollment in a specific arm during Phase 1 or from enrollment during Phase 2.

Exclusion criteria for Phase 1

Patients meeting any of the following criteria will be excluded from Phase 1:

• Prior treatment with CD137 agonists or immune checkpoint blockade therapy, including anti-CTLA-4, anti-PD-1, and anti-PD-L1 therapeutic antibodies.

• Treat with the investigational therapy within 28 days prior to the start of the investigational treatment.

• Patients who received local liver therapy (e.g., radiofrequency ablation, percutaneous ethanol or acetic acid injection, cryoablation, high-intensity focused ultrasound, transarterial chemoembolization, transarterial embolization, etc.) within 28 days prior to the start of the study, or who have not recovered from any side effects of such procedures.

• Untreated or incompletely treated esophageal and/or gastric varices with bleeding or a high risk of bleeding.

• Patients must undergo esophagogastric-duodenal endoscopy (EGD) and all variceal sizes (from smallest to largest) must be assessed and treated according to local standards of care prior to enrollment. Patients who underwent EGD within 6 months prior to starting study treatment do not need to repeat the procedure.

• Previous bleeding events due to esophageal and/or gastric varices within 6 months prior to the start of the study treatment.

• Adverse events from previous cancer therapies have not yet subsided to Grade 1 or better, except for any grade of hair loss.

• Inadequate blood pressure control is defined as systolic blood pressure (BP) > 150 mmHg and/or diastolic blood pressure > 100 mmHg (the average of at least three of two or more readings). Antihypertensive treatment is permitted to achieve these parameters.

• History of hypertensive crisis or hypertensive encephalopathy.

• Significant vascular disease within 6 months prior to the start of treatment (e.g., aortic aneurysm requiring surgical repair or recent peripheral artery thrombosis).

• A history of hemoptysis (≥2.5 mL of bright red blood per episode) within one month prior to the start of the study treatment.

• Evidence of bleeding diathesis or significant coagulation disorder (in the absence of therapeutic anticoagulation).

• Currently or recently (≤10 days before the start of study treatment) treated with aspirin (>325 mg/day) or with clopidogrel, dipyridamole, ticlopidine or cilostazol.

• Current or recent (≤10 days prior to study treatment initiation) use of full-dose oral or parenteral anticoagulants or thrombolytics for therapeutic (not prophylactic) purposes. Prophylactic anticoagulation is permitted for the development of intravenous access devices, provided that the drug activity causes an INR <1.5 x ULN and the aPTT is within the normal range within 14 days prior to study treatment initiation. For prophylactic use of anticoagulants or thrombolytic therapy, the approved dose described on the local label may be used.

• Perform a core biopsy or other minor surgical procedure within 3 days prior to the start of the study treatment, excluding the placement of vascular access devices.

• A history of abdominal or tracheoesophageal fistula, gastrointestinal (GI) perforation, or intra-abdominal abscess within 6 months prior to the start of the study treatment.

• Patients with a history of intestinal obstruction and/or clinical signs or symptoms of GI obstruction, including sub-occlusive or obstructive syndromes associated with an underlying disease, or who require routine parenteral hydration, parenteral nutrition, or tube feeding prior to the initiation of study treatment. Patients with signs or symptoms of sub-occlusive or obstructive syndromes or intestinal obstruction at the time of initial diagnosis may be enrolled if they have received definitive (surgical) treatment to resolve their symptoms.

• Evidence of free air in the abdomen that cannot be explained by paracentesis or recent surgery.

• Critically ill, non-healing or lacerated wounds, active ulcers, or untreated fractures.

• Grade 2 proteinuria, as demonstrated by ≥2+ protein in urine strip analysis and ≥1.0g protein in 24-hour urine collection. All patients with ≥2+ protein in urine strip analysis at screening must undergo 24-hour urine collection to obtain protein. Patients with <2+ protein in urine strip analysis are eligible for the study.

• Metastatic disease involving major airways or blood vessels, or large central mediastinal tumors (<30mm from the carina). Patients with portal vein or hepatic vein invasion may be included.

• History of intra-abdominal inflammatory processes within 6 months prior to the initiation of treatment, including but not limited to peptic ulcer disease, diverticulitis, or colitis.

• Radiation therapy within 28 days prior to the start of study treatment or abdominal/pelvic radiation therapy within 60 days prior to the start of study treatment, except for palliative radiation therapy to bone lesions within 7 days prior to the start of study treatment.

• Major surgery, open biopsy, or significant trauma within 28 days prior to the start of the study treatment; or abdominal surgery, abdominal intervention, or severe abdominal trauma within 60 days prior to the start of the study treatment; or anticipated need for major surgery during the study or failure to recover from any side effects of such surgery.

• Long-term daily use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treatment. Occasional use of NSAIDs is permitted to relieve symptoms of medical conditions such as headaches or fever.

Exclusion criteria for Phase 1 and Phase 2

Patients meeting any of the following criteria will be excluded from Phase 1 and Phase 2:

• Known fibrolamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC.

• History of hepatic encephalopathy.

• Moderate or severe ascites.

• Co-infection with HBV and HCV. Patients with a history of HCV infection but negative HCV RNA on polymerase chain reaction (PCR) are considered uninfected with HCV.

• Symptomatic, untreated, or actively progressing central nervous system (CNS) metastases. Asymptomatic patients with treated CNS lesions are eligible, provided they meet all of the following criteria:

According to RECIST v1.1, measurable disease must exist outside the CNS.

The patient has no history of intracranial hemorrhage or spinal cord hemorrhage.

- Patients who did not receive stereotactic radiotherapy within 7 days prior to the start of the study treatment, whole-brain radiotherapy within 14 days prior to the start of the study treatment, or neurosurgical resection within 28 days prior to the start of the study treatment.

The patient does not have a sustained need for corticosteroids as a treatment for CNS disease. Stable doses of anticonvulsant therapy are permitted.

- Metastasis is limited to the cerebellum or supratentorial region (i.e., no metastasis to the midbrain, pons, medulla or spinal cord).

- There is no evidence of intermediate progress between completion of CNS-targeted therapy and initiation of study treatment. Asymptomatic patients newly diagnosed with CNS metastases at screening who have received radiation therapy or surgery are eligible for the study and do not require repeat brain scans.

• History of leptomeningeal disease.

• Uncontrolled tumor-related pain. Patients requiring analgesia must be on a stable treatment regimen upon enrollment. Symptomatic lesions suitable for palliative radiotherapy (e.g., bone metastases or metastases causing nerve contact) should be treated prior to enrollment. Patients should have recovered from the effects of radiotherapy. A minimum recovery period is not required. Asymptomatic metastatic lesions that may lead to functional impairment or intractable pain if they continue to grow (e.g., epidural metastases currently unrelated to spinal cord compression) should be considered for local regional therapy (if appropriate) prior to enrollment.

• Uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage (once a month or more frequently). Patients with indwelling catheters (e.g., [insert catheter here]) are permitted.

• Uncontrolled or symptomatic hypercalcemia (ionized calcium > 1.5 mmol/L, calcium > 12 mg/dL, or corrected calcium > ULN).

• Active or history of autoimmune diseases or immunodeficiency, including but not limited to myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, antiphospholipid syndrome, Wegener's granulomatosis, Sjögren's syndrome, Guillain-Barré syndrome, or multiple sclerosis, except in the following cases:

Patients with a history of autoimmune-related hypothyroidism and who are receiving a stable dose of thyroid replacement hormone are eligible for this study.

Patients with type 1 diabetes who are receiving an insulin regimen are eligible for this study.

- Patients with eczema, psoriasis, chronic simple lichen simplex, or vitiligo with only dermatological manifestations (e.g., excluding patients with psoriatic arthritis) are eligible for this study if they meet all of the following criteria:

- The rash must cover <10% of the body surface area.

- The disease is well controlled at baseline and only requires the use of ineffective topical corticosteroids.

- No acute exacerbations of underlying conditions requiring psoralen plus UVA radiation, methotrexate, retinoids, biologics, oral calcineurin inhibitors, or potent or oral corticosteroids have occurred in the past 12 months.

• A history of idiopathic pulmonary fibrosis, organizing pneumonia (e.g., bronchiolitis obliterans), drug-induced lung inflammation, or idiopathic lung inflammation, or evidence of active lung inflammation on chest computed tomography. A history of radiation-induced pneumonia (fibrosis) in a radiation field is permissible.

• Active tuberculosis (TB) with a positive record of purified protein derivative (PPD) skin test or TB blood test, confirmed by a positive chest X-ray within 3 months prior to the start of study treatment. Patients with a positive PPD skin test or TB blood test followed by a negative chest X-ray may be eligible for this study.

• Patients must have significant cardiovascular disease (such as New York Heart Association Class II or higher heart disease, myocardial infarction, or stroke), unstable arrhythmia, or unstable angina within 3 months prior to starting the study treatment.

• Had major surgery (excluding diagnostic procedures) within 4 weeks prior to the start of the study treatment or is expected to need major surgery during the study.

• Patients with a history of malignant tumors other than HCC within the 5 years prior to screening, but with negligible risk of metastasis or death (e.g., 5-year OS rate >90%), such as adequately treated cervical carcinoma in situ, non-melanoma skin cancer, localized prostate cancer, ductal carcinoma in situ, or stage I uterine cancer.

• A serious infection occurred within 4 weeks prior to the initiation of study treatment, including but not limited to hospitalization due to complications of infection, bacteremia, or severe pneumonia, or any active infection that may affect patient safety.

• Treat with therapeutic oral or IV antibiotics within 2 weeks prior to the start of the study treatment.

Patients receiving prophylactic antibiotics (e.g., to prevent urinary tract infections or exacerbations of chronic obstructive pulmonary disease) are eligible for this study.

• Previous allogeneic stem cell or solid organ transplantation.

• Any other disease, metabolic disorder, physical examination findings, or clinical laboratory findings that preclude the use of the investigational drug, may affect the interpretation of the results, or may put the patient at high risk of treatment complications.

• Pregnant or breastfeeding, or planning to become pregnant during the study.

• Treat with a live attenuated vaccine within 4 weeks prior to the start of the study treatment.

• History of severe allergic reactions to chimeric antibodies, humanized antibodies, or fusion proteins.

• It is known to be hypersensitive to Chinese hamster ovary cell products or recombinant human antibodies.

• Known allergy or hypersensitivity to any investigational drug or its excipients.

• Treat with systemic immunostimulants (including, but not limited to, interferon and interleukin-2) within 4 weeks prior to the start of study treatment or within 5 drug elimination half-lives (whichever is longer).

• Treatment with systemic immunosuppressants (including but not limited to corticosteroids, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-tumor necrosis factor agents) within 2 weeks prior to the start of study treatment, or anticipated to require systemic immunosuppressants during study treatment, except in the following circumstances:

Patients receiving acute, low-dose systemic immunosuppressants or a single pulse dose of systemic immunosuppressants (e.g., 48-hour corticosteroids for contrast agent allergy) are eligible.

Patients receiving mineralocorticoids (e.g., fludrocortisone), corticosteroids for chronic obstructive pulmonary disease or asthma, or low-dose corticosteroids for the treatment of orthostatic hypotension or adrenal insufficiency are eligible for this study.

• Grade 3 bleeding or bleeding events occurring within 8 weeks prior to the start of the study treatment

• Patients entering Phase 2: At the time of consent, immunotherapy-related adverse events have not yet precipitated to Grade 1 or better or baseline, except for patients with persistent endocrine events that are adequately managed with complementary therapies.

Exclusion criteria for arms containing RO7247669

Patients meeting any of the following criteria were excluded from arms containing RO7247669 during Phase 1:

• Previous treatment with anti-lymphocyte activation gene 3 (LAG-3) agents.

• Within 6 months of initial drug administration, the left ventricular ejection fraction (LVEF) assessed by transthoracic echocardiography (TTE) or multi-gated acquisition (MUGA) scan (TTE is the preferred test) is <50%.

• Troponin T (TnT) or Troponin I (TnI) > institutional ULN. Patients with TnT or TnI levels between >1 and <2x ULN are eligible if the repeated level over 24 hours is ≤1x ULN. Patients with repeated levels over 24 hours between 1x and <2x ULN may undergo cardiac evaluation and be considered for treatment.

Study completion and duration

The study is defined as ending on the date the last patient completes their final visit, including survival follow-up via telephone or at the clinic. The total length of the study (from the screening of the first patient to the study endpoint) is expected to be approximately 3–5 years.

Basic principles of RO7247669 dosage and schedule

RO7247669 was administered at a fixed dose of 600 mg Q3W (600 mg on day 1 of each 21-day cycle), 2100 mg Q2W (2100 mg on days 1 and 15 of each 28-day cycle), or 1200 mg Q3W (1200 mg on day 1 of each 21-day cycle) to determine the optimal dose of RO7247669 in combination with bevacizumab for the treatment of patients with HCC. The fixed dosing regimen of 600 mg Q3W was selected based on existing clinical pharmacokinetic, efficacy, and safety data from the NP41300 study. During the dose escalation phase A of the study, RO7247669 was well tolerated in patients, and no specific safety issues associated with RO7247669 were identified. No drug-induced tumor thrombosis (DLT) was observed up to the maximum dose of 2100 mg every two weeks (Q2W), and no median drug-induced tumor thrombosis (MTD) was identified. Antitumor activity was observed from the 600 mg Q2W dose as measured by radiographic PR. The pharmacokinetics of RO7247669 were dose-linear within the dose range tested in study NP41300. Intratumoral PD-1 and LAG3 target binding was further modeled using estimated target properties. It was estimated that a dose and schedule of RO7247669 600 mg Q3W would result in >90% PD-1 and LAG3 target binding at the tumor site throughout the treatment period. Furthermore, RO7247669 was well-tolerated in good laboratory-standard toxicology and dose-range searching monkey toxicology studies up to a maximum dose of 100 mg/kg. The toxicological results are consistent with those reported in studies using commercially available CPIs in cynomolgus monkeys.

Basic principles of bevacizumab dosage and schedule

In the Q3W treatment arm, bevacizumab is administered at a dose of 15 mg/kg Q3W (15 mg/kg on day 1 of each 21-day cycle), which is the approved dose of bevacizumab.

In the Q2W treatment arm, bevacizumab is administered at a dose of 10 mg/kg Q2W (10 mg/kg on days 1 and 15 of each 28-day cycle), which is the approved dose of bevacizumab.

Control arm (atezo + bev)

Patients in the atezolizumab plus bevacizumab (Atezo+bev) arm received treatment as outlined in Table 28 until unacceptable toxicity or loss of clinical benefit was determined based on a comprehensive assessment of imaging and biochemical data, local biopsy results (if any), and clinical status (e.g., worsening symptoms, such as disease-related pain). Treatment is recommended to be initiated no later than 7 days after randomization; however, the first dose of study treatment should not be administered within 3 days of a core biopsy or other surgical procedure.

Table 28. Treatment regimen for Atezo + BEV arm

Atezo + Bev = Atezolizumab plus bevacizumab.

^On day 1 of each cycle, bevacizumab is administered at least 5 minutes after the completion of atezolizumab infusion.

RO7247669 2100mg Q2W+bev

Patients in the RO7247669 2100 Q2W+bev arm will receive treatment as outlined in Table 29 until unacceptable toxicity or loss of clinical benefit is determined based on a comprehensive assessment of imaging and biochemical data, local biopsy results (if any), and clinical status (e.g., worsening symptoms, such as disease-related pain). Treatment is recommended to be initiated no later than 7 days after randomization; however, the first dose of study treatment should not be administered within 3 days of a core biopsy or other surgical procedure.

Table 29. Treatment regimen for RO7247669 2100mg Q2W + bev arm

Q2W = every 2 weeks. RO7247669+Bev = RO7247669 plus bevacizumab.

^On day 1 of cycle 1, bevacizumab is administered at least 90 minutes after the completion of the RO7247669 infusion. If the first infusion is tolerated and there is no infusion-related response (IRR), bevacizumab is administered at least 60 minutes after the RO7247669 infusion. If the second infusion is tolerated and there is no IRR, bevacizumab is administered at least 30 minutes after all subsequent RO7247669 infusions.

RO7247669 1200mg Q3W+bev

Patients in the RO7247669 1200 Q3W+bev arm received treatment as outlined in Table 30 until unacceptable toxicity or loss of clinical benefit was determined by the investigators after a comprehensive assessment of imaging and biochemical data, local biopsy results (if any), and clinical status (e.g., worsening symptoms, such as disease-related pain). Treatment is recommended to be initiated no later than 7 days after randomization; however, the first dose of study treatment should not be administered within 3 days of a core biopsy or other surgical procedure.

Table 30. Treatment regimen for RO7247669 1200mg Q3W + bev arm

Q3W = every 3 weeks. RO7247669+Bev = RO7247669 plus bevacizumab.

^On day 1 of cycle 1, bevacizumab is administered at least 90 minutes after the completion of the RO7247669 infusion. If the first infusion is tolerated and there is no infusion-related response (IRR), bevacizumab is administered at least 60 minutes after the RO7247669 infusion. If the second infusion is tolerated and there is no IRR, bevacizumab is administered at least 30 minutes after all subsequent RO7247669 infusions.

RO7247669 600mg Q3W+bev

Patients in the RO7247669 600Q3W+bev arm received treatment as outlined in Table 31 until unacceptable toxicity or loss of clinical benefit was determined by the investigators after a comprehensive assessment of imaging and biochemical data, local biopsy results (if any), and clinical status (e.g., worsening symptoms, such as disease-related pain). Treatment is recommended to be initiated no later than 7 days after randomization; however, the first dose of study treatment should not be administered within 3 days of a core biopsy or other surgical procedure.

Table 31. Treatment regimen for RO7247669 600mg Q3W + bev arm

Q3W = every 3 weeks. RO7247669+bev = RO7247669 plus bevacizumab.

^On day 1 of cycle 1, bevacizumab is administered at least 90 minutes after the completion of the RO7247669 infusion. If the first infusion is tolerated and there is no infusion-related response (IRR), bevacizumab is administered at least 60 minutes after the RO7247669 infusion. If the second infusion is tolerated and there is no IRR, bevacizumab is administered at least 30 minutes after all subsequent RO7247669 infusions.

B. Statistical methods

Preliminary analysis

The primary efficacy endpoint was the objective response rate (ORR) during Phase 1, as shown in Table 26. ORR was determined according to RECIST v1.1. Patients with missing or no response assessments were classified as nonresponders. The ORR for each arm, i.e., the proportion of patients with complete or partial response, and the 95% CI (Clopper-Pearson method), were calculated. The normal approximation of a binomial distribution was also used to calculate the difference in ORR between the experimental and control arms, and the 95% CI.

Determining the sample size

This study was designed to obtain preliminary efficacy, safety, and pharmacokinetic (PK) data on the administration of immunotherapy-based treatment combinations to patients with advanced hepatocellular carcinoma (HCC). Cohort 1 consisted of patients with locally advanced or metastatic HCC who had not received prior systemic therapy for their disease. During the study, approximately 170 to 320 patients were randomized to the control arm and the experimental arm.

Mid-term analysis

Interim analyses are expected to be conducted during the study, with the earliest (Phase 1) interim analysis occurring when at least one experimental arm has completed initial enrollment and patients have been followed up for at least 6 weeks. Based on the interim analysis of clinical activity of the experimental arm compared to the control arm, posterior probabilities can be used to guide further enrollment of the treatment arm. If the interim analysis indicates that the experimental arm has higher activity than the control arm, 20 additional patients may be enrolled for that arm.

An interim analysis was also conducted after approximately 15 patients had been enrolled in the Phase 2 treatment arm and followed up for at least 6 weeks. Further enrollment for that arm was discontinued if no clinical activity was observed.

C. Background and Basic Principles

The basic principles of patient groups

This study included patients with locally advanced or metastatic HCC who had not received prior systemic therapy for their disease, regardless of PD-L1 expression or the cause of HCC. This broad cohort is similar to that included in two studies that evaluated the combination of atezolizumab and bevacizumab in patients with HCC: study GO30140 (an initial phase Ib study) and study YO40245 (a randomized phase III study), which demonstrated statistically significant and clinically meaningful benefits of this combination compared to sorafenib.

Advanced HCC is an incurable disease with a high level of unmet medical need. Sorafenib is currently approved for first-line treatment of patients with advanced HCC and is considered the standard of care in this disease context. However, the prognosis remains poor, with a median overall survival (OS) of 6.5–10.7 months, and treatment is associated with significant toxicity. Despite recent demonstrations of the clinical benefit of atezolizumab plus bevacizumab, there remains a persistent need for more effective and better-tolerated combination therapies for patients with locally advanced or metastatic HCC.

Patients included in this study also needed to have adequate liver function, defined as Child-Pugh A. Patients with Child-Pugh B or C liver function had an increased risk of death due to underlying cirrhosis, which could obscure the appropriate assessment of treatment-related antitumor efficacy; therefore, these patients were excluded from the study. As noted by the expert panel convened by the American Association for the Study of Liver Diseases (AASLD), the proposed study cohort is the patient group recommended for initial studies of new agents or combinations of agents against HCC (Llovet et al., N Engl J Med, 359:378-380, 2008).

The basic principle of immunotherapy-based treatment following initial radiological advancements.

In studies of immunotherapeutic agents, complete remission, partial remission, and disease stabilization all occurred after radiographic evidence of a significant increase in tumor burden. This initial increase in tumor burden due to immune cell infiltration in the presence of a T-cell response is termed pseudoprogression (Hales et al., Ann Oncol, 21:1944-1951, 2010). Evidence of response following tumor growth has been observed in several tumor types. Furthermore, in some responding patients with radiographic evidence of progression, biopsies of new lesions or newly growing areas within existing lesions showed immune cells but no surviving cancer cells. Because a response can occur after pseudoprogression, this study allowed patients randomized to the immunotherapy-based treatment group to continue combination therapy after significant radiographic progression according to RECIST v1.1, provided the benefit-risk ratio was deemed favorable.

The basic principle of using HCC-specific modified RECIST

For advanced HCC, RECIST was found to be weakly associated with clinical benefits demonstrated in the SHARP trial and other sorafenib clinical studies (Llovet et al., N Engl J Med, 359:378-380, 2008; Liu et al., Clin Cancer Res, 20:1623-1631, 2014; Takada et al., BMC Res Notes, 8:609, 2015). Similar results were observed after localized therapies for HCC, such as radiofrequency ablation and chemoembolization. These therapies reduce tumor vascularization and induce necrosis, but do not necessarily cause changes in the overall tumor size. To provide a general framework for the design of HCC clinical trials, AASLD proposed a modified standard (HCC mRECIST), which quantifies only the feasible portion of the tumor to provide improved assessment endpoints (Llovet et al., J Natl Cancer Inst, 100:698-711, 2008; Lencioni and Llovet, Semin Liver Des, 30:52-60, 2010; Lencioni et al., J Hepatol, 66:1166-1172, 2017).

Given bevacizumab's anti-angiogenic modality, exploratory efficacy endpoints included analyses based on HCC mRECIST. These analyses allowed for the assessment of HCC mRECIST as a measure of efficacy relative to standard RECIST v1.1 in patients with locally advanced or metastatic HCC.

Background of liver cancer

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 diagnosis, most patients with primary liver cancer present with advanced disease, a stage where curative therapies are not recommended. The WHO estimates that more than one million people will die from liver cancer by 2030, highlighting this as a significant global public health issue (Villanueva, N Engl J Med, 380:1450-1462, 2019).

Background of hepatocellular carcinoma

Most hepatocellular carcinoma (HCC) occurs in patients with underlying liver disease, primarily due to hepatitis B virus (HBV) or hepatitis C virus (HCV) infection or alcohol abuse. HBV infection accounts for the majority of HCC cases globally; however, in Western countries and Japan, HCV is the leading cause of HCC (Villanueva, N Engl J Med, 380:1450-1462, 2019). Universal HBV vaccination and widespread implementation of direct-acting antiviral agents against HCV may alter the etiological landscape of HCC. However, the incidence of non-alcoholic fatty liver disease (NAFLD), a risk factor for HCC, is increasing globally, and NAFLD is rapidly becoming a leading cause of liver cancer in Western countries (Villanueva, N Engl J Med, 380:1450-1462, 2019).

Hepatocellular carcinoma (HCC) is a highly lethal disease, with the highest mortality-to-morbidity ratio (0.98) among all solid tumors (Kamangar et al., J Clin Oncol, 24:2137-2150, 2006). Due to late onset of symptoms, up to 80% of patients presenting with HCC for the first time have advanced, unresectable, or metastatic disease. It is a medically complex and difficult-to-treat disease because most patients with HCC have underlying cirrhosis, requiring simultaneous management of both the malignancy and the cirrhosis. In the United States, the 5-year overall survival (OS) rate for patients with HCC is 17%, dropping dramatically to only 3% if distant metastases are present (Siegel et al., CA Cancer J Clin, 66:7-30, 2016). In China, the 5-year OS rate for patients with HCC is 10.1% (Chen et al., CA Cancer J Clin, 66:115-132, 2016).

First-line treatment for advanced hepatocellular carcinoma

Sorafenib is an oral multi-kinase inhibitor and is currently considered the global standard of care for first-line treatment of patients with advanced hepatocellular carcinoma (HCC). The efficacy of sorafenib has been demonstrated in two large, multicenter, randomized, double-blind, placebo-controlled phase III trials: the Sorafenib HCC Assessment Randomization Protocol (SHARP) trial and a trial conducted in the Asia-Pacific region. Both studies demonstrated a survival benefit of sorafenib compared to placebo. In the SHARP trial, the median overall survival (OS) for sorafenib was 10.7 months, compared to 7.9 months for placebo; in the Asia-Pacific trial, the median OS was 6.5 months versus 4.2 months. A benefit was also demonstrated in the median time to radiographic progression: 5.5 months versus 2.8 months in the SHARP trial, and 2.8 months versus 1.4 months in the Asia-Pacific trial. The objective response rate (ORR) in the SHARP trial (according to RECIST v1.0) was 2.3% (7 out of 299 patients), and the ORR in the Asia Pacific trial was 3.3% (5 out of 150 patients). The shorter OS and duration of benefit in the Asia Pacific trial may be due to the fact that patients had more advanced disease at recruitment, and may also be due to regional differences in etiology and supportive care (Llovet et al., NEngl J Med, 359:378-380, 2008; Cheng et al., Eur J Cancer, 48:1452-1465, 2009; Cheng et al., Lancet Oncol, 10:25-34, 2012).

Although both Phase III studies reported survival benefits, the overall benefit-risk ratio of sorafenib was not high due to known toxicities. Common adverse events reported in both sorafenib studies included hand-foot skin reaction, diarrhea, hypertension, weight loss, fatigue, anorexia, alopecia, nausea, and rash/desquamation. Since the approval of sorafenib, several head-to-head negative Phase III studies have been conducted comparing it to sorafenib (including sunitinib, brinibril, and linivab) (Cheng et al., J Clin Oncol, 31:4067-4075, 2013; Johnson et al., J Clin Oncol, 31:3517-3524, 2013; Cainap et al., J Clin Oncol, 33:172-179, 2015). Although first-line treatment with lenvatinib (a multi-target receptor tyrosine kinase inhibitor) has shown to be non-inferior to sorafenib in terms of overall survival (OS), the clinically meaningful difference in OS compared to care has remained elusive until recently (Kudo et al. 2018).

Study YO40245 (IMbrave150) was a randomized phase III trial evaluating atezolizumab plus bevacizumab in patients with advanced or metastatic HCC compared to sorafenib as first-line therapy. This study was the first to demonstrate, in a head-to-head comparison with sorafenib, a novel treatment combination that showed statistically and clinically significant improvements in overall survival (OS) and progression-free survival (PFS). At the time of preliminary analysis, the atezolizumab plus bevacizumab arm had a 42% reduction in the risk of death compared to the sorafenib arm (stratified hazard ratio (HR) = 0.58 (95% CI: 0.42 to 0.79); p = 0.0006; median OS, not estimable (NE), was 13.24 months). Independent review of PFS according to RECIST v1.1 also demonstrated statistically significant and clinically meaningful improvements favoring combination therapy (stratified HR = 0.59 (95% CI: 0.47 to 0.76); p < 0.0001; median PFS, 6.83 vs. 4.27 months). Overall, in HCC, the atezolizumab plus bevacizumab combination is generally well-tolerated and has manageable toxicities, with a safety profile consistent with known risks in individual studies and with underlying disease (Finn et al., N Engl J Med, 382:1894-1905, 2020).

Research basic principles

Cancer immunotherapy (CIT) has demonstrated significant clinical efficacy, with notable survival benefits observed in a variety of advanced malignancies. Currently, the prevailing CIT approach involves targeting T-cell suppressor factors such as PD-L1/PD-1 to circumvent immune escape mechanisms and revitalize anti-tumor responses. While these targets have yielded significant clinical success across various cancer indications, ongoing research indicates that a series of stepwise events is essential for generating a sustained anti-tumor immune response (Chen and Mellman, Immunity, 39:1-10, 2013). Each event is crucial for an effective response, and each is also susceptible to several tumor immune escape mechanisms. Therefore, identifying and mitigating the various factors leading to a lack of effective anti-cancer immune responses is critical for disseminating cancer immunotherapy and advancing the field of CIT, most likely through combination targeted therapy regimens.

The current phase Ib/II umbrella study is designed to accelerate the development of CIT portfolios by identifying early signs and building proof-of-concept clinical data in patients with advanced hepatocellular carcinoma.

This study assesses the importance of simultaneously targeting multiple immune evasion mechanisms through immune cell initiation and activation, tumor infiltration, and/or recognition of tumor cells for elimination. To improve the confidence level of clinical signal detection in the experimental arm, a control arm was included in the study. Furthermore, patients experiencing disease progression during the initial treatment regimen (Phase 1) may qualify for continued treatment with a different regimen (Phase 2), which could contribute to a scientific understanding of immune evasion mechanisms in patients who fail to respond or experience disease progression during treatment with CIT regimens.

The study initially recruited patients with locally advanced or metastatic HCC who had not received prior systemic therapy for their disease. Despite recent demonstrations of the clinical benefit of atezolizumab plus bevacizumab, a significant unmet medical need remains for patients with unresectable locally advanced or metastatic HCC, requiring further evaluation of novel and more effective treatment combinations.

Table 32 summarizes the targets and proposed mechanisms of action for each investigational drug (IMP).

Table 32. Classification of Targets and Proposed Mechanisms of Action of Drugs in Experimental Studies

IMP = Research Drug; LAG-3 = Lymphocyte Activation Gene-3; NK = Natural Killer; VEGF = Vascular Endothelial Growth Factor.

^Wallin et al., Nat Commun, 7:12624, 2016.

D. Safety assessment

Safety assessment consists of monitoring and recording adverse events, including serious adverse events and adverse events of particular concern, performing safety laboratory assessments as specified in the protocol, measuring vital signs as specified in the protocol, and conducting other tests as specified in the protocol that are deemed essential to the safety assessment of the study.

According to ICH guidelines for good clinical practice, an adverse event is defined as any adverse medical event that occurs in subjects using a drug in a clinical study, regardless of its causal relationship. Therefore, an adverse event can be any of the following:

• Any adverse or unexpected signs (including abnormal laboratory test results), symptoms, or illnesses that are temporarily associated with the use of the drug, whether or not they are considered to be related to the drug.

• Any new disease or worsening of an existing disease (a worsening of the characteristics, frequency, or severity of a known disease).

• Recurrence of intermittent medical conditions (e.g., headache) that were not present at baseline.

• Any worsening of symptom-related laboratory values or other clinical tests (e.g., ECG, X-ray) or that leads to changes in or termination of study treatment or concomitant treatment.

• Adverse events related to the intervention specified in the protocol, including adverse events that occurred prior to the allocation of study treatment (e.g., screening invasive procedures, such as biopsy).

E. Statistical considerations and analysis plan

Final study analyses are based on patient data collected upon study termination. Unless otherwise specified, efficacy analyses are based on the efficacy-evaluable population, defined as all patients receiving at least one dose of each drug in their assigned treatment regimen, and safety analyses are based on the safety-evaluable population, defined as all patients receiving any amount of study treatment.

The analysis results are summarized according to the actual treatment regimens received by patients and by stage (stage 1 or stage 2). Data are described and summarized by sample size. Continuous variables are summarized using mean, standard deviation, median, and range. Categorical variables are summarized using counts and percentages. If the sample size is small, lists are used instead of tables.

New baseline values were established for the Phase 2 efficacy and safety analysis.

Primary therapeutic endpoint

The primary efficacy endpoint is the objective response rate (ORR) during Phase 1, as defined above. ORR is determined according to RECIST v1.1. Patients with missing or non-response assessments will be classified as non-responders.

ORR, i.e., the proportion of patients achieving complete or partial remission, and 95% CI (Clopper-Pearson method), were calculated for each arm. A normal approximation of the binomial distribution was also used to calculate the ORR difference between the experimental and control arms, and 95% CI.

Secondary efficacy endpoints

Secondary efficacy endpoints were PFS, OS, OS at a specific time point (e.g., 6 months), duration of response (DOR), and disease control during phase 1, as defined above. PFS, DOR, and disease control were determined according to RECIST v1.1.

DOR is derived for patients with evaluable efficacy of complete or partial remission. For patients who did not have a recorded disease progression or death during the study, PFS and DOR were censored on the last day of tumor assessment. Patients still alive at the time of OS analysis were censored on the last date on which their survival was known.

The Kaplan-Meier method was used to estimate the median PFS, OS, and DOR, and a 95% CI was constructed using the Brookmeyer and Crowley methods. The Kaplan-Meier method was also used to estimate the OS rate at specific time points, and a 95% CI was calculated based on the Greenwood estimate against variance. Disease control rate (proportion of patients with stable disease for ≥12 weeks), partial response, or complete response were calculated for each treatment arm, and a 95% CI was estimated using the Clopper-Pearson exact method.

Exploratory therapeutic endpoints

The exploratory efficacy endpoints were ORR, PFS, DOR, and disease control during Phase 1 as determined by HCC mRECIST; and ORR, PFS, DOR, and disease control during Phase 2 as determined by RECIST v1.1 and HCC mRECIST. ORR, PFS, DOR, and disease control were analyzed using the same methods described above. DOR was obtained for patients achieving complete or partial remission that could be evaluated for efficacy.

Detailed research information on F.RO7247669+bev arm-specificity

Background of RO7247669

RO7247669 is a novel fragment-crystallizable (Fc)-silenced IgG1-based bispecific antibody (bsAb) in a 1+1 format, incorporating monovalent binding to two immune checkpoint proteins: PD-1 and lymphocyte activation gene-3 (LAG-3). RO7247669 is engineered to target dysfunctional tumor antigen-specific T lymphocytes (expressing PD-1 and LAG-3) to establish or reconstitute an effective anti-tumor immune response in cancer patients, potentially eliciting better therapeutic responses than existing therapies. Furthermore, RO7247669 is engineered to prevent binding to the Fc-γ receptor, potentially circumventing tumor-associated macrophage resistance mechanisms observed in IgG4-based anti-PD-1 antibodies such as pembrolizumab and nivolumab (Arlauckas et al., Sci Transl Med, 9:389, 2017).

Clinical evaluation of RO7247669 is being conducted as a single agent in patients with or without prior checkpoint inhibitor (CPI) exposure in a first-in-human dose-finding study (NP41300).

Background of bevacizumab

Bevacizumab is a recombinant humanized monoclonal antibody that recognizes all isoforms of vascular endothelial growth factor (VEGF). It exerts a direct anti-angiogenic effect by binding to and scavenging VEGF in the tumor microenvironment. Additional antitumor activity may act on tumor angiogenesis, interstitial pressure, and vascular permeability, thereby providing enhanced chemotherapy delivery to tumor cells (Jain, Nat Med, 7:987-989, 2001).

Bevacizumab is approved for first-line and second-line treatment of metastatic colorectal cancer (mCRC), advanced non-small cell lung cancer (NSCLC), metastatic breast cancer, advanced renal cell carcinoma (RCC), and ovarian cancer, as well as the treatment of recurrent glioblastoma. Bevacizumab is currently being tested in combination with atezolizumab in Phase I-III clinical trials. Bevacizumab has been generally well-tolerated, and adverse events have been managed.

The basic principle of RO7247669 600mg Q3W+BEV arm

PD-1/PD-L1 pathway

Encouraging clinical data in the field of tumor immunotherapy demonstrate that therapies focused on enhancing T-cell anti-cancer responses can produce significant survival benefits in patients with advanced malignancies (Hodi et al., N Engl J Med, 363:711-723, 2010; Kantoff et al., N Engl J Med, 363:411-422, 2010; Chen et al., Clin Cancer Res, 18:6580-6587, 2012).

The PD-1/PD-L1 pathway serves as an immune checkpoint to temporarily suppress the immune response in the presence of chronic antigens, such as chronic infection or cancer. PD-1 is an inhibitory receptor expressed on activated and depleted T cells, including tumor-infiltrating CD8+ T cells that recognize mutated tumor antigens (neoantigens). Binding of PD-L1 to PD-1 inhibits T cell proliferation, activation, cytokine production, and cytolytic activity, resulting in a state of inactivated and depleted T cells (Butte et al., Immunity, 27:111-122, 2007; Yang et al., J Immunol, 187:1113-1119, 2011).

Treatment targeting the PD-1/PD-L1 pathway (as a single agent and in combination with chemotherapy and other targeted agents) to enhance anti-tumor T-cell responses has been clinically validated in a variety of solid tumors. Cancer immunotherapy (CIT) agents, particularly immune CPIs, have had a significant impact on the treatment of patients with advanced malignancies in recent years. However, despite their significant clinical efficacy, it has become clear that they are not sufficiently active as single therapies for many patients. To date, treatment with single-agent CPIs targeting the PD-1/PD-L1 pathway has shown minimal activity in patients with hepatocellular carcinoma (HCC).

LAG-3 pathway

LAG-3 is an immune checkpoint protein involved in the regulation of antitumor immunity and chronic infection. LAG-3 is expressed on a subset of activated T cells, B cells, natural killer (NK) cells, and tolerogenic plasmacytoid dendritic cells (DCs), and constitutively expressed on regulatory T cells (Huard et al., Immunogenetics, 39:213-217, 1994). Structurally similar to CD4, LAG-3 is a member of the Ig superfamily and binds to major histocompatibility complex class II (MHC-II). The interaction between LAG-3 and MHC-II inhibits T cell proliferation, activation, cytolysis, and the production of pro-inflammatory cytokines (Goldberg and Drake, Curr Top Microbiol Immunol, 344:269-278, 2011). The effect of LAG-3 expression on regulatory T cells remains controversial. An earlier report concluded that LAG-3 promotes T-regulatory cell-mediated immunosuppression (Camisaschi et al., J Immunol, 184:6545-6551, 2010). However, a more recent report by the same authors describes LAG-3 as limiting T-regulatory cell-mediated immunosuppression (Zhang et al., Sci Immunol, 2eaah4569, 2017).

LAG-3 has been reported to be expressed in a variety of tumor types, including breast cancer, ovarian cancer, NSCLC, melanoma, RCC, prostate cancer, and HCC, and is associated with poor prognosis (Matsuzaki et al., Proc Natl Acad Sci USA, 107:7875-7800, 2010; Baitsch et al., J Clin Invest, 121:2350-2360, 2011; Thommen et al., Cancer Immunol Res, 31344-55, 2015; He et al., Cancer Sci, 107:1193-1197, 2016; Norstrom et al., Oncotarget, 7:23581-23593, 2016). Clinical evaluation of anti-LAG-3 agents as a single agent and in combination with other CPIs is being conducted in several early studies in patients with advanced solid tumors (Long et al., Genes Cancer, 9(5-6):176-189, 2018).

Preliminary data indicate that anti-LAG-3 therapy, as a single agent and in combination with anti-PD-1 therapy, is well tolerable and its safety profile is consistent with that of other CPIs (Ascierto et al., Ann Onco, 28(Supplement 5): c611-612, 2017; Hong et al., J Clin Oncol, 36: 3012, 2018; Stratton et al., Society for Immunotherapy of Cancer (SITC) Abstract P325 2018).

Emerging clinical data highlight the role of the LAG-3 pathway in the pathogenesis of HCC. Compared to peripheral blood lymphocytes, tumor-infiltrating lymphocytes (TILs) in patients with HCC infected with hepatitis B virus showed significantly elevated LAG-3 expression, which was associated with severe functional T cell deficiency at the tumor site (Li et al., Immunol Lett, 150:1116-1122, 2013). Similar elevations in LAG-3 expression on TILs have been reported in patients with HCC who have not received prior systemic therapy for their disease, and no relationship has been identified between LAG-3 expression and any disease-related features, including viral status (Yarchoan et al., Clin Cancer Res, 23:7333-7339, 2017).

LAG-3 expression has recently been identified as a prognostic factor for poor survival in patients with HCC (in addition to vascular invasion, tumor size, and cirrhosis) (Guo et al., J Transl Med, 18:306, 2020).

In conclusion, targeting LAG-3 may represent an attractive strategy for treating patients with HCC.

VEGF pathway

VEGF-A is a pro-angiogenic molecule produced by endothelial cells, tumor cells, and tumor-associated macrophages. In addition to promoting tumor angiogenesis, increasing evidence suggests that the VEGF pathway also plays an important role in cancer immune evasion by exerting and maintaining an immunosuppressive tumor microenvironment through multiple mechanisms.

VEGF-A inhibits the maturation of dendritic cells (DCs), promotes the expression of inhibitory immune checkpoint molecules on intratumoral CD8+ T cells, and induces the expression of Fas ligand (FasL) on endothelial cells, thereby gaining the ability to kill effector CD8+ T cells rather than regulatory T cells (Gabrilovich et al., Nat Med, 2:1096-1103, 1996; Huang et al., Blood, 110:624-631, 2007; Motz et al., Nat Med, 20:607-615, 2014; Voron et al., J Exp Med, 212:139-148, 2015). Experiments using activated endothelial cells have also shown that VEGF may reduce lymphocyte adhesion to the blood vessel wall in the tumor microenvironment, thereby helping to reduce the recruitment of immune cells at the tumor site (Bouzin et al., J Immunol, 178:1505-1511, 2007). Furthermore, VEGF recruits macrophages to the tumor microenvironment with M2 polarization, which is typically involved in wound healing. These M2 tumor-associated macrophages ultimately contribute to the establishment and maintenance of an immunosuppressive microenvironment (Chen and Mellman, Immunity, 39:1-10, 2013).

Targeting the VEGF pathway with agents such as bevacizumab has been clinically validated, demonstrating antitumor activity in patients with various advanced malignancies. However, when administered as a single agent to patients with HCC, bevacizumab has shown only minimal activity (Siegel et al., J Clin Oncol, 26:2992-2998, 2008; Boige et al., Oncologist, 17:1063-1072, 2012).

Combination therapy of anti-PD-1/LAG-3 bispecific antibody and anti-VEGF agent

Durable clinical benefits are limited to a small number of patients treated with single-agent PD-L1/PD-1 inhibitors. Therapies targeting mechanisms that antagonize PD-L1/PD-1 therapy are needed to improve outcomes for patients with solid tumors. Strong scientific fundamentals and emerging clinical data suggest that combination PD-1, LAG-3, and VEGF inhibition may offer clinical benefit across multiple tumor types.

Anti-VEGF agents promote tumor angiogenesis, thereby increasing the accessibility of therapeutic agents (Jain, Nat Med, 7:987-989, 2001). Furthermore, bevacizumab can restore and/or maintain the antigen-presenting capacity of dendritic cells (DCs), thereby enhancing T-cell infiltration in tumors (Oelkrug and Ramage, Clin Exp Immunol, 178:1-8, 2014; Wallin et al., Nat Commun, 7:12624, 2016). Administration of anti-VEGF-A has been shown to attenuate the expression of FasL in tumor endothelial cells and produce a significantly increased influx of tumor-rejecting CD8+ T cells, thereby inducing tumor growth inhibition (Motz et al., Nat Med, 20:607-615, 2014). Anti-VEGF therapy can also reduce the frequency of myeloid-derived suppressor cells and decrease the production of repressive cytokines (Roland et al., PLoS One, 4:e7669, 2009). Furthermore, VEGF-A has been shown to induce the expression of PD-1 and other inhibitory immune checkpoint proteins (including TIM-3 and LAG-3) on CD8+ T cells, which can be reversed by VEGF inhibition (Voron et al., J Exp Med, 212:139-148, 2015).

The immunomodulatory effects of bevacizumab are expected to increase CD8+ T cell recruitment and alleviate intratumoral immunosuppression, thereby enhancing the efficacy of immunotherapy. In fact, clinical data have already demonstrated its beneficial anti-angiogenic and immunomodulatory effects in the context of PD-L1/PD-1 pathway blockade. Early studies have demonstrated that combination therapy with anti-PD-1 therapies nivolumab and pembrolizumab, along with various anti-angiogenic agents, can elicit responses in patients with metastatic urothelial carcinoma, RCC, ovarian cancer, and endometrial cancer (Apolo et al., J Clin Oncol, 35(Supplement 6):293, 2017; Lee et al., Ann Oncol, 28(Supplement 5):v295-v329, 2017; Makker et al., J Clin Oncol, 35(Supplement 15):5598, 2017; Liu et al., JAMA Oncol, 5:1731-1738, 2019; Dudek et al., J Clin Oncol, 38:1138-1145, 2020). The activity of combination therapy with the anti-PD-L1 therapy atezolizumab and bevacizumab has also been demonstrated in multiple large randomized phase III clinical trials in patients with NSCLC, RCC, and HCC (Socinski et al., N Engl J Med, 378:2288-2301, 2018; Rini et al., Lancet, 393:2404-2415, 2019; Finn et al., N Engl J Med, 382:1894-1905, 2020). Resistance to PD-L1/PD-1 blockade may lead to the expression of multiple co-suppressive immune checkpoints on the surface of effector T cells. LAG-3 is frequently co-expressed with PD-1 on tumor-infiltrating lymphocytes (TILs), and dual blockade of PD-1 and LAG-3 has been shown to enhance CD8+ T cell effector function and strengthen anti-tumor immunity in non-clinical models. In mice with colon cancer, fibrosarcoma, or ovarian cancer, blocking these two receptors resulted in tumor remission in approximately 80% of the animals, compared to 10% to 40% as a single agent (Woo et al., Cancer Res, 72:917-927, 2012; Huang et al., Oncotarget, 6:27359-27377, 2015). TILs from patients with ovarian cancer showed that antigen-specific CD8+ T cells co-expressing PD-1 and LAG-3 exhibited greater impairment in their ability to respond to homologous antigen stimulation compared to CD8+ T cells expressing a checkpoint molecule (Matsuzaki et al., Proc Natl Acad Sci USA, 107:7875-7880, 2010). In patients with NSCLC, overexpression of LAG-3 on the TIL is associated with PD-1/PD-L1 expression and is associated with a higher risk of recurrence and poor survival outcomes (He et al., J Thorac Oncol, 12:814-823, 2017). In clinical samples from patients with untreated HCC, most showed co-staining of LAG-3 and PD-1, with higher expression in the TIL compared to background liver tissue (Yarchoan et al., Clin Cancer Res, 23:7333-7339, 2017). Based on these data, it is speculated that the combination of bsAb RO7247669, which simultaneously targets the PD-1 and LAG-3 pathways, with the immunomodulatory effects of bevacizumab, may enhance the anti-tumor immune response, thereby producing improved and more durable clinical benefits in patients with HCC.

Other embodiments

Although the invention has been described in considerable detail above by way of example and instance for the purpose of clear understanding, such description and instance should not be construed as limiting the scope of the invention.

Claims (129)

1. A method for treating a subject with cancer, the method comprising administering to the subject one or more dosing cycles a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to LAG3, wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks. 2. The method according to claim 1, wherein the cancer is a solid tumor. 3. The method according to claim 1 or 2, wherein the cancer is locally advanced or metastatic. 4. The method according to any one of claims 1 to 3, wherein the cancer is skin cancer, liver cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, or esophageal cancer. 5. The method according to claim 4, wherein the skin cancer is melanoma. 6. The method of claim 4 or 5, wherein the skin cancer is a previously untreated, unresectable or metastatic melanoma. 7. The method according to claim 5, wherein 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 described herein is not a mucosal melanoma or a uveal melanoma. 8. The method according to claim 5, wherein the liver cancer is hepatocellular carcinoma (HCC). 9. The method according to claim 5, wherein the lung cancer is non-small cell lung cancer (NSCLC). 10. The method of claim 5, wherein the renal cell carcinoma is renal cell carcinoma (RCC). 11. The method of claim 5, wherein the bladder cancer is metastatic urothelial carcinoma (mUC). 12. The method of claim 5, wherein the breast cancer is triple-negative breast cancer (TNBC). 13. The method of claim 5, wherein the esophageal cancer is esophageal squamous cell carcinoma (ESCC). 14. A method for treating a subject with melanoma, the method comprising administering to the subject one or more dosing cycles a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to 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. 15. The method of claim 14, wherein the subject does not have ocular melanoma. 16. A method for treating a subject with liver cancer, the method comprising administering to the subject one or more dosing cycles a bispecific antibody targeting PD-1 and LAG3, the bispecific antibody comprising a first antigen-binding domain specifically binding to PD-1 and a second antigen-binding domain specifically binding to LAG3, wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks. 17. The method of claim 16, wherein the liver cancer is hepatocellular carcinoma (HCC). 18. The method of claim 17, wherein the HCC is locally advanced, metastatic, and/or unresectable. 19. The method according to any one of claims 16 to 18, wherein the subject has not previously received systemic anticancer therapy. 20. The method according to any one of claims 1 to 19, wherein the length of each of the one or more administration cycles is 21 days. 21. The method of claim 20, wherein the method comprises administering the bispecific antibody to the subject on day 1 of each of the one or more dosing cycles. 22. The method according to any one of claims 1 to 21, wherein the method comprises administering the bispecific antibody intravenously to the subject. 23. The method according to any one of claims 1 to 22, wherein the method further comprises administering bevacizumab to the subject every three weeks at a dose of about 15 mg/kg. 24. The method of claim 23, wherein each of the one or more dosing cycles is 21 days in length and the method comprises administering the bevacizumab to the subject on day 1 of each of the one or more dosing cycles. 25. The method of claim 23 or 24, wherein the bevacizumab is administered intravenously. 26. The method of any one of claims 1 to 25, wherein the subject has not previously been treated for metastatic or unresectable disease. 27. The method according to any one of claims 1 to 25, wherein the subject has not previously been treated with an anticancer therapy including an immunomodulator. 28. The method according to any one of claims 1 to 25, wherein the subject has not previously been treated with anti-LAG3 therapy. 29. The method according to any one of claims 1 to 28, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds to PD-1, the first antigen-binding domain comprising: a VH domain, which comprises: (i) The HVR-H1 sequence, which contains the amino acid sequence of SEQ ID NO:25; (ii) The HVR-H2 sequence, which contains the amino acid sequence GGR; and (iii) The HVR-H3 sequence, comprising the amino acid sequence of SEQ ID NO:26; and The VL struct contains: (i) The HVR-L1 sequence, which contains the amino acid sequence of SEQ ID NO:27; (ii) The HVR-L2 sequence, which contains the amino acid sequence RSS; and (iii) The HVR-L3 sequence, which contains the amino acid sequence of SEQ ID NO:28. 30. The method of claim 29, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: a VH domain, which comprises: (i) The HVR-H1 sequence, which contains 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) The HVR-H3 sequence, comprising the amino acid sequence of SEQ ID NO:33; and The VL struct contains: (i) The HVR-L1 sequence, which contains the amino acid sequence of SEQ ID NO:34; (ii) The HVR-L2 sequence, comprising the amino acid sequence of SEQ ID NO:35; and (iii) The HVR-L3 sequence, which contains the amino acid sequence of SEQ ID NO:36. 31. The method according to claim 29 or 30, wherein 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. 32. The method according to any one of claims 29 to 31, wherein the bispecific antibody is a full-length antibody. 33. The method of claim 32, wherein the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain, the Fc domain being IgG, optionally wherein the IgGFc domain is an IgG1 Fc domain or an IgG4 Fc domain. 34. The method of claim 33, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to the Fc receptor, optionally wherein the Fc receptor is an Fcγ receptor. 35. The method according to any one of claims 29 to 34, wherein the bispecific antibody targeting PD-1 and LAG3 comprises: (a) Fc domain belonging to the human IgG1 subclass, containing amino acid mutations L234A, L235A, and P329G (according to Kabat EU index number); and/or (b) An Fc domain comprising modifications that promote association between a first subunit and a second subunit of the Fc domain. 36. The method according to any one of claims 33 to 35, wherein the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (according to Kabat EU index number), and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, and Y407V (according to Kabat EU index number). 37. The method according to any one of claims 29 to 36, wherein the bispecific antibody targeting PD-1 and LAG3 comprises: an Fc domain, a first Fab fragment comprising the first antigen-binding domain, and a second Fab fragment comprising the second antigen-binding domain. 38. The method of claim 37, wherein in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3, variable domains VL and VH are interchanged, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain, optionally wherein in the first Fab fragment, the variable domains VL and VH are interchanged. 39. The method according to claim 37 or 38, wherein in the constant domain CL of one of the Fab fragments, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number), optionally wherein in the constant domain CL of the second Fab fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number). 40. The method according to any one of claims 29 to 39, wherein the bispecific antibody comprises: a first heavy chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 39; a first light chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 40; a second heavy chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 41; and The second light chain comprises an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:42. 41. The method of claim 40, 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. 42. The method according to any one of claims 2 to 41, wherein the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in the tumor. 43. The method according to any one of claims 1 to 42, wherein the subject is a human being. 44. 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 to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks. 45. The bispecific antibody according to claim 44 for use in a method, wherein the cancer is a solid tumor. 46. The bispecific antibody according to claim 44 or 45 for use in a method, wherein the cancer is locally advanced or metastatic. 47. The bispecific antibody for use in a method according to any one of claims 44 to 46, wherein the cancer is skin cancer, liver cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, or esophageal cancer. 48. The bispecific antibody according to claim 47 for use in a method, wherein the skin cancer is melanoma. 49. The bispecific antibody according to claim 47 or 48 for use in a method, wherein the skin cancer is a previously untreated, unresectable or metastatic melanoma. 50. The bispecific antibody according to claim 48 for use in a method, wherein 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 described herein is not a mucosal melanoma or a uveal melanoma. 51. The bispecific antibody according to claim 47 for use in a method, wherein the liver cancer is hepatocellular carcinoma (HCC). 52. The bispecific antibody according to claim 47 for use in the method, wherein the lung cancer is non-small cell lung cancer (NSCLC). 53. The bispecific antibody according to claim 47 for use in a method, wherein the renal cell carcinoma is renal cell carcinoma (RCC). 54. The bispecific antibody according to claim 47 for use in a method, wherein the bladder cancer is metastatic urothelial carcinoma (mUC). 55. The bispecific antibody according to claim 47 for use in a method, wherein the breast cancer is triple-negative breast cancer (TNBC). 56. The bispecific antibody according to claim 47 for use in a method, wherein the esophageal cancer is esophageal squamous cell carcinoma (ESCC). 57. 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 to PD-1 and a second antigen-binding domain that specifically binds to 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. 58. The bispecific antibody of claim 57 for use in a method, wherein the patient does not have ocular melanoma. 59. 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, and wherein the method comprises administering the bispecific antibody to the subject at a fixed dose of 600 mg every three weeks. 60. The bispecific antibody according to claim 59 for use in a method, wherein the liver cancer is HCC. 61. The bispecific antibody according to claim 60 for use in a method, wherein the HCC is locally advanced, metastatic, and/or unresectable. 62. The bispecific antibody for use in a method according to any one of claims 59 to 61, wherein the subject has not previously received systemic anticancer therapy. 63. The bispecific antibody for use in a method according to any one of claims 44 to 62, wherein the length of each of the one or more dosing cycles is 21 days. 64. The bispecific antibody of claim 63 for use in a method, wherein the method comprises administering the bispecific antibody to the subject on day 1 of each of the one or more dosing cycles. 65. The bispecific antibody for use in a method according to any one of claims 44 to 64, wherein the method comprises administering the bispecific antibody intravenously to the subject. 66. The bispecific antibody for use in a method according to any one of claims 44 to 65, wherein the method further comprises administering bevacizumab to the subject every three weeks at a dose of about 15 mg/kg. 67. The bispecific antibody of claim 66 for use in a method, wherein the length of each of the one or more dosing cycles is 21 days and the method comprises administering the bevacizumab to the subject on day 1 of each of the one or more dosing cycles. 68. The bispecific antibody for use in a method according to claim 66 or 67, wherein the bevacizumab is administered intravenously. 69. The bispecific antibody for use in a method according to any one of claims 44 to 68, wherein the subject has not previously been treated for metastatic or unresectable disease. 70. The bispecific antibody for use in a method according to any one of claims 44 to 69, wherein the subject has not previously been treated with an anticancer therapy comprising an immunomodulator. 71. The bispecific antibody for use in a method according to any one of claims 44 to 70, wherein the subject has not previously been treated with anti-LAG3 therapy. 72. The bispecific antibody for use in a method according to any one of claims 44 to 71, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds to PD-1, the first antigen-binding domain comprising: a VH domain comprising: (i) The HVR-H1 sequence, which contains the amino acid sequence of SEQ ID NO:25; (ii) The HVR-H2 sequence, which contains the amino acid sequence GGR; and (iii) The HVR-H3 sequence, comprising the amino acid sequence of SEQ ID NO:26; and The VL struct contains: (i) The HVR-L1 sequence, which contains the amino acid sequence of SEQ ID NO:27; (ii) The HVR-L2 sequence, which contains the amino acid sequence RSS; and (iii) The HVR-L3 sequence, which contains the amino acid sequence of SEQ ID NO:28. 73. The bispecific antibody of claim 72 for use in a method, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: a VH domain comprising: (i) The HVR-H1 sequence, which contains 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) The HVR-H3 sequence, comprising the amino acid sequence of SEQ ID NO:33; and The VL struct contains: (i) The HVR-L1 sequence, which contains the amino acid sequence of SEQ ID NO:34; (ii) The HVR-L2 sequence, comprising the amino acid sequence of SEQ ID NO:35; and (iii) The HVR-L3 sequence, which contains the amino acid sequence of SEQ ID NO:36. 74. The bispecific antibody for use in a method according to claim 72 or 73, wherein 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. 75. The bispecific antibody for use in a method according to any one of claims 72 to 74, wherein the bispecific antibody is a full-length antibody. 76. The bispecific antibody of claim 75 for use in a method, wherein the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain, the Fc domain being IgG, optionally wherein the IgG Fc domain is an IgG1 Fc domain or an IgG4 Fc domain. 77. The bispecific antibody of claim 76 for use in a method, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to the Fc receptor, optionally wherein the Fc receptor is an Fcγ receptor. 78. The bispecific antibody for use in a method according to any one of claims 72 to 77, wherein the bispecific antibody targeting PD-1 and LAG3 comprises: (a) Fc domain belonging to the human IgG1 subclass, containing amino acid mutations L234A, L235A, and P329G (according to Kabat EU index number); and/or (b) An Fc domain comprising modifications that promote association between a first subunit and a second subunit of the Fc domain. 79. The bispecific antibody for use in a method according to any one of claims 76 to 78, wherein the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (according to Kabat EU index number), and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, and Y407V (according to Kabat EU index number). 80. The bispecific antibody for use in a method according to any one of claims 72 to 79, wherein the bispecific antibody targeting PD-1 and LAG3 comprises: an Fc domain, a first Fab fragment comprising the first antigen-binding domain, and a second Fab fragment comprising the second antigen-binding domain. 81. The bispecific antibody of claim 80 for use in a method, wherein in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3, variable domains VL and VH are interchanged, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain, optionally wherein in the first Fab fragment, the variable domains VL and VH are interchanged. 82. The bispecific antibody for use in a method according to claim 80 or 81, wherein in the constant domain CL of one of the Fab fragments, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number), optionally wherein in the constant domain CL of the second Fab fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number). 83. The bispecific antibody for use in a method according to any one of claims 44 to 82, wherein the bispecific antibody comprises: a first heavy chain comprising the same as SEQ ID The sequence of NO:39 comprises an amino acid sequence having at least 95% sequence identity; a first light chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:40; a second heavy chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:41; and a second light chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:42. 84. The bispecific antibody of claim 83 for use in a method, 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. 85. The bispecific antibody for use in a method according to any one of claims 44 to 84, wherein the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in the tumor. 86. The bispecific antibody for use in a method according to any one of claims 44 to 85, wherein the subject is a human. 87. 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 to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and wherein the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks. 88. The use according to claim 87, wherein the cancer is a solid tumor. 89. The use according to claim 87 or 88, wherein the cancer is locally advanced or metastatic. 90. The use according to any one of claims 87 to 89, wherein the cancer is skin cancer, liver cancer, lung cancer, kidney cancer, bladder cancer, breast cancer, or esophageal cancer. 91. The use according to claim 90, wherein the skin cancer is melanoma. 92. The use according to claim 90 or 91, wherein the skin cancer is a previously untreated, unresectable or metastatic melanoma. 93. The use according to claim 91, wherein 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 described herein is not a mucosal melanoma or a uveal melanoma. 94. The use according to claim 90, wherein the liver cancer is hepatocellular carcinoma (HCC). 95. The use according to claim 90, wherein the lung cancer is non-small cell lung cancer (NSCLC). 96. The use according to claim 90, wherein the renal cell carcinoma is renal cell carcinoma (RCC). 97. The use according to claim 90, wherein the bladder cancer is metastatic urothelial carcinoma (mUC). 98. The use according to claim 90, wherein the breast cancer is triple-negative breast cancer (TNBC). 99. The use according to claim 90, wherein the esophageal cancer is esophageal squamous cell carcinoma (ESCC). 100. Use of a bispecific antibody targeting PD-1 and LAG3 in the manufacture of a medicament for treating a subject with melanoma, 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, wherein the bispecific antibody is administered 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. 101. The use according to claim 100, wherein the subject does not have ocular melanoma. 102. 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, wherein the bispecific antibody is administered to the subject at a fixed dose of 600 mg every three weeks. 103. The use according to claim 102, wherein the liver cancer is HCC. 104. The use according to claim 103, wherein the HCC is locally advanced, metastatic, and/or unresectable. 105. The use according to any one of claims 102 to 104, wherein the subject has not previously received systemic anticancer therapy. 106. The use according to any one of claims 87 to 105, wherein the length of each of the one or more administration cycles is 21 days. 107. The use according to claim 106, wherein the bispecific antibody is administered to the subject on day 1 of each of the one or more dosing cycles. 108. The use according to any one of claims 87 to 107, wherein the bispecific antibody is administered intravenously to the subject. 109. The use according to any one of claims 87 to 108, wherein bevacizumab is administered to the subject every three weeks at a dose of about 15 mg/kg. 110. The use according to claim 109, wherein the length of each of the one or more dosing cycles is 21 days and the bevacizumab is administered to the subject on day 1 of each of the one or more dosing cycles. 111. The use according to claim 109 or 110, wherein the bevacizumab is administered intravenously. 112. The use according to any one of claims 87 to 111, wherein the subject has not previously been treated for metastatic or unresectable disease. 113. The use according to any one of claims 87 to 111, wherein the subject has not previously been treated with an anticancer therapy including an immunomodulator. 114. The use according to any one of claims 87 to 111, wherein the subject has not previously been treated with anti-LAG3 therapy. 115. The use according to any one of claims 87 to 114, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds to PD-1, the first antigen-binding domain comprising: a VH domain, which comprises: (i) The HVR-H1 sequence, which contains the amino acid sequence of SEQ ID NO:25; (ii) The HVR-H2 sequence, which contains the amino acid sequence GGR; and (iii) The HVR-H3 sequence, comprising the amino acid sequence of SEQ ID NO:26; and The VL struct contains: (i) The HVR-L1 sequence, which contains the amino acid sequence of SEQ ID NO:27; (ii) The HVR-L2 sequence, which contains the amino acid sequence RSS; and (iii) The HVR-L3 sequence, which contains the amino acid sequence of SEQ ID NO:28. 116. The use according to claim 115, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a second antigen-binding domain that specifically binds to LAG3, the second antigen-binding domain comprising: a VH domain, which comprises: (i) The HVR-H1 sequence, which contains 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) The HVR-H3 sequence, comprising the amino acid sequence of SEQ ID NO:33; and The VL struct contains: (i) The HVR-L1 sequence, which contains the amino acid sequence of SEQ ID NO:34; (ii) The HVR-L2 sequence, comprising the amino acid sequence of SEQ ID NO:35; and (iii) The HVR-L3 sequence, which contains the amino acid sequence of SEQ ID NO:36. 117. The use according to claim 115 or 116, wherein the first antigen-binding domain comprises: a VH domain comprising the amino acid sequence of SEQ ID NO:29; and The second antigen-binding domain comprises a VL domain containing the amino acid sequence of SEQ ID NO:30, and the second antigen-binding domain comprises: 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. 118. The use according to any one of claims 115 to 117, wherein the bispecific antibody is a full-length antibody. 119. The use according to claim 118, wherein the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain, the Fc domain being IgG, optionally wherein the IgG Fc domain is an IgG1 Fc domain or an IgG4 Fc domain. 120. The use according to claim 119, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to the Fc receptor, optionally wherein the Fc receptor is an Fcγ receptor. 121. The use according to any one of claims 115 to 120, wherein the bispecific antibody targeting PD-1 and LAG3 comprises: (a) Fc domain belonging to the human IgG1 subclass, containing amino acid mutations L234A, L235A, and P329G (according to Kabat EU index number); and/or (b) An Fc domain comprising modifications that promote association between a first subunit and a second subunit of the Fc domain. 122. The use according to any one of claims 119 to 121, wherein the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (according to Kabat EU index number), and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, and Y407V (according to Kabat EU index number). 123. The use according to any one of claims 115 to 122, wherein the bispecific antibody targeting PD-1 and LAG3 comprises: an Fc domain, a first Fab fragment comprising the first antigen-binding domain, and a second Fab fragment comprising the second antigen-binding domain. 124. The use according to claim 123, wherein in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3, the variable domains VL and VH are interchanged, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain, optionally wherein in the first Fab fragment, the variable domains VL and VH are interchanged. 125. The use according to claim 123 or 124, wherein in the constant domain CL of one of the Fab fragments, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number), optionally wherein in the constant domain CL of the second Fab fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat EU index number), and in the constant domain CH1, the amino acids at positions 147 and 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number). 126. The use according to any one of claims 115 to 125, wherein the bispecific antibody comprises: a first heavy chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:39; a first light chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:40; a second heavy chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:41; and a second light chain comprising an amino acid sequence having at least 95% sequence identity with the sequence of SEQ ID NO:42. 127. The use according to claim 126, 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. 128. The use according to any one of claims 87 to 127, wherein the bispecific antibody achieves at least 90% LAG3 receptor occupancy (RO) in the tumor. 129. The use according to any one of claims 87 to 128, wherein the subject is a human being.