WO2024163009A1 - Methods and compositions for treating urothelial bladder cancer - Google Patents

Abstract

This invention relates to methods and compositions for use in treating a urothelial bladder cancer (UC) in a subject by administering to the subject a bispecific antibody targeting programmed cell death protein 1 (PD-1) and lymphocyte activation gene-3 (LAG3) (PD1-LAG3) or (a) a bispecific antibody targeting PD-1 and LAG3 and (b) an anti-TIGIT antagonist antibody.

Description

METHODS AND COMPOSITIONS FOR TREATING UROTHELIAL BLADDER CANCER

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 31 , 2023, is named 51177-048WO1_Sequence_Listing_1_31_23 and is 59,022 bytes in size.

FIELD OF THE INVENTION

This invention relates to methods and compositions for use in treating a urothelial bladder cancer (UC) in a subject by administering to the subject a bispecific antibody targeting programmed cell death protein 1 (PD-1) and lymphocyte activation gene-3 (LAG3) (PD1-LAG3) or (a) a bispecific antibody targeting PD-1 and LAG3 and (b) an anti-TIGIT antagonist antibody.

BACKGROUND OF THE INVENTION

Bladder cancer is the 10th most common cancer worldwide, with approximately 573,000 new cases and 213,000 deaths in 2020. Notably, bladder cancer is three times more common in men, for whom it is the 6th most common cancer cause and the 9th leading cause of death. Urothelial carcinoma represents the most common histologic subtype in developed countries. While most patients with bladder cancer present with localized disease, about 25% of patients present with the muscle-invasive disease at initial diagnosis, which carries a significantly worse prognosis. Metastatic disease is associated with limited survival, and cures remain rare.

Cisplatin-based chemotherapy has been the standard of care for treatment-naive patients with advanced bladder cancer since the 1990s, leading to a median survival of approximately 15 months. Frontline carboplatin-based chemotherapy is commonly offered to patients who cannot tolerate cisplatin due to renal dysfunction, neuropathy, hearing loss, or heart failure; however, the median overall survival (OS) with carboplatin-based chemotherapy is thought to be worse than with cisplatin-based regimens. Avelumab therapy was recently approved for maintenance treatment of patients with advanced bladder cancer who did not progress on platinum-based chemotherapy, based on the results of the Javelin Bladder 100 study that demonstrated an improvement in OS for patients that received avelumab compared to the best supportive care group. Patients ineligible for any platinum-based chemotherapy are commonly treated with single-agent immune CPIs (pembrolizumab, atezolizumab), single-agent chemotherapy, or best supportive care.

Cancer immunotherapy agents, particularly immune checkpoint inhibitors, have made a significant impact on the treatment of patients with advanced malignancies in recent years, including urothelial bladder cancer. However, despite these advances, many patients still do not respond to currently available immune therapies, have a suboptimal response, or become resistant to current PD-1/PD-L1-directed therapies. There continues to be a need for novel agents.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides a method for treating a subject having an urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD-1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), wherein (i) the subject is ineligible for platinum-based chemotherapy or (ii) the subject has not previously been treated with a cancer immunotherapy.

In some aspects, the bispecific antibody is administered as a monotherapy.

In another aspect, the disclosure provides a method for treating a subject having an urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of (a) an anti-TIGIT antagonist antibody; and (b) a bispecific antibody targeting PD-1 and LAG3 comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein (i) the subject is ineligible for platinum-based chemotherapy or (ii) the subject has not previously been treated with a cancer immunotherapy.

In some aspects, the subject is ineligible for platinum-based chemotherapy. In some aspects, the subject has not previously been treated with a cancer immunotherapy. In some aspects, the subject is ineligible for platinum-based chemotherapy and has not previously been treated with a cancer immunotherapy.

In some aspects, the method comprises administering to the subject the anti-TIGIT antagonist antibody at a fixed dose of 600 mg every three weeks.

In some aspects, the method comprises administering to the subject the bispecific antibody at a fixed dose of 600 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 comprises administering to the subject the bispecific antibody on Day 1 of each of the one or more dosing cycles.

In some aspects, the method comprises administering to the subject the anti-TIGIT antagonist antibody on Day 1 of each of the one or more dosing cycles.

In some aspects, the method comprises administering to the subject the bispecific antibody before the anti-TIGIT antagonist antibody.

In some aspects, the anti-TIGIT antagonist antibody is administered at least 60 minutes after the bispecific antibody in a first dosing cycle.

In some aspects, the method comprises one or more additional dosing cycles, and the anti-TIGIT antagonist antibody is administered at least 30 minutes after the bispecific antibody in the one or more additional dosing cycles.

In some aspects, the method comprises administering to the subject the bispecific antibody intravenously.

In some aspects, the method comprises administering to the subject the anti-TIGIT antagonist antibody intravenously.

In some aspects, the urothelial bladder cancer is locally advanced or metastatic. In some aspects, (a) the clinical stage of the locally advanced urothelial bladder cancer is (i) T4b, any N or (ii) any T, N2-3; or (b) the clinical stage of the metastatic urothelial bladder cancer is M1 and/or Stage IV.

In some aspects, the urothelial bladder cancer is a carcinoma.

In some aspects, the subject is ineligible for platinum-based chemotherapy based on having (a) an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and < 30 mL/min/1 .73 m²; (b) an ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1 .73 m² and < 45 mL/min/1 .73 m²; or (c) an ECOG Performance Status of 0-2 with Grade > 2 neuropathy. In some aspects, the GFR is assessed by calculation through use of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

In some aspects, the subject has not previously been treated with a chemotherapy for locally advanced, metastatic, or recurrent urothelial bladder cancer.

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises a first antigenbinding domain that specifically binds to PD-1 comprising a heavy chain variable (VH) domain comprising

(i) a hypervariable region H1 (HVR-H1) sequence comprising the amino acid sequence of SEQ ID NO: 1 ,

(ii) an HVR-H2 sequence comprising the amino acid sequence GGR, and (iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; and a light chain variable (VL) domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; (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: 4.

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG. In some aspects, the IgG Fc domain is an lgG1 Fc domain or an lgG4 Fc domain.

In some aspects, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor. In some aspects, the Fc receptor is an Fey receptor.

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises a second antigenbinding domain that specifically binds to LAG-3 comprising a VH domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, (ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and (iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; and a VL domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, (ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and (iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12.

In some aspects, the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14.

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

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

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises a first Fab fragment comprising said first antigen binding domain specifically binding to PD1 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and a second Fab fragment comprising the second antigen binding domain specifically binding to LAG3 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, and an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index).

In some aspects, in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3 the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In some aspects, in the first Fab fragment the variable domains VL and VH are replaced by each other.

In some aspects, in the constant domain CL of one of the Fab fragments the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In some aspects, in the constant domain CL of the second Fab fragment the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In some aspects, the bispecific antibody comprises a first heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15, a first light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16, a second heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17, and a second light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18. In some aspects, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18.

In some aspects, the anti-TIGIT antagonist antibody comprises the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 38); an HVR-H2 sequence comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 39); an HVR-H3 sequence comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO:

40); an HVR-L1 sequence comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO:

41); an HVR-L2 sequence comprising the amino acid sequence of WASTRES (SEQ ID NO: 42); and an HVR-L3 sequence comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 43).

In some aspects, the anti-TIGIT antagonist antibody comprises (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44 or 45; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46; or (c) a VH domain as in (a) and a VL domain as in (b). In some aspects, the anti-TIGIT antagonist antibody is a monoclonal antibody, a human antibody, or a full-length antibody.

In some aspects, the anti-TIGIT antagonist antibody is a human, monoclonal full-length lgG1 subclass antibody comprising a human lgG1 Fc region, a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 44, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 46.

In some aspects, the anti-TIGIT antagonist antibody exhibits effector function.

In some aspects, the anti-TIGIT antagonist antibody is tiragolumab.

In some aspects, the anti-TIGIT antagonist antibody is an antibody fragment that binds TIGIT selected from the group consisting of Fab, Fab’, Fab’-SH, Fv, single chain variable fragment (scFv), and (Fab’)2 fragments.

In some aspects, the anti-TIGIT antagonist antibody is an IgG class antibody. In some aspects, the IgG class antibody is an lgG1 subclass antibody.

In some aspects, a PD-L1 expression level of a tumor sample obtained from the subject has been determined. In some aspects, the PD-L1 expression level is a protein expression level, and the protein expression level has been determined by an immunohistochemical (IHC) assay comprising staining with an anti-PD-L1 antibody suitable for staining. In some aspects, the anti-PD-L1 antibody suitable for staining is the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8.

In some aspects, the protein expression level of PD-L1 is determined using a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, a Ventana SP142 IHC assay, or a pharmDx 28-8 IHC assay.

In some aspects, the tumor sample obtained from the subject has been determined to be PD-L1- positive.

In some aspects, the tumor sample obtained from the subject has been determined to be PD-L1- negative.

In some aspects, the method results in an increase in objective response rate (ORR) in a population of subjects treated according to the method as compared to a reference ORR. In some aspects, the reference ORR is an ORR of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in progression-free survival (PFS) as compared to a reference PFS. In some aspects, the reference PFS is a PFS of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in overall survival (OS) as compared to a reference OS. In some aspects, the reference OS is an OS of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in duration of response (DOR) as compared to a reference DOR. In some aspects, the reference DOR is a DOR of a population of subjects who have received a control therapy.

In some aspects, the method results in (a) an increase in PFS rate at 6 months as compared to a reference PFS rate at 6 months; or (b) an increase in PFS rate at 12 months as compared to a reference PFS rate at 12 months. In some aspects, the reference PFS rate is a PFS rate of a population of subjects who have received a control therapy. In some aspects, the method results in (a) an increase in OS rate at 6 months as compared to a reference OS rate at 6 months; (b) an increase in OS rate at 12 months as compared to a reference OS rate at 12 months; or (c) an increase in OS rate at 18 months as compared to a reference OS rate at 18 months. In some aspects, the reference OS rate is an OS rate of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in disease control rate (DCR) as compared to a reference DCR. In some aspects, the reference DCR is a DCR of a population of subjects who have received a control therapy.

In some aspects, the control therapy is a treatment comprising atezolizumab, and the control therapy does not comprise the bispecific antibody or an anti-TIGIT antagonist antibody.

In another aspect, the disclosure provides a method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

In another aspect, the disclosure provides a method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

In another aspect, the disclosure provides a method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of (a) an anti-TIGIT antagonist antibody and (b) a bispecific antibody targeting PD-1 and LAG3 comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

In another aspect, the disclosure provides a method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of (a) an anti-TIGIT antagonist antibody and (b) a bispecific antibody targeting PD-1 and LAG3 comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

In some aspects, the anti-TIGIT antagonist antibody is tiragolumab.

In some aspects, the subject is a human.

In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having an urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody, wherein (i) the subject is ineligible for platinum-based chemotherapy or (ii) the subject has not previously been treated with a cancer immunotherapy.

In some aspects, the bispecific antibody is to be administered as a monotherapy.

In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having an urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, 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 dosing regimen comprises administering to the subject one or more dosing cycles of (a) the anti-TIGIT antagonist antibody and (b) the bispecific antibody, wherein (i) the subject is ineligible for platinum-based chemotherapy; or (ii) the subject has not previously been treated with a cancer immunotherapy.

In some aspects, the subject is ineligible for platinum-based chemotherapy. In some aspects, the subject has not previously been treated with a cancer immunotherapy. In some aspects, the subject is ineligible for platinum-based chemotherapy and has not previously been treated with a cancer immunotherapy.

In some aspects, the method comprises administering to the subject the anti-TIGIT antagonist antibody at a fixed dose of 600 mg every three weeks.

In some aspects, the method comprises administering to the subject the bispecific antibody at a fixed dose of 600 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 comprises administering to the subject the bispecific antibody on Day 1 of each of the one or more dosing cycles.

In some aspects, the method comprises administering to the subject the anti-TIGIT antagonist antibody on Day 1 of each of the one or more dosing cycles.

In some aspects, the method comprises administering to the subject the bispecific antibody before the anti-TIGIT antagonist antibody.

In some aspects, the anti-TIGIT antagonist antibody is to be administered at least 60 minutes after the bispecific antibody in a first dosing cycle. In some aspects, the method comprises one or more additional dosing cycles, and the anti-TIGIT antagonist antibody is to be administered at least 30 minutes after the bispecific antibody in the one or more additional dosing cycles. In some aspects, the method comprises administering to the subject the bispecific antibody intravenously.

In some aspects, the method comprises administering to the subject the anti-TIGIT antagonist antibody intravenously.

In some aspects, the urothelial bladder cancer is locally advanced or metastatic.

In some aspects, (a) the clinical stage of the locally advanced urothelial bladder cancer is (i) T4b, any N or (ii) any T, N2-3; or (b) the clinical stage of the metastatic urothelial bladder cancer is M1 and/or Stage IV.

In some aspects, the urothelial bladder cancer is a carcinoma.

In some aspects, the subject is ineligible for platinum-based chemotherapy based on having (a) an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and < 30 mL/min/1 .73 m²; (b) an ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1 .73 m² and < 45 mL/min/1 .73 m²; or (c) an ECOG Performance Status of 0-2 with Grade > 2 neuropathy. In some aspects, the GFR is assessed by calculation through use of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

In some aspects, the subject has not previously been treated with a chemotherapy for locally advanced, metastatic, or recurrent urothelial bladder cancer.

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises a first antigenbinding domain that specifically binds to PD-1 comprising a heavy chain variable (VH) domain comprising (I) a hypervariable region H1 (HVR-H1) sequence comprising the amino acid sequence of SEQ ID NO: 1 , (ii) an HVR-H2 sequence comprising the amino acid sequence GGR, and (iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; and a light chain variable (VL) domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; (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: 4.

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG. In some aspects, the IgG Fc domain is an lgG1 Fc domain or an lgG4 Fc domain.

In some aspects, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor. In some aspects, the Fc receptor is an Fey receptor.

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises a second antigenbinding domain that specifically binds to LAG-3 comprising a VH domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, (ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and (iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; and a VL domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, (ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and (iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12.

In some aspects, the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14. In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises (a) an Fc domain of human IgG 1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index); and/or (b) an Fc domain comprising a modification promoting the association of the first and second subunit of the Fc domain.

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

In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises a first Fab fragment comprising said first antigen binding domain specifically binding to PD1 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and a second Fab fragment comprising the second antigen binding domain specifically binding to LAG3 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, and an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index).

In some aspects, in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3 the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In some aspects, in the first Fab fragment the variable domains VL and VH are replaced by each other.

In some aspects, in the constant domain CL of one of the Fab fragments the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In some aspects, in the constant domain CL of the second Fab fragment the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In some aspects, the bispecific antibody comprises a first heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15, a first light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16, a second heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17, and a second light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18. In some aspects, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18. In some aspects, the anti-TIGIT antagonist antibody comprises the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 38); an HVR-H2 sequence comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 39); an HVR-H3 sequence comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO:

40); an HVR-L1 sequence comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO:

41); an HVR-L2 sequence comprising the amino acid sequence of WASTRES (SEQ ID NO: 42); and an HVR-L3 sequence comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 43).

In some aspects, the anti-TIGIT antagonist antibody comprises (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44 or 45; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46; or (c) a VH domain as in (a) and a VL domain as in (b).

In some aspects, the anti-TIGIT antagonist antibody is a monoclonal antibody, a human antibody, or a full-length antibody.

In some aspects, the anti-TIGIT antagonist antibody is a human, monoclonal full-length lgG1 subclass antibody comprising a human lgG1 Fc region, a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 44, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 46.

In some aspects, the anti-TIGIT antagonist antibody exhibits effector function.

In some aspects, the anti-TIGIT antagonist antibody is tiragolumab.

In some aspects, the anti-TIGIT antagonist antibody is an antibody fragment that binds TIGIT selected from the group consisting of Fab, Fab’, Fab’-SH, Fv, single chain variable fragment (scFv), and (Fab’)2 fragments.

In some aspects, the anti-TIGIT antagonist antibody is an IgG class antibody. In some aspects, the IgG class antibody is an lgG1 subclass antibody.

In some aspects, a PD-L1 expression level of a tumor sample obtained from the subject has been determined. In some aspects, the PD-L1 expression level is a protein expression level, and the protein expression level has been determined by an immunohistochemical (IHC) assay comprising staining with an anti-PD-L1 antibody suitable for staining. In some aspects, the anti-PD-L1 antibody suitable for staining is the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8.

In some aspects, the protein expression level of PD-L1 is determined using a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, a Ventana SP142 IHC assay, or a pharmDx 28-8 IHC assay.

In some aspects, the tumor sample obtained from the subject has been determined to be PD-L1- positive.

In some aspects, the tumor sample obtained from the subject has been determined to be PD-L1- negative.

In some aspects, the method results in an increase in objective response rate (ORR) in a population of subjects treated according to the method as compared to a reference ORR. In some aspects, the reference ORR is an ORR of a population of subjects who have received a control therapy. In some aspects, the method results in an increase in progression-free survival (PFS) as compared to a reference PFS. In some aspects, the reference PFS is a PFS of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in overall survival (OS) as compared to a reference OS. In some aspects, the reference OS is an OS of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in duration of response (DOR) as compared to a reference DOR. In some aspects, the reference DOR is a DOR of a population of subjects who have received a control therapy.

In some aspects, the method results in (a) an increase in PFS rate at 6 months as compared to a reference PFS rate at 6 months; or (b) an increase in PFS rate at 12 months as compared to a reference PFS rate at 12 months. In some aspects, the reference PFS rate is a PFS rate of a population of subjects who have received a control therapy.

In some aspects, the method results in (a) an increase in OS rate at 6 months as compared to a reference OS rate at 6 months; (b) an increase in OS rate at 12 months as compared to a reference OS rate at 12 months; or (c) an increase in OS rate at 18 months as compared to a reference OS rate at 18 months. In some aspects, the reference OS rate is an OS rate of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in disease control rate (DOR) as compared to a reference DCR. In some aspects, the reference DCR is a DCR of a population of subjects who have received a control therapy.

In some aspects, the control therapy is a treatment comprising atezolizumab, and the control therapy does not comprise the bispecific antibody or an anti-TIGIT antagonist antibody.

In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a locally advanced or metastatic urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody; the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and the subject is ineligible for platinum- based chemotherapy.

In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a locally advanced or metastatic urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody; the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and the subject has not previously been treated with a cancer immunotherapy.

In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a locally advanced or metastatic urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of (a) the anti-TIGIT antagonist antibody and (b) the bispecific antibody; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a locally advanced or metastatic urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of (a) the anti-TIGIT antagonist antibody and (b) the bispecific antibody; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

In some aspects, the anti-TIGIT antagonist antibody is tiragolumab.

In some aspects, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 is a flow chart showing the design of the BO44157 phase II clinical trial of RO7247669 alone or in combination with tiragolumab compared with atezolizumab in patients with previously untreated, locally advanced or metastatic urothelial bladder cancer (UC). 1 L = first line; CIT = cancer immunotherapy; ECOG PS = Eastern Cooperative Oncology Group Performance Status; G = grade; GFR = glomerular filtration rate; mllC = metastatic urothelial bladder cancer; PD-L1 = programmed deathligand 1 .

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides therapeutic methods and compositions for treatment of urothelial bladder cancer (UC). Compositions, uses, and kits involving such combinations and/or dosing regimens are also provided herein. I. Definitions

The following abbreviations are used herein:

The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

The term “TIGIT” or “T-cell immunoreceptor with Ig and ITIM domains” as used herein refers to any native TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIG9, VSTM3, and WUCAM. The term encompasses “full- length,” unprocessed TIGIT (e.g., full-length human TIGIT having the amino acid sequence of SEQ ID NO: 59), as well as any form of TIGIT that results from processing in the cell (e.g., processed human TIGIT without a signal sequence, having the amino acid sequence of SEQ ID NO: 60). The term also encompasses naturally occurring variants of TIGIT, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human TIGIT may be found under UniProt Accession Number Q495A1.

As used herein, “tiragolumab" is a fully human lgG1/kappa MAb-derived in Open Monoclonal Technology (OMT) rats that binds TIGIT and comprises the heavy chain sequence of SEQ ID NO: 47 and the light chain sequence of SEQ ID NO: 48. Tiragolumab comprises two N-linked glycosylation sites (N306) in the Fc domain. Tiragolumab is also described in WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Proposed INN: List 117, Vol. 31 , No. 2, published June 9, 2017 (see page 343).

The term “anti-TIGIT antagonist antibody” refers to an antibody or an antigen-binding fragment or variant thereof that is capable of binding TIGIT with sufficient affinity such that it substantially or completely inhibits the biological activity of TIGIT. For example, an anti-TIGIT antagonist antibody may block signaling through PVR, PVRL2, and/or PVRL3 so as to restore a functional response by T-cells (e.g., proliferation, cytokine production, target cell killing) from a dysfunctional state to antigen stimulation. For example, an anti-TIGIT antagonist antibody may block signaling through PVR without impacting PVR- CD226 interaction. It will be understood by one of ordinary skill in the art that in some instances, an anti- TIGIT antagonist antibody may antagonize one TIGIT activity without affecting another TIGIT activity. For example, an anti-TIGIT antagonist antibody for use in certain of the methods or uses described herein is an anti-TIGIT antagonist antibody that antagonizes TIGIT activity in response to one of PVR interaction, PVRL3 interaction, or PVRL2 interaction, e.g., without affecting or minimally affecting any of the other TIGIT interactions. In one aspect, the extent of binding of an anti-TIGIT antagonist antibody to an unrelated, non-TIGIT protein is less than about 10% of the binding of the antibody to TIGIT as measured, e.g., by a radioimmunoassay (RIA). In certain aspects, an anti-TIGIT antagonist antibody that binds to TIGIT has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10^s M or less, e.g., from 10^s M to 10¹³ M, e.g., from 10⁹ M to 10¹³ M). In certain aspects, an anti-TIGIT antagonist antibody binds to an epitope of TIGIT that is conserved among TIGIT from different species or an epitope on TIGIT that allows for cross-species reactivity. In some aspects, the anti-TIGIT binding antibody has intact Fc-mediated effector function (e.g., tiragolumab, vibostolimab, etigilimab, EOS084448, or TJ-T6). In some aspects, the anti-TIGIT binding antibody has enhanced Fc- mediated effector function (e.g., SGN-TGT). In other aspects, the anti-TIGIT binding antibody lacks Fc- mediated effector function (e.g., domvanalimab, BMS-986207, ASP8374, or COM902). In some aspects, the anti-TIGIT binding antibody is an lgG1 class antibody (e.g., tiragolumab, vibostolimab, domvanalimab, BMS-986207, etigilimab, BGB-A1217, SGN-TGT, EOS084448 (EOS-448), TJ-T6, or AB308). In other aspects, the anti-TIGIT binding antibody is an lgG4 class antibody (e.g., ASP8374 or COM902). In one aspect, the anti-TIGIT antagonist antibody is tiragolumab.

The term “PD-1 axis binding antagonist” refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partners, so as to remove T-cell dysfunction resulting from signaling on the PD-1 signaling axis, with a result being to restore or enhance T-cell function (e.g., proliferation, cytokine production, and/or target cell killing). As used herein, a PD-1 axis binding antagonist includes a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist. In some instances, the PD-1 axis binding antagonist includes a PD-L1 binding antagonist or a PD-1 binding antagonist. 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 decreases, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L1 with either one or more of its binding partners, such as PD-1 and/or B7-1 . In some instances, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1 and/or B7-1 . In some instances, the PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate 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 instance, a PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD- L1 so as to render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some instances, the PD-L1 binding antagonist binds to PD-L1 . In some instances, a 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, IMC-001 , KL-A167, APL-502, cosibelimab, lodapolimab, FAZ053, TG-1501 , BGB-A333, BCD-135, AK- 106, LDP, GR1405, HLX20, MSB2311 , RC98, PDL-GEX, KD036, KY1003, YBL-007, and HS-636. In some aspects, the anti-PD-L1 antibody is atezolizumab, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab). In one specific aspect, the PD-L1 binding antagonist is MDX-1105. In another specific aspect, the PD-L1 binding antagonist is MEDI4736 (durvalumab). In another specific aspect, the PD-L1 binding antagonist is MSB0010718C (avelumab). In other aspects, the PD-L1 binding antagonist may be a small molecule, e.g., GS-4224, INCB086550, MAX-10181 , INCB090244, CA-170, or ABSK041 , which in some instances may 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 decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and/or PD-L2. PD-1 (programmed death 1) is also referred to in the art as “programmed cell death 1 ,” “PDCD1 ,” “CD279,” and “SLEB2.” An exemplary human PD-1 is shown in UniProtKB/Swiss-Prot Accession No. Q15116. In some instances, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one instance, a PD-1 binding antagonist reduces the negative co- stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some instances, the PD-1 binding antagonist binds to PD-1 . In some instances, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody). Exemplary anti-PD-1 antagonist antibodies include nivolumab, pembrolizumab, MEDI-0680, PDR001 (spartalizumab), REGN2810 (cemiplimab), BGB-108, prolgolimab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, sasanlimab, penpulimab, CS1003, HLX10, SCT-I10A, zimberelimab, balstilimab, genolimzumab, Bl 754091 , cetrelimab, YBL-006, BAT1306, HX008, budigalimab, AMG 404, CX-188, JTX-4014, 609A, Sym021 , LZM009, F520, SG001 , AM0001 , ENUM 244C8, ENUM 388D4, STI-1110, AK-103, and hAb21. In a specific aspect, a PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific aspect, a PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific aspect, a PD-1 binding antagonist is a PD-L2 Fc fusion protein, e.g., AMP-224. In another specific aspect, a PD-1 binding antagonist is MEDI- 0680. In another specific aspect, a PD-1 binding antagonist is PDR001 (spartalizumab). In another specific aspect, a PD-1 binding antagonist is REGN2810 (cemiplimab). In another specific aspect, a PD-1 binding antagonist is BGB-108. In another specific aspect, a PD-1 binding antagonist is prolgolimab. In another specific aspect, a PD-1 binding antagonist is camrelizumab. In another specific aspect, a PD-1 binding antagonist is sintilimab. In another specific aspect, a PD-1 binding antagonist is tislelizumab. In another specific aspect, a PD-1 binding antagonist is toripalimab. Other additonal exemplary PD-1 binding antagonists include BION-004, CB201 , AUNP-012, ADG104, and LBL-006. In a particular aspect, a PD-1 binding antagonist antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6.

The term “PD-L2 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1 . PD-L2 (programmed death ligand 2) is also referred to in the art as “programmed cell death 1 ligand 2,” “PDCD1 LG2,” “CD273,” “B7-DC,” “Btdc,” and “PDL2.” An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot Accession No. Q9BQ51 . In some instances, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1 . Exemplary PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1 . In one aspect, a PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some aspects, the PD-L2 binding antagonist binds to PD-L2. In some aspects, a PD-L2 binding antagonist is an immunoadhesin. In other aspects, a PD-L2 binding antagonist is an anti- PD-L2 antagonist antibody.

The terms “programmed death ligand 1” and “PD-L1” refer herein to native sequence human PD- L1 polypeptide. Native sequence PD-L1 polypeptides are provided under Uniprot Accesion No. Q9NZQ7. For example, the native sequence PD-L1 may have the amino acid sequence as set forth in Uniprot Accesion No. Q9NZQ7-1 (isoform 1) (SEQ ID NO: 61). In another example, the native sequence PD-L1 may have the amino acid sequence as set forth in Uniprot Accesion No. Q9NZQ7-2 (isoform 2). In yet another example, the native sequence PD-L1 may have the amino acid sequence as set forth in Uniprot Accesion No. Q9NZQ7-3 (isoform 3). PD-L1 is also referred to in the art as “programmed cell death 1 ligand 1 ,” “PDCD1 LG1 ,” “CD274,” “B7-H,” and “PDL1

The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or“EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human lgG1 EU antibody.

The term “cancer” refers to a disease caused by an uncontrolled division of abnormal cells in a part of the body. In one instance, the cancer is a urothelial bladder cancer (UC), e.g., a locally advanced or metastatic UC. Cancers include solid tumor cancers and non-solid tumor cancers and 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 particular 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), e.g., BCG-refractory NMIBC), MIBC urothelial bladder cancer (UBC); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); cancer of the urinary tract; lung cancer, such as small cell lung cancer (SCLC), which includes extensive stage SCLC (ES-SCLC); nonsmall cell lung cancer (NSCLC), which includes squamous NSCLC or non-squamous NSCLC, including locally advanced unresectable NSCLC (e.g., Stage I II B NSCLC), or recurrent or metastatic NSCLC (e.g., Stage IV NSCLC), adenocarcinoma of the lung, or squamous cell cancer (e.g., epithelial squamous cell cancer (e.g., squamous 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 cancer (HNSCC); ovarian cancer (OC); esophageal cancer; cancer of the peritoneum; hepatocellular cancer; gastric cancer (GC) (e.g., gastroesophageal junction (GEJ) cancer) or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; glioblastoma; cancer of the urinary tract; hepatoma; breast cancer (e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC (e.g., early TNBC (eTNBC)), which are estrogen receptors (ER-), progesterone receptors (PgR-), and HER2 (HER2-) negative); prostate cancer, such as castration-resistant prostate cancer (CRPC); cancer of the peritoneum; hepatocellular cancer; gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)); glioblastoma; cervical cancer (e.g., a Stage IVB, metastatic, recurrent, or persistent cervical cancer, e.g., a metastatic and/or recurrent PD-L1- positive cervical carcinoma); ovarian cancer; hepatoma; colon cancer; rectal cancer; colorectal cancer (CRC; e.g., CRC with microsatellite-stable (MSS) and microsatellite instability (MSI) low (MSI-Low)); endometrial or uterine carcinoma; salivary gland carcinoma; prostate cancer; vulval cancer; thyroid cancer; hepatic carcinoma; anal carcinoma; penile carcinoma; melanoma, including superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, and nodular melanomas; multiple myeloma and B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myologenous leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), Meigs’ syndrome, brain cancer, head and neck cancer, and associated metastases.

In some instances, the cancer (e.g., urothelial bladder cancer (UC), e.g., locally advanced or metastatic UC) is a tumor having a tumor microenvironment comprising LAG3-expressing CD8+ T cells.

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

The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer," “cancerous,” “cell proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as referred to herein. A “tumor cell,” as used herein, refers to any tumor cell present in a tumor or a sample thereof. Tumor cells may be distinguished from other cells that may be present in a tumor sample, for example, stromal cells and tumor-infiltrating immune cells, using methods known in the art and/or described herein.

“Tumor immunity” refers to the process in which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is “treated” when such evasion is attenuated, and the tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

As used herein, by “metastasis” is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through 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. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.

As used herein, “treating” comprises effective cancer treatment with an effective 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 a chemotherapeutic agent, e.g., nab- paclitaxel). Treating herein includes, inter alia, adjuvant therapy, neoadjuvant therapy, non-metastatic cancer therapy (e.g., locally advanced cancer therapy), and metastatic cancer therapy. The treatment may be first-line treatment (e.g., the patient may be previously untreated or not have received prior systemic therapy), or second line or later treatment.

Herein, an “effective amount” refers to the amount of a therapeutic agent (e.g., a a bispecific antibody targeting PD-1 and LAG3) or a combination of therapeutic agents (e.g., a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, e.g., tiragolumab), that achieves a therapeutic result. In some examples, the effective amount of a therapeutic agent or a combination of therapeutic agents is the amount of the agent or of the combination of agents that achieves a clinical endpoint of improved progression-free survival (PFS), improved PFS rate (e.g., improved PFS rate at 12 months), improved objective response rate (ORR), improved overall survival (OS), improved OS rate (e.g., improved OS rate at 12 months), improved pathologic response rate (PRR), improved disease control rate (DCR), a complete response (CR), a pathological complete response (pCR), a partial response (PR), improved survival (e.g., disease-free survival (DFS), and/or and/or improved duration of response (DOR).

As used herein, “complete response” and “CR” refer to disappearance of all target lesions.

As used herein, “partial response" and “PR” refer to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD prior to treatment, in the absence of CR.

As used here, “progressive disease” and “PD” refer to at least a 20% increase in the SLD of target lesions, taking as reference the smallest sum of diameters at prior timepoints, including baseline. The appearance of one or more new lesions may also be considered PD. As used herein, “stable disease” and “SD” refers to neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum.

As used herein, “disease control rate” and “DCR" refer to the percentage of patients cancer who have achieved CR, PR, and stable disease (SD). For example, DCR may be defined as the proportion of patients with SD for >12 weeks or a CR or PR, as determined by the investigator according to RECIST v1.1.

As used herein, “overall response rate,” “objective response rate,” and “ORR” refer interchangeably to the sum of CR rate and PR rate. For example, objective response may be defined as a CR or PR per Response Evaluation Criteria in Solid Tumors (RECIST) v.1 .1 , as determined by investigator assessment and confirmed by repeat assessment > 4 weeks after initial documentation. In another example, ORR may be defined as the proportion of patients with CR or PR on two consecutive occasions >4 weeks apart, as determined by the investigator according to RECIST v1 .1 .

As used herein, “progression-free survival" and “PFS” refer to the length of time during and after treatment during which the cancer does not get worse. PFS may include the amount of time patients have experienced a CR or a PR, as well as the amount of time patients have experienced stable disease. For example, PFS may be defined as the time from the first study treatment to the first occurrence of progression or death from any cause, whichever occurs first, per RECIST v.1 .1 as determined by the investigator. In another example, PFS may be defined as the time from study enrollment to the first occurrence of progression or death from any cause, whichever occurs first, per RECIST v.1 .1 as determined by the investigator.

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

As used herein, the term “duration of response” and “DOR” refer to a length of time from documentation of a tumor response until disease progression or death from any cause, whichever occurs first. For example, DOR may be defined as the time from the first occurrence of a documented objective response to the time of the first documented disease progression or death from any cause, whichever occurs first, per RECIST v1 .1 as determined by the investigator.

As used herein, the term “chemotherapeutic agent” refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include EGFR inhibitors (including small molecule inhibitors (e.g., erlotinib (TARCEVA®, Genentech/OSI Pharm.); PD 183805 (Cl 1033, 2-propenamide, N- [4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3’-Chloro-4’-fluoroanilino)-7-methoxy-6-(3- morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)- quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4- d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1 H-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 (TYKERB®, GSK572016 or N-[3-chloro-4-[(3 fluorophenyl)methoxy]phenyl]- 6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine)); a tyrosine kinase inhibitor (e.g., an EGFR inhibitor; a small molecule HER2 tyrosine kinase inhibitor such as TAK165 (Takeda); CP- 724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; PKI-166 (Novartis); pan-HER inhibitors such as canertinib (CI- 1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 (ISIS Pharmaceuticals) which inhibit Raf-1 signaling; non-HER-targeted tyrosine kinase inhibitors such as imatinib mesylate (GLEEVEC®, Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoding nucleic acid); quinoxalines (U.S. Patent No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI- 1033 (Pfizer); Affinitac (ISIS 3521 ; Isis/Lilly); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1 C11 (Imclone); and rapamycin (sirolimus, RAPAMUNE®)); proteasome inhibitors such as bortezomib (VELCADE®, Millennium Pharm.); disulfiram; epigallocatechin gallate; salinosporamide A; carfilzomib; 17-AAG (geldanamycin); radicicol; lactate dehydrogenase A (LDH-A); fulvestrant (FASLODEX®, AstraZeneca); letrozole (FEMARA®, Novartis), finasunate (VATALANIB®, Novartis); oxaliplatin (ELOXATIN®, Sanofi); 5-FU (5-fluorouracil); leucovorin; lonafamib (SCH 66336); sorafenib (NEXAVAR®, Bayer Labs); AG1478, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5a-reductases including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat dolastatin; aldesleukin, talc duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin y1 and calicheamicin w1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP-16); ifosfamide; mitoxantrone; novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, prodrugs, and derivatives of any of the above.

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

The term “cytotoxic agent” as used herein refers to any agent that is detrimental to cells (e.g., causes cell death, inhibits proliferation, or otherwise hinders a cellular function). Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At^{21 1}, I¹³¹ , 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeutic agents; enzymes and fragments thereof such as nucleolytic enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid biosynthesis, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism. In one instance, the cytotoxic agent is a platinum-based chemotherapeutic agent (e.g., carboplatin or cisplatin). In one instance, the cytotoxic agent is an antagonist of EGFR, e.g., N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g., erlotinib). In one instance the cytotoxic agent is a RAF inhibitor, e.g., 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 term “patient” or “subject” refers to a human patient or subject. For example, the patient or subject may be an adult.

The term “antibody” herein specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. In one instance, the antibody is a full-length monoclonal antibody. In one instance, the antibody is a bispecific antibody.

The term IgG “isotype” or “subclass” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, y, e, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non- covalent association of the antibody with one or more other proteins or peptides.

The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms refer to an antibody comprising an Fc region.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C- terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C- terminal amino acids of the heavy chain are glycine (G446) and lysine (K447). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including an Fc region are denoted herein without the C-terminal lysine (Lys447) if not indicated otherwise. In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody disclosed herein, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447). In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody disclosed herein, comprises an additional C-terminal glycine residue (G446). In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody disclosed herein, comprises an additional C-terminal lysine residue (K447). In one embodiment, the Fc region contains a single amino acid substitution N297A of the heavy chain. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

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

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

The term “hypervariable region” or“HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”).

Generally, antibodies comprise six CDRs: three in the VH (CDR-H1 , CDR-H2, CDR-H3), and three in the VL (CDR-L1 , CDR-L2, CDR-L3). Exemplary CDRs herein include:

(a) hypervariable loops occurring 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 occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31 -35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et aL, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

(c) antigen contacts occurring 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 indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

“Framework” or “FR” refers to variable domain residues other than complementary determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2- CDR-H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4.

The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e g., residues 82a, 82b, and 82c, etc., according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen. The term “bispecific” antibody as used herein means that the antibody is able to specifically bind to at least two distinct antigens, for example two binding sites each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) binding to different antigens or to different epitopes on the same antigen. Such a bispecific antibody is an 1 +1 format. Other bispecific antibody formats are 2+1 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, a bispecific antibody comprises two antigen binding sites, each of which is specific for a different antigen. In one instance, the bispecific antibody is a bispecific antibody comprising an Fc domain.

As used herein, a “PD-L1 -positive tumor cell fraction” is the percentage of viable tumor cells showing partial or complete membrane staining (exclusive of cytoplasmic staining) at any intensity relative to all viable tumor cells present in a sample, following staining of the sample in the context of an immunohistochemical (IHC) assay, e.g., an IHC assay staining for PD-L1 using the antibody SP142, SP263, 22C3, or 28-8. Accordingly, a PD-L1 -positive tumor cell fraction may be calculated using the PD- L1 IHC SP142 (Ventana) assay, for example, by the formula PD-L1 -positive tumor cell fraction = (number of PD-L1 -positive tumor cells)/(total number of PD-L1 -positive and PD-L1 negative tumor cells), wherein PD-L1 cytoplasmic staining of tumor cells and all non-tumor cells (e.g., tumor-infiltrating immune cells, normal cells, necrotic cells, and debris) are excluded from evaluation and scoring. It will be appreciated that any given diagnostic PD-L1 antibody may correspond with a particular IHC assay protocol and/or scoring terminology that can be used to derive a PD-L1 -positive tumor cell fraction. For example, a PD- L1-positive tumor cell fraction can be derived from a tumor cell sample stained with SP263, 22C3, SP142, or 28-8 using OPTIVIEW® detection on Benchmark ULTRA, EnVision Flex on AutostainerLink 48, OPTIVIEW® detection and amplification on Benchmark ULTRA, or EnVision Flex on AutostainerLink 48, respectively.

As used herein, the “Ventana SP142 IHC assay” is conducted 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” is conducted 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” is conducted 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” is conducted 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 “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

As used herein, “in combination with” refers to administration of one treatment modality in addition to another treatment modality, for example, a treatment regimen that includes administration of a bispecific antibody targeting programmed cell death protein 1 (PD-1) and lymphocyte activation gene-3 (LAG3) and an anti-TIGIT antagonist antibody (e.g., tiragolumab). As such, “in combination with” refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the patient.

A drug that is administered “concurrently” with one or more other drugs is administered during a treatment cycle occurring at the same time, during the same treatment cycle, and/or on the same day of treatment as the one or more other drugs, and, optionally, at the same time as the one or more other drugs. For instance, for cancer therapies given every 3 weeks, the concurrently administered drugs may be each administered on day 1 of a 3-week cycle. For dosing regimens comprising dosing cycles of two or more agents with different administration frequencies (e.g., dosing regimens comprising dosing cycles of (a) an agent that is administered every three weeks and (b) an agent that is administered once a week for three weeks, followed by 1 week off), dosing regimens are concurrent if they occur over the same period of time (e.g., the dosing cycles of the two or more agents with different administration frequencies begin on the same day).

As used herein, the term “adverse event” or “AE” refers to any unfavorable and unintended sign

(including an abnormal laboratory finding), symptom, or disease temporally 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 may be classified by “grade," as defined by the National Cancer Institute Common Terminology Criteria for Adverse Events v4.0 or v5.0 (NIH CTCAE). In some aspects, the AE is a low-grade AE, e.g., a Grade 1 or Grade 2 AE. Grade 1 includes AEs that are asymptomatic or have mild symptoms. Grade 2 includes AEs that are moderate and limit age-appropriate instrumental activities of daily living (e.g., preparing meals, shopping for groceries or clothes) and that indicate local or noninvasive intervention. In other instances, the AE is a high-grade AE, e.g., a Grade 3, Grade 4, or Grade 5 AE. In some instances, the AE is a Grade 3 or a Grade 4 AE. Grade 3 includes AEs that are severe or medically significant, but not immediately life-threatening, and that indicate hospitalization or prolongation of hospitalization. Grade 4 includes AEs that have life-threatening consequences and indicate urgent intervention. Grade 5 includes AEs that result in or relate to death.

As used herein, the term “treatment-related AE” refers to an AE that is judged by an investigator to have occurred as a result of a treatment, e.g., a bispecific antibody targeting PD-1 and LAG3 and/or an anti-TIGIT antagonist antibody, e.g., tiragolumab.

The term “valent” as used within the current application denotes the presence of a specified number of binding domains in an antigen binding molecule. As such, the terms “bivalent,” “tetravalent,” and “hexavalent” denote the presence of two binding domains, four binding domains, and six binding domains, respectively, in an antigen binding molecule. The bispecific antibodies according to the invention are at least “bivalent” and may be “trivalent” or “multivalent” (e.g., “tetravalent” or “hexavalent”). In a particular aspect, the antibodies of the present invention have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e., that the antibody is trivalent or multivalent).

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibodies formed from antibody fragments and single domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571 ,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding domains that 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 Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g., U.S. Patent No. 6,248,516 B1). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full-length antibodies. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. As used herein, Thus, the term “Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteins from the antibody hinge region. Fab’-SH are Fab’ fragments wherein the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.

The term “cross-Fab fragment” or “xFab fragment" or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e., the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). This crossover Fab molecule is also referred to as CrossFab <VLVH). On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab molecule is also referred to as CrossFab (CLCHD.

A “single chain Fab fragment” or“scFab” is a polypeptide consisting 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 said antibody domains and said linker have one of the following orders in 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 said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CH1 domain. In addition, these single chain Fab molecules might be further stabilized by generation of interchain disulfide bonds via 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).

A “crossover single chain Fab fragment” or “x-scFab” is a is a polypeptide consisting 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 said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CL- linker-VL-CH1 and b) VL-CH1 -linker- VH-CL; wherein VH and VL form together an antigen-binding domain which binds specifically to an antigen and wherein said linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules might be further stabilized by generation of interchain disulfide bonds via 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).

A “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, e.g., described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full-length antibodies.

A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. The first single domains were derived from the variable domain of the antibody heavy chain from camelids (nanobodies or VHH fragments). Furthermore, the term single-domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks. Fibronectin is a scaffold which can be engineered to bind to antigen. Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the p-sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest. For further details see Protein Eng. Des. Sei. 18, 435- 444 (2005), US20080139791 , W02005056764 and US6818418B1. Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site. For further details see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges - examples of microproteins include KalataBI and conotoxin and knottins. The microproteins have a loop which can beengineered to include upto 25 amino acids without affecting the overall fold of the microprotein. For further details of engineered knottin domains, see W02008098796.

The term “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,” “a bispecific antibody that specifically binds PD-1 and LAG3,” “bispecific antigen binding molecule specific for PD-1 and LAG3” or an “anti-PD-1/anti-LAG3 antibody” are used interchangeably herein and refer to a bispecific antibody that is capable of binding PD-1 and LAG3 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-1 and LAG3. In some aspects, the bispecific antibody targeting PD-1 and LAG3 comprises (a) a first antigen-binding domain that specifically binds to PD-1 comprising a heavy chain variable (VH) domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 1 , (ii) an HVR-H2 sequence comprising the amino acid sequence GGR, and (iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; and a light chain variable (VL) domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; (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: 4 and (b) a second antigen-binding domain that specifically binds to LAG3 comprising a VH domain comprising (i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, (ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and (iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; and a VL domain comprising (i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, (ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and (iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12. In some aspects, the first antigenbinding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14.

In some aspects, the bispecific antibody that specifically binds PD-1 and LAG3 is RO7247669. The RO7247669 antibody was first described in WO 2018/185043, which is hereby incorporated by reference in its entirety. RO7247669 is a novel, fragment crystallizable (Fc)-silent lgG1 -based bispecific antibody (BsAb) in a 1 + 1 format, that incorporates monovalent binding to the checkpoint receptors, PD-1 and lymphocyte activation gene 3 (LAG3). Use of a natural IgG-like monovalent heterodimeric lgG1 format allows the antibody to simultaneously bind to PD-1 and LAG3. The RO7247669 BsAb is engineered to preferentially bind to T-cells that co-express both PD-1 and LAG3, or to a lesser extent either PD-1 or LAG3 alone. Monovalent binding to LAG3 reduces internalization of the antibody (Ab) upon binding to the T-cell surface, and the retention time of RO7247669 on the T-cell surface is higher when simultaneously bound to PD-1 and LAG3. PGLALA mutations have been introduced into the lgG1 - based Fc region of RO7247669 to avoid drug-shaving and thus tumor-associated macrophage resistance mechanisms that have been observed with lgG4-based antibodies such as KEYTRUDA® (pembrolizumab) and OPDIVO® (nivolumab) (Arlauckas et al., Sei Transl Med, 9: eaal3604, 2017; Shen et al., EurJ Pharm Sci, 157:105629, 2021).

The term “PD-1 ,” also known as Programmed cell death protein 1 , is a type I membrane protein of 288 amino acids that was 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 regulators and has two ligands, PD-L1 (B7-H1 , CD274) and PD-L2 (B7-DC, CD273). The protein's structure includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, which suggests that PD-1 negatively regulates TCR signals. This is consistent with binding of SHP-1 and SHP-2 phosphatases to the cytoplasmic tail of PD-1 upon ligand binding. While PD-1 is not expressed on naive T cells, it is upregulated following T cell receptor (TCR)-mediated activation and is observed on both activated and exhausted T cells (Agata et aL, Int. Immunology 8 (1996), 765-772). These exhausted T-cells have a dysfunctional phenotype and are unable to respond appropriately. Although PD-1 has a relatively wide expression pattern its most important role is likely as a coinhibitory receptor on T cells (Chinai et al, Trends in Pharmacological Sciences 36 (2015), 587-595). Current therapeutic approaches thus focus on blocking the interaction of PD-1 with its ligands to enhance T cell response. The terms “Programmed Death 1 ,” “Programmed Cell Death 1 ,” “Protein PD-1 ,” “PD-1 ,” PD1 ,” “PDCD1 ,” “hPD-1” and “hPD-1” can be used interchangeably, and include variants, isoforms, species homologs of human PD-1 , and analogs having at least one common epitope with PD-1 . The amino acid sequence of human PD-1 is shown in UniProt (www.uniprot.org) accession no. Q15116 (SEQ ID NO: 19).

The terms “LAG3” or “Lag-3” or “Lymphocyte activation gene-3” or “CD223” as used herein refer to any native LAG3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed LAG3 as well as any form of LAG3 resulting from processing in the cell. The term also encompasses naturally occurring variants of LAG3, e.g., splice variants or allelic variants. In one preferred embodiment the term “LAG3” refers to human LAG3. The amino acid sequence of an exemplary processed (without signal sequences) LAG3 is shown in SEQ ID NO: 20. The amino acid sequence of an exemplary Extracellular Domain (ECD) LAG3 is shown in SEQ ID NO: 21.

The terms “anti-LAG3 antibody” and “an antibody that binds to LAG3” refer to an antibody that is capable of binding LAG3 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting LAG3. In one aspect, the extent of binding of an anti-LAG3 antibody to an unrelated, non-LAG3 protein is less than about 10% of the binding of the antibody to LAG3 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to LAG3 has a dissociation constant (KD) of 1uM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10^s M or less, e.g., from 10⁸ M to 10¹³ M, e.g., from 10⁹ M to 10¹³ M). In certain aspects, an anti- LAG3 antibody binds to an epitope of LAG3 that is conserved among LAG3 from different species. In one preferred embodiment, an “anti-LAG3 antibody,” “an antibody that specifically binds to human LAG3,” and “an antibody that binds to human LAG3” refers to an antibody specifically binding to the human LAG3 antigen or its Extracellular Domain (ECD) with a binding affinity of a Ko-value of 1 .0 x 10⁸ mol/l or lower, in one embodiment of a Ko-value of 1 .0 x 10⁹ mol/l or lower, in one embodiment of a Ko-value of 1.0 x 10⁹ mol/l to 1 .0 x 10¹³ mol/l. In this context the binding affinity is determined with a standard binding assay, such as surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden) e.g., using the LAG3 extracellular domain. The term “anti-LAG3 antibody” also encompasses bispecific antibodies that are capable of binding LAG3 and a second antigen.

The “knob-into-hole” technology is described e.g., 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). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain. In a further specific embodiment, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

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

An “activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcyRllla (CD16a), FcyRI (CD64), FcyRlla (CD32), and FcaRI (CD89). A particular activating Fc receptor is human FcyRllla (see UniProt accession no. P08637, version 141).

The term “peptide linker” refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non- immunogenic linker peptides are, for example, (G4S)_n, (SG4)n or G4(SG4)n peptide linkers, wherein “n” is generally a number between 1 and 10, typically between 2 and 4, in particular 2, i.e. the peptides selected from the group consisting of GGGGS (SEQ ID NO: 22) GGGGSGGGGS (SEQ ID NO: 23), SGGGGSGGGG (SEQ ID NO: 24) and GGGGSGGGGSGGGG (SEQ ID NO: 25), but also include the sequences GSPGSSSSGS (SEQ ID NO: 26), (G4S)₃ (SEQ ID NO: 27), (G4S)₄ (SEQ ID NO: 28), GSGSGSGS (SEQ ID NO: 29), GSGSGNGS (SEQ ID NO: 30), GGSGSGSG (SEQ ID NO: 31), GGSGSG (SEQ ID NO: 32), GGSG (SEQ ID NO: 33), GGSGNGSG (SEQ ID NO: 34), GGNGSGSG (SEQ ID NO: 35) and GGNGSG (SEQ ID NO: 36). Peptide linkers of particular interest are (G4S) (SEQ ID NO: 22), (G4S)₂ or GGGGSGGGGS (SEQ ID NO: 23), (G4S)₃ (SEQ ID NO: 27) and (G₄S)4 (SEQ ID NO: 29), more particularly (G₄S)2 (SEQ ID NO: 23) or GGGGSGGGGS (SEQ ID NO: 23).

By “fused to” or “connected to” is meant that the components (e.g., an antigen-binding domain and a Fc domain) are linked by peptide bonds, either directly or via one or more peptide linkers.

II. Therapeutic Methods and Compositions for Urothelial Bladder Cancer

A. Methods comprising a bispecific antibody targeting PD-1 and LAG3

In one aspect, the disclosure provides a method for treating a subject having an urothelial bladder cancer (UC) (e.g., a locally advanced or metastatic UC), the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD-1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), wherein (i) the subject is ineligible for platinum-based chemotherapy or (ii) the subject has not previously been treated with a cancer immunotherapy. In some aspects, the bispecific antibody is administered as a monotherapy.

In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a UC (e.g., a locally advanced or metastatic UC), wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody, wherein (i) the subject is ineligible for platinum-based chemotherapy or (ii) the subject has not previously been treated with a cancer immunotherapy, e.g., wherein the bispecific antibody is to be administered as a monotherapy.

The urothelial bladder cancer (UC) may be, e.g., a locally advanced or metastatic UC. In some aspects, (a) the clinical stage of the locally advanced urothelial bladder cancer is (i) T4b, any N or (ii) any T, N2-3; or (b) the clinical stage of the metastatic urothelial bladder cancer is M1 and/or Stage IV. In some aspects, the urothelial bladder cancer is a carcinoma, e.g, a transitional cell carcinoma.

Exemplary bispecific antibodies targeting PD-1 and LAG3 are provided in Section IV, below. A particular example for a bispecific antibody targeting PD-1 and LAG3 is PD1-LAG3 as defined herein. In some aspects, the method comprises administering the bispecific antibody (e.g., a bispecific antibody targeting PD-1 and LAG3 as provided in Section IV) at a fixed dose of 600 mg every three weeks (Q3W). In some aspects, the length of each of the one or more dosing cycles of the bispecific antibody is 21 days. In some aspects, the method comprises administering (e.g., intravenously administering) to the subject the bispecific antibody on Day 1 of each of the one or more dosing cycles. For example, the bispecific antibody may be administered by intravenous infusion over about 60 minutes or over about 30 minutes. In some aspects, the bispecific antibody is administered by intravenous infusion over a longer duration in a first cycle and over a shorter duration in a second or further dosing cycle: for example, in some aspects, the bispecific antibody is administered over 60 (± 15) minutes (e.g., over about 60 minutes) in a first dosing cycle and is administered over 30 (± 10) minutes (e.g., over about 30 minutes) in the one or more additional dosing cycles.

In some aspects, the subject is ineligible for platinum-based chemotherapy. For example, the subject may be determined to be ineligible for platinum-based chemotherapy based on having (a) an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and < 30 mL/min/1 .73 m²; (b) an ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1.73 m² and < 45 mL/min/1.73 m²; or (c) an ECOG Performance Status of 0-2 with Grade > 2 neuropathy. In some aspects, the GFR is assessed by calculation through use of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation:

CKD-EPI = 142 x (serum creatinine/A)^B x 0.9938^As^e x (1.012 if female) Where A and B are the following:

■ Female serum creatinine < 0.7: A = 0.7 and B = -0.241

■ Female serum creatinine > 0.7: A = 0.7 and B = -1.2

■ Male serum creatinine < 0.9: A = 0.9 and B = -0.302

■ Male serum creatinine > 0.9: A = 0.9 and B = -1 .2.

In some aspects, the subject has not previously been treated with a cancer immunotherapy. In some aspects, the subject has not previously been treated with a chemotherapy for locally advanced, metastatic, or recurrent urothelial bladder cancer. In some aspects, the subject is ineligible for platinum-based chemotherapy and has not previously been treated with a cancer immunotherapy.

In some aspects, the dosing regimen comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 15, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more than 50 dosing cycles of the bispecific antibody (e.g., comprises at least 5, at least 10, or at least 45 dosing cycles of the bispecific antibody).

In some aspects, a PD-L1 expression level of a tumor sample obtained from the subject has been determined. In some aspects, the US is a PD-L1 -positive UC. In other aspects, the UC is a PD-L1- negative UC. Exemplary methods for assessing PD-L1 expression level are provided, e.g., in Section III, below. In some aspects, the PD-L1 expression level is a protein expression level, and the protein expression level has been determined by an immunohistochemical (IHC) assay comprising staining with an anti-PD-L1 antibody suitable for staining (e.g., has been determined using a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, a Ventana SP142 IHC assay, or a pharmDx 28-8 IHC assay). In some aspects, the anti-PD-L1 antibody suitable for staining is the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8.

For example, in one aspect, the disclosure provides a method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy. In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in such a method.

In another aspect, the disclosure provides a method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy. In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in such a method.

In some aspects, the method results in an increase in objective response rate (ORR) (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15- 20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) in a population of subjects treated according to the method as compared to a reference ORR. In some aspects, the reference ORR is an ORR of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in progression-free survival (PFS) (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e g., an increase of 1-5%, 5-10%, 10-15%, 15- 20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) as compared to a reference PFS. In some aspects, the reference PFS is a PFS of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in overall survival (OS) (e.g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80- 85%, 85-90%, 90-95%, 95-100%, or more than 100%) as compared to a reference OS. In some aspects, the reference OS is an OS of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in duration of response (DOR) (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15- 20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) as compared to a reference DOR. In some aspects, the reference DOR is a DOR of a population of subjects who have received a control therapy.

In some aspects, the method results in (a) an increase in PFS rate (e.g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25- 30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) at 6 months as compared to a reference PFS rate at 6 months; or (b) an increase in PFS rate (e.g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) at 12 months as compared to a reference PFS rate at 12 months. In some aspects, the reference PFS rate is a PFS rate of a population of subjects who have received a control therapy.

In some aspects, the method results in (a) an increase in OS rate (e.g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25- 30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) at 6 months as compared to a reference OS rate at 6 months; (b) an increase in OS (e.g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50- 55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) rate at 12 months as compared to a reference OS rate at 12 months; or (c) an increase in OS rate (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,

65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, ID- 15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) at 18 months as compared to a reference OS rate at 18 months. In some aspects, the reference OS rate is an OS rate of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in disease control rate (DCR) (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15- 20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) as compared to a reference DCR. In some aspects, the reference DCR is a DCR of a population of subjects who have received a control therapy.

In some aspects, the control therapy is a treatment comprising atezolizumab, and the control therapy does not comprise the bispecific antibody or an anti-TIGIT antagonist antibody. In some aspects, the control therapy is atezolizumab monotherapy.

In some aspects, the subject is a human.

B. Methods comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody

In another aspect, the disclosure provides a method for treating a subject having an urothelial bladder cancer (UC) (e.g., a locally advanced or metastatic UC), the method comprising administering to the subject one or more dosing cycles of (a) an anti-TIGIT antagonist antibody; and (b) a bispecific antibody targeting PD-1 and LAG3 comprising a first antigen-binding domain that specifically binds to PD- 1 and a second antigen-binding domain that specifically binds to LAG3, wherein (i) the subject is ineligible for platinum-based chemotherapy or (ii) the subject has not previously been treated with a cancer immunotherapy.

In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a UC (e.g., a locally advanced or metastatic UC), wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, 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 dosing regimen comprises administering to the subject one or more dosing cycles of (a) the anti-TIGIT antagonist antibody and (b) the bispecific antibody, wherein (i) the subject is ineligible for platinum-based chemotherapy; or (ii) the subject has not previously been treated with a cancer immunotherapy.

The urothelial bladder cancer (UC) may be, e.g., a locally advanced or metastatic UC. In some aspects, (a) the clinical stage of the locally advanced urothelial bladder cancer is (i) T4b, any N or (ii) any T, N2-3; or (b) the clinical stage of the metastatic urothelial bladder cancer is M1 and/or Stage IV. In some aspects, the urothelial bladder cancer is a carcinoma, e.g, a transitional cell carcinoma.

Exemplary bispecific antibodies targeting PD-1 and LAG3 are provided in Section IV, below. A particular example for a bispecific antibody targeting PD-1 and LAG3 is PD1-LAG3 as defined herein. In some aspects, the method comprises administering the bispecific antibody (e.g., a bispecific antibody targeting PD-1 and LAG3 as provided in Section IV) at a fixed dose of 600 mg every three weeks (Q3W). In some aspects, the length of each of the one or more dosing cycles of the bispecific antibody is 21 days. In some aspects, the method comprises administering (e.g., intravenously administering) to the subject the bispecific antibody on Day 1 of each of the one or more dosing cycles. For example, the bispecific antibody may be administered by intravenous infusion over about 60 minutes or over about 30 minutes. In some aspects, the bispecific antibody is administered by intravenous infusion over a longer duration in a first cycle and over a shorter duration in a second or further dosing cycle: for example, in some aspects, the bispecific antibody is administered over 60 (± 15) minutes (e.g., over about 60 minutes) in a first dosing cycle and is administered over 30 (± 10) minutes (e.g., over about 30 minutes) in the one or more additional dosing cycles.

Exemplary anti-TIGIT antagonist antibodies are provided in Section V, below. In some aspects, the method comprises administering (e.g., intravenously administering) the anti-TIGIT antagonist antibody (e.g., anti-TIGIT antagoniost antibody as provided in Section V, e.g., tiragolumab) at a fixed dose of 600 mg every three weeks (Q3W). In some aspects, the length of each of the one or more dosing cycles of the bispecific antibody is 21 days. In some aspects, the method comprises administering (e.g., intravenously administering) to the subject the anti-TIGIT antagonist antibody on Day 1 of each of the one or more dosing cycles.

On days on which the bispecific antibody and the anti-TIGIT antagonist antibody are administered on the same day (e.g., on the first day of the dosing regimen), the bispecific antibody may be administered before the anti-TIGIT antagonist antibody. For example, in some aspects, the anti-TIGIT antagonist antibody is administered at least 60 minutes after the bispecific antibody (e.g., in a first dosing cycle). In some aspects in which the method comprises one or more additional dosing cycles, the anti- TIGIT antagonist antibody may be administered at least 30 minutes after the bispecific antibody in the one or more additional dosing cycles. Alternatively, in some aspects, the anti-TIGIT antagonist antibody is administered before the bispecific antibody or the anti-TIGIT antagonist antibody and the bispecific antibody are co-administered.

In some aspects, the subject is ineligible for platinum-based chemotherapy. For example, the subject may be determined to be ineligible for platinum-based chemotherapy based on having (a) an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and < 30 mL/min/1 .73 m²; (b) an ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1.73 m² and < 45 mL/min/1.73 m²; or (c) an ECOG Performance Status of 0-2 with Grade > 2 neuropathy. In some aspects, the GFR is assessed by calculation through use of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation:

CKD-EPI = 142 x (serum creatinine/A)^B x 0.9938^A9^e x (1.012 if female) Where A and B are the following:

■ Female serum creatinine < 0.7: A = 0.7 and B = -0.241

■ Female serum creatinine > 0.7: A = 0.7 and B = -1.2

■ Male serum creatinine < 0.9: A = 0.9 and B = -0.302

■ Male serum creatinine > 0.9: A = 0.9 and B = -1 .2. In some aspects, the subject has not previously been treated with a cancer immunotherapy. In some aspects, the subject has not previously been treated with a chemotherapy for locally advanced, metastatic, or recurrent urothelial bladder cancer.

In some aspects, the subject is ineligible for platinum-based chemotherapy and has not previously been treated with a cancer immunotherapy.

In some aspects, the dosing regimen comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 15, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more than 50 dosing cycles of the bispecific antibody (e.g., comprises at least 5, at least 10, or at least 45 dosing cycles of the bispecific antibody). In some aspects, the dosing regimen comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 15, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more than 50 dosing cycles of the anti-TIGIT antagonist antibody. In some aspects, the dosing regimen comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 15, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more than 50 dosing cycles (e.g., at least 5, at least 10, or at least 45 dosing cycles) of (a) the bispecific antibody and (b) the anti-TIGIT antagonist antibody.

In some aspects, a PD-L1 expression level of a tumor sample obtained from the subject has been determined. In some aspects, the US is a PD-L1 -positive UC. In other aspects, the UC is a PD-L1- negative UC. Exemplary methods for assessing PD-L1 expression level are provided, e.g., in Section III, below. In some aspects, the PD-L1 expression level is a protein expression level, and the protein expression level has been determined by an immunohistochemical (IHC) assay comprising staining with an anti-PD-L1 antibody suitable for staining (e.g., has been determined using a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, a Ventana SP142 IHC assay, or a pharmDx 28-8 IHC assay). In some aspects, the anti-PD-L1 antibody suitable for staining is the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8.

For example, in one aspect, the disclosure provides a method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of (a) an anti-TIGIT antagonist antibody (e.g., tiragolumab) and (b) a bispecific antibody targeting PD-1 and LAG3 comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy. In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 and/or an anti-TIGIT antagonist antibody for use in such a method.

In another aspect, the disclosure provides a method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of (a) an anti-TIGIT antagonist antibody (e.g., tiragolumab) and (b) a bispecific antibody targeting PD-1 and LAG3 comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy. In another aspect, the disclosure provides a bispecific antibody targeting PD-1 and LAG3 and/or an anti-TIGIT antagonist antibody for use in such a method.

In some aspects, the method results in an increase in objective response rate (ORR) (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e g., an increase of 1-5%, 5-10%, 10-15%, 15- 20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) in a population of subjects treated according to the method as compared to a reference ORR. In some aspects, the reference ORR is an ORR of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in progression-free survival (PFS) (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15- 20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) as compared to a reference PFS. In some aspects, the reference PFS is a PFS of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in overall survival (OS) (e.g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80- 85%, 85-90%, 90-95%, 95-100%, or more than 100%) as compared to a reference OS. In some aspects, the reference OS is an OS of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in duration of response (DOR) (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15- 20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) as compared to a reference DOR. In some aspects, the reference DOR is a DOR of a population of subjects who have received a control therapy.

In some aspects, the method results in (a) an increase in PFS rate (e.g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25- 30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) at 6 months as compared to a reference PFS rate at 6 months; or (b) an increase in PFS rate (e.g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) at 12 months as compared to a reference PFS rate at 12 months. In some aspects, the reference PFS rate is a PFS rate of a population of subjects who have received a control therapy. In some aspects, the method results in (a) an increase in OS rate (e g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25- 30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) at 6 months as compared to a reference OS rate at 6 months; (b) an increase in OS (e.g., an increase of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50- 55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) rate at 12 months as compared to a reference OS rate at 12 months; or (c) an increase in OS rate (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10- 15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) at 18 months as compared to a reference OS rate at 18 months. In some aspects, the reference OS rate is an OS rate of a population of subjects who have received a control therapy.

In some aspects, the method results in an increase in disease control rate (DCR) (e.g., an increase of at least 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, e.g., an increase of 1-5%, 5-10%, 10-15%, 15- 20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, or more than 100%) as compared to a reference DCR. In some aspects, the reference DCR is a DCR of a population of subjects who have received a control therapy.

In some aspects, the control therapy is a treatment comprising atezolizumab, and the control therapy does not comprise the bispecific antibody or an anti-TIGIT antagonist antibody. In some aspects, the control therapy is atezolizumab monotherapy.

In some aspects, the subject is a human.

III. Assessment of PD-L1 Expression

The expression of PD-L1 may be assessed in a subject treated according to any of the methods and compositions for use described herein. The methods and compositions for use may include determining the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the subject having a cancer (e.g., a urothelial bladder cancer (UC), e.g., a locally advanced or metastatic UC). In other examples, the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the subject has been determined prior to initiation of treatment or after initiation of treatment. PD-L1 expression may be determined using any suitable approach. For example, PD-L1 expression may be determined as described in U.S. Patent Publication Nos. US 20180030138 A1 and US 20180037655 A1 . Any suitable tumor sample may be used, e.g., a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample.

In some aspects, the PD-L1 expression level in a cancer (e.g., a UC) or a sample therefrom is a protein expression level, and the protein expression level has been determined by an immunohistochemical (IHC) assay comprising staining with an anti-PD-L1 antibody suitable for staining (e.g., has been determined using a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, a Ventana SP142 IHC assay, or a pharmDx 28-8 IHC assay). In some aspects, the anti-PD-L1 antibody suitable for staining is the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8.

For example, PD-L1 expression may be determined in terms of the percentage of a tumor sample comprised by tumor-infiltrating immune cells expressing a detectable expression level of PD-L1 , as the percentage of tumor-infiltrating immune cells in a tumor sample expressing a detectable expression level of PD-L1 , and/or as the percentage of tumor cells in a tumor sample expressing a detectable expression level of PD-L1. It is to be understood that in any of the preceding examples, the percentage of the tumor sample comprised by tumor-infiltrating immune cells may be in terms of the percentage of tumor area covered by tumor-infiltrating immune cells in a section of the tumor sample obtained from the subject, for example, as assessed by IHC using an anti-PD-L1 antibody (e.g., the SP142 antibody). Any suitable anti- PD-L1 antibody may be used, including, e.g., SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28-8 (Dako), E1 L3N (Cell Signaling Technology), 4059 (ProSci, Inc.), h5H1 (Advanced Cell Diagnostics), and 9A11. In some examples, the anti-PD-L1 antibody is SP142. In other examples, the anti-PD-L1 antibody is SP263.

In some examples, a tumor sample obtained from the subject has a detectable expression level of PD-L1 in less than 1 % of the tumor cells in the tumor sample, in 1% or more of the tumor cells in the tumor sample, in from 1% to less than 5% of the tumor cells in the tumor sample, in 5% or more of the tumor cells in the tumor sample, in from 5% to less than 50% of the tumor cells in the tumor sample, or in 50% or more of the tumor cells in the tumor sample.

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

In some aspects, the cancer (e.g., UC, e.g., locally advanced or metastatic UC) of a subject treated according to any of the methods provided herein has a PD-L1 -positive tumor cell (TC) fraction or tumor-infiltrating immune cell (IC) fraction of < 5%. In some aspects, the esophageal cancer has a PD-L1- positive TC fraction of <1%. In other aspects, the cancer of a subject treated according to any of the methods provided herein has a PD-L1 -positive TC fraction or IC fraction of > 5%. In some aspects, PD-L1 is detected using a Ventana SP142 IHC assay, a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, or a pharmDx 28-8 IHC assay.

In some examples, tumor samples may be scored for PD-L1 positivity in tumor-infiltrating immune cells and/or in tumor cells according to the criteria for diagnostic assessment shown in Table 1 and/or Table 2, respectively. Table 1. Tumor-infiltrating immune cell (IC) IHC diagnostic criteria

Table 2. Tumor cell (TC) IHC diagnostic criteria

IV. Bispecific antibodies targeting PD-1 and LAG3

A. Exemplary bispecific antibodies that bind to PD-1 and LAG3

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

(i) an HVR-H1 comprising the amino acid sequence of GFSFSSY (SEQ ID NO: 1),

(ii) an HVR-H2 comprising the amino acid sequence GGR, and

(iii) an HVR-H3 comprising an amino acid sequence of TGRVYFALD (SEQ ID NO: 2); and a VL domain comprising (i) an HVR-L1 comprising the amino acid sequence of SESVDTSDNSF (SEQ ID NO: 3);

(ii) an HVR-L2 comprising the amino acid sequence RSS, and

(iii) an HVR-L3 comprising the amino acid sequence of NYDVPW (SEQ ID NO: 4). In one aspect, the bispecific antibody comprises a Fc domain that is an IgG. In some aspects, the Fc domain is an lgG1 Fc domain or an lgG4 Fc domain. In some aspects, the Fc domain has reduced or even abolished effector function. In particular, the Fc domain may comprisee one or more amino acid substitutions that reduces binding to an Fc receptor, in particular towards Fey receptor.

In a further aspect, provided herein is a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises a Fc domain that is an IgG, particularly an lgG1 Fc domain or an lgG4 Fc domain and wherein the Fc domain comprises one or more amino acid substitutions that reduces binding to an Fc receptor, in particular towards Fey receptor.

In another aspect, provided herein is 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 second antigen-binding domain that specifically binds to LAG3 comprises a VH domain comprising:

(i) an HVR-H1 comprising the amino acid sequence of DYTMN (SEQ ID NO: 7),

(ii) an HVR-H2 comprising the amino acid sequence of VISWDGGGTYYTDSVKG (SEQ ID NO: 8), and

(iii) an HVR-H3 comprising an amino acid sequence of GLTDTTLYGSDY (SEQ ID NO: 9); and a VL domain comprising

(i) an HVR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 10),

(ii) an HVR-L2 comprising the amino acid sequence of AASTLQS (SEQ ID NO: 11), and

(iii) an HVR-L3 comprising the amino acid sequence of QQTYSSPLT (SEQ ID NO: 12).

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

(i) an HVR-H1 comprising the amino acid sequence of GFSFSSY (SEQ ID NO: 1),

(ii) an HVR-H2 comprising the amino acid sequence GGR, and

(iii) an HVR-H3 comprising an amino acid sequence of TGRVYFALD (SEQ ID NO: 2); and a VL domain comprising

(i) an HVR-L1 comprising the amino acid sequence of SESVDTSDNSF (SEQ ID NO: 3);

(ii) an HVR-L2 comprising the amino acid sequence RSS, and the second antigen-binding domain that specifically binds to LAG3 comprises a VH domain comprising:

(i) an HVR-H1 comprising the amino acid sequence of DYTMN (SEQ ID NO: 7),

(ii) an HVR-H2 comprising the amino acid sequence of VISWDGGGTYYTDSVKG (SEQ ID NO: 8), and

(iii) an HVR-H3 comprising an amino acid sequence of GLTDTTLYGSDY (SEQ ID NO: 9); and a VL domain comprising

(i) an HVR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 10),

(ii) an HVR-L2 comprising the amino acid sequence of AASTLQS (SEQ ID NO: 11), and

(iii) an HVR-L3 comprising the amino acid sequence of QQTYSSPLT (SEQ ID NO: 12). In a further aspect, provided herein is 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 first antigen- binding domain specifically binding to PD-1 comprises a VH domain comprising the amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYTMSWVRQAPGKGLEWVATISGGGRDIYYPDSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVLLTGRVYFALDSWGQGTLVTVSS (SEQ ID NO: 5) and a VL domain comprising the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKASESVDTSDNSFIHWYQQKPGQSPKLLIYRSSTLESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCQQNYDVPWTFGQGTKVEIK (SEQ ID NO: 6).

In another aspect, provided herein is 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 second antigen-binding domain specifically binding to LAG3 comprises a VH domain comprising the amino acid sequence of EVQLLESGGGLVQPGGSLRL SCAASGFIFDDYTMNVWRQAPGKGLEWVAVISWDGGGTYYTDSVKGRFTISRDDFKNTLY LQMNSLRAEDTAVYYCAKGLTDTTLYGSDYWGQGTLVTVSS (SEQ ID NO: 13) and a VL domain comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQ TYSSPLTFGGGTKVEIK (SEQ ID NO: 14).

In another aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the first antigen-binding domain specifically binding to PD-1 comprises a VH domain comprising the amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYTMSWVRQAPGKGLEWVATISGGGRDIYYPDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVLLTGRVYFALDSWGQGTLVTVSS (SEQ ID NO: 5) and a VL domain comprising the amino acid sequence of DIVMTQSPDSLAVSLGERATINCKASESVDTSDNSFIHWYQQKPGQSPKLLIYRSSTLESGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQNYDVPWTFGQGTKVEIK (SEQ ID NO: 6); and the second antigenbinding domain specifically binding to LAG3 comprises a VH domain comprising the amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFIFDDYTMNWVRQAPGKGLEWVAVISWDGGGTYYTDSVKGRF TISRDDFKNTLY LQMNSLRAEDTAVYYCAKGLTDTTLYGSDYWGQGTLVTVSS ( SEQ ID NO: 13) and a VL domain comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQ TYSSPLTFGGGTKVEIK (SEQ ID NO: 14).

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 specifically binding to PD-1 comprises a VH domain having at least 90% identity to (e.g., having 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to) the amino acid sequence of SEQ ID NO: 5 and a VL domain having at least 90% identity to (e.g., having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to) the amino acid sequence of SEQ ID NO: 6. In one aspect, the first antigen-binding domain specifically binding to PD-1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6. 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 specifically binding to LAG3 comprises a VH domain having at least 90% identity to (e.g., having 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to) the amino acid sequence of SEQ ID NO: 13 and a VL domain having at least 90% identity to (e.g., having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to) the amino acid sequence of SEQ ID NO: 14. In one aspect, the second antigen-binding domain specifically binding to LAG3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14.

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 specifically binding to PD-1 comprises a VH domain having at least 90% identity to (e.g., having 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to) the amino acid sequence of SEQ ID NO: 5 and a VL domain having at least 90% identity to (e.g., having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to) the amino acid sequence of SEQ ID NO: 6 and the second antigen-binding domain specifically binding to LAG3 comprises a VH domain having at least 90% identity to (e.g., having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to) the amino acid sequence of SEQ ID NO: 13 and a VL domain having at least 90% identity to (e.g., having 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identity to) the amino acid sequence of SEQ ID NO: 14.

In another aspect, provided is 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 first antigen-binding domain specifically binding to PD-1 comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain specifically binding to LAG3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14.

In a further 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. In particular, it is a humanized or chimeric antibody.

In one 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 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 (1+1 format).

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

In a particular aspect, provided is a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises a first heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15, a first light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16, a second heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17, and a second light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO:18. For example, in one aspect, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO:18 (PD1- LAG3).

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

In one aspect, the bispecific antibody comprises a first heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15, a first light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16, a second heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 37, and a second light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18. More particularly, the bispecific antibody may comprise a first heavy chain comprising an amino acid sequence of SEQ ID NO: 15, a first light chain comprising an amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising an amino acid sequence of SEQ ID NO: 37, and a second light chain comprising an amino acid sequence of SEQ ID NO: 18.

In some aspects, the bispecific antibody that specifically binds PD-1 and LAG3 is RO7247669.

Fc domain modifications reducing Fc receptor binding and/or effector function

In certain aspects, provided is a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the bispecific antibody comprises a Fc domain comprising one or more amino acid modifications that reduce binding to an Fc receptor, in particular towards Fey receptor, and reduce or abolish effector function.

In certain aspects, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e g., a human IgG 1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

The following section describes preferred aspects of the bispecific antigen binding molecules of the invention comprising Fc domain modifications reducing Fc receptor binding and/or effector function. In one aspect, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, in particular towards Fey receptor. In particular, the Fc domain is of human lgG1 subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).

The Fc domain confers favorable pharmacokinetic properties to the bispecific antibodies of the invention, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the bispecific antibodies of the invention to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Accordingly, in particular embodiments the Fc domain of the the bispecific antibodies of the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgG Fc domain, in particular an lgG1 Fc domain or an lgG4 Fc domain. More particularly, the Fc domain is an lgG1 FC domain.

In one such aspect the Fc domain (or the bispecific antigen binding molecule of the invention comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native lgG1 Fc domain (or the bispecific antigen binding molecule of the invention comprising a native lgG1 Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native lgG1 Fc domain (or the bispecific antigen binding molecule of the invention comprising a native lgG1 Fc domain). In one aspect, the Fc domain (or the bispecific antigen binding molecule of the invention comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function. In a particular aspect the Fc receptor is an Fey receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fey receptor, more specifically human FcyRllla, FcyRI or FcyRlla, most specifically human FcyRllla. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a specific aspect, the Fc receptor is an inhibitory human Fey receptor, more specifically human FcyRIIB. 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 domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native lgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the the bispecific antigen binding molecule of the invention comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greaterthan about 90% of the binding affinity of a native lgG1 Fc domain (or the the bispecific antigen binding molecule of the invention comprising a native lgG1 Fc domain) to FcRn.

In a particular aspect, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In a particular aspect, the Fc domain of the bispecific antigen binding molecule of the invention comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is 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 an Fc receptor. In another aspect, the amino acid mutation reduces the binding affinity of the Fc domain to an 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 invention comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to bispecific antibodies of the invention comprising a non-engineered Fc domain. In a particular aspect, the Fc receptor is an Fey receptor. In other aspects, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor Is an inhibitory Fc receptor. In a specific aspect, the Fc receptor is an inhibitory human Fey receptor, more specifically human FcyRIIB. In some aspects the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fey receptor, more specifically human FcyRllla, FcyRI or FcyRlla, most specifically human FcyRllla. Preferably, binding to each of these receptors is reduced. In some aspects, binding affinity to a complement component, specifically binding affinity to C1q, is also reduced. In one aspect, binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the bispecific antigen binding molecule of the invention comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the bispecific antigen binding molecule of the invention comprising said nonengineered form of the Fc domain) to FcRn. The Fc domain, or the the bispecific antigen binding molecule of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain of the bispecific antigen binding molecule of the invention is engineered to have reduced effector function, as compared to a nonengineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581). Certain antibody variants with improved or diminished binding to FcRs are described, (e.g. U.S. 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 comprises an amino acid substitution at a position of E233, L234, L235, N297, P331 and P329. In some aspects, the Fc domain comprises the amino acid substitutions L234A and L235A (“LALA”). In one such embodiment, the Fc domain is an lgG1 Fc domain, particularly a human lgG1 Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D or P331S. In more particular embodiments the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA"). The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor binding of a human IgG 1 Fc domain, as described in PCT Patent Application No. WO 2012/130831 A1. Said document also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions. Such antibody is an IgG 1 with mutations L234A and L235A or with mutations L234A, L235A and P329G (numbering according to EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

In one aspect, the bispecific antibody of the invention comprises (all positions according to EU index of Kabat) (i) a homodimeric Fc-region of the human lgG1 subclass optionally with the mutations P329G, L234A and L235A, or (ii) a homodimeric Fc-region of the human lgG4 subclass optionally with the mutations P329G, S228P and L235E, or (iii) a homodimeric Fc-region of the human lgG1 subclass optionally with the mutations P329G, L234A, L235A, I253A, H310A, and H435A, or optionally with the mutations P329G, L234A, L235A, H310A, H433A, and Y436A, or (iv) a heterodimeric Fc-region wherein one Fc-region polypeptide comprises the mutation T366W, and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or wherein one Fc-region polypeptide comprises the mutations T366W and Y349C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C, or wherein one Fc-region polypeptide comprises the mutations T366W and S354C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C, or (v) a heterodimeric Fc-region of the human lgG1 subclass wherein both Fc-region polypeptides comprise the mutations P329G, L234A and L235A and one Fc-region polypeptide comprises the mutation T366W, and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or wherein one Fc- region polypeptide comprises the mutations T366W and Y349C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C, or wherein one Fc-region polypeptide comprises the mutations T366W and S354C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

In one aspect, the Fc domain is an lgG4 Fc domain. In a more specific embodiment, the Fc domain is an lgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an lgG4 Fc domain comprising amino acid substitutions L235E and S228P and P329G. This amino acid substitution reduces in vivo Fab arm exchange of lgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). Thus, in one aspect, provided is a bispecific antibody, comprising (all positions according to EU index of Kabat) a heterodimeric Fc-region of the human lgG4 subclass wherein both Fc-region polypeptides comprise the mutations P329G, S228P and L235E and one Fc-region polypeptide comprises the mutation T366W, and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or wherein one Fc-region polypeptide comprises the mutations T366W and Y349C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C, or wherein one Fc-region polypeptide comprises the mutations T366W and S354C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs 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. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371 ,826). 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 concerning other examples of Fc region variants.

Binding to Fc receptors can be easily determined, e.g., by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. A suitable such binding assay is described herein. Alternatively, binding affinity of Fc domains or cell activating bispecific antigen binding molecules comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor. Effector function of an Fc domain, or bispecific antibodies of the invention comprising an Fc domain, can be measured by methods known in the art. A suitable assay for measuring ADCC is described herein. Other examples of in vitro assays to assess ADCC activity of a molecule of interest 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 methods may be employed (see, for example, ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wl)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

The following section describes preferred aspects of the bispecific antibodies of the invention comprising Fc domain modifications reducing Fc receptor binding and/or effector function. In one aspect, the invention relates to the bispecific comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the Fc domain comprises one or more amino acid substitution that reduces the binding affinity of the antibody to an Fc receptor, in particular towards Fey receptor. In another aspect, the invention relates to 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, wherein the Fc domain comprises one or more amino acid substitution that reduces effector function. In particular aspect, the Fc domain is of human lgG1 subclass with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index). Fc domain modifications promoting heterodimerization

The bispecific antigen binding molecules of the invention comprise different antigen-binding domains, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain may be comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antibodies of the invention in recombinant production, it will thus be advantageous to introduce in the Fc domain of the bispecific antigen binding molecules of the invention a modification promoting the association of the desired polypeptides.

Accordingly, in particular aspects the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein the Fc domain comprises a modification promoting the association of the first and second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect said modification is in the CH3 domain of the Fc domain.

In a specific aspect said modification is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole" modification in the other one of the two subunits of the Fc domain. Thus, the invention relates to a bispecific antibody comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding site that specifically binds to LAG3, wherein the first subunit of the Fc domain comprises knobs and the second subunit of the Fc domain comprises holes according to the knobs into holes method. In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).

The knob-into-hole technology is described e.g., 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). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).

Accordingly, in one aspect, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen binding molecules of the invention an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis. In a specific aspect, in the CH3 domain of the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one aspect, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).

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

But also other knobs-in-holes technologies as described by EP 1 870 459, can be used alternatively or additionally. In one embodiment the multispecific antibody comprises the mutations R409D and K370E in the CH3 domain of the “knobs chain” and the mutations D399K and E357K in the CH3 domain of the “hole-chain” (numbering according to Kabat EU index).

In one aspect, the bispecific antibody comprises a T366W mutation in the CH3 domain of the “knobs chain” and the mutations T366S, L368A and Y407V in the CH3 domain of the “hole chain” and additionally the mutations R409D and K370E in the CH3 domain of the “knobs chain” and the mutations D399K and E357K in the CH3 domain of the “hole chain” (numbering according to the Kabat EU index).

In one aspect, the bispecific antibody comprises the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains, or the multispecific antibody comprises the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains and additionally the mutations R409D and K370E in the CH3 domain of the “knobs chain” and the mutations D399K and E357K in the CH3 domain of the “hole chain” (numbering according to the Kabat EU index).

In an alternative aspect, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g., as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.

Apart from the “knob-into-hole technology” other techniques for modifying the CH3 domains of the heavy chains of a multispecific antibody to enforce heterodimerization are known in the art. These technologies, especially the ones 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 contemplated herein as alternatives to the “knob-into-hole technology” in combination with a bispecific antibody. In one aspect, in the bispecific antibody the approach described in EP 1870459 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3-domain-interface between both, the first and the second heavy chain.

Accordingly, in this aspect in the tertiary structure of the multispecific antibody the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface that is located between the respective antibody CH3 domains, wherein the respective amino acid sequences of the CH3 domain of the first heavy chain and the amino acid sequence of the CH3 domain of the second heavy chain each comprise a set of amino acids that is located within said interface in the tertiary structure of the antibody, wherein from the set of amino acids that is located in the interface in the CH3 domain of one heavy chain a first amino acid is substituted by a positively charged amino acid and from the set of amino acids that is located in the interface in the CH3 domain of the other heavy chain a second amino acid is substituted by a negatively charged amino acid. The bispecific antibody according to this aspect is herein also referred to as “CH3(+/-)-engineered bispecific antibody” (wherein the abbreviation

stands for the oppositely charged amino acids that were introduced in the respective CH3 domains).

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

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

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

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

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

In another aspect, in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by K and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index). Additionally at least one of the following substitutions is comprised in the CH3 domain of the other heavy chain: the amino acid Y at position 349 is substituted by E, the amino acid Y at position 349 is substituted by D and the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index). In one embodiment the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index). In one aspect, the approach described in WO 2012/058768 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one aspect, in the CH3 domain of one heavy chain the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted by A and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index). In another embodiment, in addition to the aforementioned substitutions, in the CH3 domain of the other heavy chain at least one of the amino acids at positions 411 (originally T), 399 (originally D), 400 (originally S), 405 (originally F), 390 (originally N) and 392 (originally K) is substituted (numbering according to Kabat EU index). Preferred substitutions are:

- substituting the amino acid T at position 411 by an amino acid selected from N, R, Q, K, D, E and W (numbering according to Kabat EU index),

- substituting the amino acid D at position 399 by an amino acid selected from R, W, Y, and K (numbering according to Kabat EU index),

- substituting the amino acid S at position 400 by an amino acid selected from E, D, R and K (numbering according to Kabat EU index),

- substituting the amino acid F at position 405 by an amino acid selected from I, M, T, S, V and W (numbering according to Kabat EU index;

- substituting the amino acid N at position 390 by an amino acid selected from R, K and D (numbering according to Kabat EU index; and

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

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

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

In one aspect, the approach described in WO 2011/090762 is used to support heterodimerization of the first heavy chain and the second heavy chain of the bispecific antibody. WO 2011/090762 relates to amino acid modifications according to the “knob-into-hole” (KiH) technology. In one embodiment in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by W, and in the CH3 domain of the other heavy chain the amino acid Y at position 407 is substituted by A (numbering according to Kabat EU index). In another embodiment in the CH3 domain of one heavy chain the amino acid T at position 366 is substituted by Y, and in the CH3 domain of the other heavy chain the amino acid Y at position 407 is substituted by T (numbering according to Kabat EU index).

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

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

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

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

/. Modifications in the Fab domains

In one aspect, the invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD-1 and a second Fab fragment that specifically binds to LAG3, wherein in one of the Fab fragments either the variable domains VH and VL or the constant domains CH1 and CL are exchanged. The bispecific antibodies are prepared according to the Crossmab technology. Multispecific antibodies with a domain replacement/exchange in one binding arm (CrossMabVH- VL or CrossMabCH-CL) are described in detail in W02009/080252, W02009/080253 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191 . They clearly reduce the byproducts caused by the mismatch of a light chain against a first antigen with the wrong heavy chain against the second antigen (compared to approaches without such domain exchange).

In a particular aspect, the invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD-1 and a second Fab fragment that specifically binds to LAG3, wherein in one of the Fab fragments the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In a particular aspect, the bispecific antibody is one, wherein in the first Fab fragment comprising the antigen-binding domain that specifically binds to PD-1 the variable domains VL and VH are replaced by each other.

In another aspect, and to further improve correct pairing, the bispecific antibody comprising a first Fab fragment that specifically binds to PD-1 and a second Fab fragment that specifically binds to LAG3, can contain different charged amino acid substitutions (so-called “charged residues”). These modifications are introduced in the crossed or non-crossed CH1 and CL domains. Such modifiactions are described e.g., in WO2015/150447, WO2016/020309 and PCT/EP2016/073408.

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

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

In a further aspect, the bispecific antibody is a bivalent antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other.

The antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain under a) are isolated chains.

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

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

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

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

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

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

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

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

In one aspect, the bispecific antibody is a bivalent antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced by each other, and wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other.

The antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain und a) are isolated chains. In the antibody under b) within the light chain the variable light chain domain VL is replaced by the variable heavy chain domain VH of said antibody, and the constant light chain domain CL is replaced by the constant heavy chain domain CH1 of said antibody; and within the heavy chain the variable heavy chain domain VH is replaced by the variable light chain domain VL of said antibody, and the constant heavy chain domain CH1 is replaced by the constant light chain domain CL of said antibody.

In one aspect, the bispecific antibody is a bivalent antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced by each other.

The antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain under a) are isolated chains. In the antibody under b) within the light chain the constant light chain domain CL is replaced by the constant heavy chain domain CH1 of said antibody; and within the heavy chain the constant heavy chain domain CH1 is replaced by the constant light chain domain CL of said antibody.

In one aspect, the bispecific antibody is a bispecific antibody comprising a) a full-length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains, and b) one, two, three or four single chain Fab fragments specifically binding to a second antigen, wherein said single chain Fab fragments under b) are fused to said full-length antibody under a) via a peptide linker at the C- or N- terminus of the heavy or light chain of said 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 chains of said 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 chains of said 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 chains of said 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 a peptide linker at the C-terminus of each heavy or light chain of said 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 a peptide linker at the C-terminus of each heavy chain of said 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 a peptide linker at the C-terminus of each light chain of said full-length antibody.

In one aspect, the bispecific antibody is a trivalent antibody comprising a) a full-length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains, b) a first polypeptide consisting of ba) an antibody heavy chain variable domain (VH), or bb) an antibody heavy chain variable domain (VH) and an antibody constant domain 1 (CH1), wherein said first polypeptide is fused with the N-terminus of its VH domain via a peptidic linker to the C-terminus of one of the two heavy chains of said full-length antibody, c) a second polypeptide consisting of ca) an antibody light chain variable domain (VL), or cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), wherein said second polypeptide is fused with the N-terminus of the VL domain via a peptide linker to the C-terminus of the other of the two heavy chains of said full-length antibody, and wherein 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 specifically binding to a 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 an interchain disulfide bridge by introduction of a disulfide bond between the following positions:

(i) heavy chain variable domain position 44 to light chain variable domain position 100, or

(ii) heavy chain variable domain position 105 to light chain variable domain position 43, or

(iii) heavy chain variable domain position 101 to light chain variable domain position 100 (numbering always according to Kabat EU index).

Techniques to introduce unnatural disulfide bridges for stabilization are described, e.g., 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 polypeptides under b) and c) is between heavy chain variable domain position 44 and light chain variable domain position 100. In one embodiment the optional disulfide bond between the variable domains of the polypeptides under b) and c) is between heavy chain variable domain position 105 and light chain variable domain position 43 (numbering always according to Kabat). In one embodiment a trivalent, bispecific antibody without said optional disulfide stabilization between the variable domains VH and VL of the single chain Fab fragments is preferred.

In one aspect, the bispecific antibody is a trispecific or tetraspecific antibody, comprising a) a first light chain and a first heavy chain of a full-length antibody which specifically binds to a first antigen, and b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody which specifically binds to a second antigen, wherein the variable domains VL and VH are replaced by each other, and/or wherein the constant domains CL and CH1 are replaced by each other, and c) wherein one to four antigen-binding domains which specifically bind to one or two further antigens (i.e., to a third and/or fourth antigen) are fused via a peptide linkerto the C- or N-terminus of the light chains or heavy chains of a) and/or b).

The antibody under a) does not contain a modification as reported under b) and the heavy chain and the light chain und a) are isolated chains.

In one aspect, the trispecific or tetraspecific antibody comprises under c) one or two antigenbinding domains which specifically bind to one or two further antigens.

In one aspect, the antigen-binding domains are selected from the group of a scFv fragment and a scFab fragment.

In one aspect, the antigen-binding domains are scFv fragments.

In one aspect, the antigen-binding domains are scFab fragments.

In one aspect, the antigen-binding domains are fused to the C-terminus of the heavy chains of a) and/or b).

In one aspect, the trispecific or tetraspecific antibody comprises under c) one or two antigenbinding domains which specifically bind to one further antigen.

In one aspect, the trispecific or tetraspecific antibody comprises under c) two identical antigenbinding domains which specifically bind to a third antigen. In one preferred embodiment such two identical antigen-binding domains are fused both via the same peptidic linkerto the C-terminus of the heavy chains of a) and b). In one preferred embodiment the two identical antigen-binding domains are either a scFv fragment or a scFab fragment.

In one aspect, the trispecific or tetraspecific antibody comprises under c) two antigen-binding domains which specifically bind to a third and a fourth antigen. In one embodiment said two antigenbinding domains are fused both via the same peptide connector to the C-terminus of the heavy chains of a) and b). In one preferred embodiment said two antigen-binding domains are either a scFv fragment or a scFab fragment.

In one aspect, the bispecific antibody is a bispecific, tetravalent antibody comprising a) two light chains and two heavy chains of an antibody, which specifically bind to a first antigen

(and comprise two Fab fragments), b) two additional Fab fragments of an antibody, which specifically bind to a second antigen, wherein said additional Fab fragments are fused both via a peptidic linker either to the C- or N-termini of the heavy chains of a), and wherein in the Fab fragments the following modifications were performed

(i) in both Fab fragments of a), or in both Fab fragments of b), the variable domains VL and VH are replaced by each other, and/or the constant domains CL and CH1 are replaced by each other, or

(ii) in both Fab fragments of a) the variable domains VL and VH are replaced by each other, and the constant domains CL and CH1 are replaced by each other, and in both Fab fragments of b) the variable domains VL and VH are replaced by each other, or the constant domains CL and CH1 are replaced by each other, or

(iii) in both Fab fragments of a) the variable domains VL and VH are replaced by each other, or the constant domains CL and CH1 are replaced by each other, and in both Fab fragments of b) the variable domains VL and VH are replaced by each other, and the constant domains CL and CH1 are replaced by each other, or

(iv) in both Fab fragments of a) the variable domains VL and VH are replaced by each other, and in both Fab fragments of b) the constant domains CL and CH1 are replaced by each other, or

(v) in both Fab fragments of a) the constant domains CL and CH1 are replaced by each other, and in both Fab fragments of b) the variable domains VL and VH are replaced by each other.

In one aspect, said additional Fab fragments are fused both via a peptidic linker either to the C- termini of the heavy chains of a), orto the N-termini of the heavy chains of a).

In one aspect, said additional Fab fragments are fused both via a peptidic linker to the C-termini of the heavy chains of a).

In one aspect, said additional Fab fragments are fused both via a peptide linker to the N-termini of the heavy chains of a).

In one aspect, in the Fab fragments the following modifications are performed: in both Fab fragments of a), or in both Fab fragments of b), the variable domains VL and VH are replaced by each other, and/or the constant domains CL and CH1 are replaced by each other.

In one aspect, the bispecific antibody is a tetravalent antibody comprising: a) a (modified) heavy chain of a first antibody, which specifically binds to a first antigen and comprises a first VH-CH1 domain pair, wherein to the C terminus of said heavy chain the N-terminus of a second VH-CH1 domain pair of said first antibody is fused via a peptide linker, b) two light chains of said first antibody of a), c) a (modified) heavy chain of a second antibody, which specifically binds to a second antigen and comprises a first VH-CL domain pair, wherein to the C-terminus of said heavy chain the N-terminus of a second VH-CL domain pair of said second antibody is fused via a peptide linker, and d) two (modified) light chains of said second antibody of c), each comprising a CL-CH1 domain pair. In one aspect, the bispecific antibody comprises a) the heavy chain and the light chain of a first full-length antibody that specifically binds to a first antigen, and b) the heavy chain and the light chain 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 under a) does not contain a modification as reported under b) and the heavy chain and the light chain are isolated chains.

In one aspect, the bispecific antibody comprises a) a full-length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains, and b) an Fv fragment specifically binding to a second antigen comprising a VH2 domain and a VL2 domain, wherein both domains are connected to each other via a disulfide bridge, wherein only either the VH2 domain or the VL2 domain is fused via a peptide linker to the heavy or light chain of the full-length antibody specifically binding to a first antigen.

In the bispecific antibody the heavy chains and the light chains under a) are isolated chains.

In one aspect, the other of the VH2 domain or the VL2 domain is not fused via a peptide linker to the heavy or light chain of the full-length antibody specifically binding to a first antigen.

In all aspects as reported herein the first light chain comprises a VL domain and a CL domain and the first heavy chain comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

In one aspect, the bispecific antibody is a trivalent antibody comprising a) two Fab fragments that specifically binds to a first antigen, b) one CrossFab fragment that specifically binds to a second antigen in which the CH1 and the CL domain are exchanged for each other, c) one Fc-region comprising a first Fc-region heavy chain and a second Fc region heavy chain, wherein the C-terminus of CH1 domains of the two Fab fragments are connected to the N-terminus of the heavy chain Fc-region polypeptides, and wherein the C-terminus of the CL domain of the CrossFab fragment is connected to the N-terminus of the VH domain of one of the Fab fragments.

In one aspect, the bispecific antibody is a trivalent antibody comprising a) two Fab fragments that specifically binds to a first antigen, b) one CrossFab fragment that specifically binds to a second antigen in which the CH1 and the CL domain are exchanged for each other, c) one Fc-region comprising a first Fc-region heavy chain and a second Fc region heavy chain, wherein the C-terminus of CH1 domain of the first Fab fragment is connected to the N-terminus of one of the heavy chain Fc-region polypeptides and the C-terminus of the CL-domain of the CrossFab fragment is connected to the N-terminus of the other heavy chain Fc-region polypeptide, and wherein the C-terminus of the CH1 domain of the second Fab fragment is connected 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, the bispecific antibody comprises a) a full-length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains, and b) a Fab fragment specifically binding to a second antigen comprising a VH2 domain and a VL2 domain comprising 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 said antibody, and within the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of said antibody wherein the heavy chain Fab fragment is inserted between the CH1 domain of one of the heavy chains of the full-length antibody and the respective Fc-region of the full-length antibody, and the N- terminus of the light chain Fab fragment is conjugated to the C-terminus of the light chain of the full-length antibody that is paired with the heavy chain of the full-length antibody into which the heavy chain Fab fragment has been inserted.

In one aspect, the bispecific antibody comprises a) a full-length antibody specifically binding to a first antigen and consisting of two antibody heavy chains and two antibody light chains, and b) a Fab fragment specifically binding to a second antigen comprising a VH2 domain and a VL2 domain comprising 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 said antibody, and within the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of said antibody and wherein the C-terminus of the heavy chain fragment of the Fab fragment is conjugated to the N-terminus of one of the heavy chains of 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 that pairs with the heavy chain of the full-length antibody to which the heavy chain fragment of the Fab fragment is conjugated.

B. Dosing of bispecific antibodies that bind to PD-1 and LAG3

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

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 as defined herein is suitably administered to the subject at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g., 0.1 mg/kg - 10 mg/kg) of the bispecific antibody can be an initial candidate dosage for administration to the subject, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the bispecific antibody would be in the range from about 0.005 mg/kg to about 10 mg/kg. In other examples, a dose may also comprise from about 1 pg/kg body weight, about 5 pg/kg body weight, about 10 pg/kg body weight, about 50 pg/kg body weight, about 100 pg/kg body weight, about 200 pg/kg body weight, about 350 pg/kg body weight, about 500 pg/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 per administration, and any range derivable therein. In examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 pg/kg body weight to about 500 mg/kg body weight etc., can be administered, based on the numbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the subject. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the subject receives from about two to about twenty, or e.g. about six doses of the fusion protein). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

In one aspect, the bispecific antibody targeting PD-1 and LAG3 is administered to the subject at a fixed dose of about 300 mg, of about 500 mg, of about 600 mg, of about 700 mg, of about 800 mg, of about 900 mg, of about 1000 mg, of about 1100 mg or of about 1200 mg every three weeks (Q3W) e.g., at a fixed dose administered on Day 1 (± 1 day) of a three-week dosing cycle). In one particular aspect, the bispecific antibody targeting PD-1 and LAG3 is administered to the subject at a fixed dose of about 600 mg every three weeks (Q3W), e.g., at a fixed dose of 600 mg Q3W (e.g., administered on Day 1 (± 1 day) of a three-week dosing cycle). In particular, the bispecific antibody targeting PD-1 and LAG3 is administered to the subject at a fixed dose of about 600 mg every three weeks (Q3W) on Day 1 of the three-week dosing cycle.

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

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

V. Anti-TIGIT Antagonist Antibodies

The invention provides anti-TIGIT antagonist antibodies useful for treating cancer in a subject (e.g., a human) having a cancer.

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

In certain instances, the anti-TIGIT antagonist antibody includes at least one, two, three, four, five, or six HVRs selected from: (a) an HVR-H1 comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 38); (b) an HVR-H2 comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 39); (c) an HVR-H3 comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 40); (d) an HVR-L1 comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 41), (e) an HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 42); and/or (f) an HVR-L3 comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 43), or a combination of one or more of the above HVRs and one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 38-43.

In some instances, anti-TIGIT antagonist antibodies may include (a) an HVR-H1 comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 38); (b) an HVR-H2 comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 39); (c) an HVR-H3 comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 40); (d) an HVR-L1 comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 41); (e) an HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 42); and (f) an HVR-L3 comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 43). In some instances, the anti-TIGIT antagonist antibody has a VH domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVK GRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 44) or an amino acid sequence having at least 90% sequence identity (e.g., at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVK GRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSS (SEQ ID NO: 45); 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) to, or the sequence of, DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSG SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIK (SEQ ID NO: 46). In some instances, the anti-TIGIT antagonist antibody has a VH domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO: 44 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) to, or the sequence of, SEQ ID NO: 46. In some instances, the anti-TIGIT antagonist antibody has a VH domain comprising the amino acid sequence of SEQ ID NO: 44 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46. In some instances, the anti-TIGIT antagonist antibody has a VH domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO: 45 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) to, or the sequence of, SEQ ID NO: 46. In some instances, the anti-TIGIT antagonist antibody has a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46.

In some instances, the anti-TIGIT antagonist antibody includes a heavy chain and a light chain sequence, wherein: (a) the heavy chain comprises the amino acid sequence: EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVK GRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47); and (b) the light chain comprises the amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSG SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC (SEQ ID NO: 48).

In some instances, the anti-TIGIT antagonist antibody further comprises at least one, two, three, or four of the following light chain variable region framework regions (FRs): an FR-L1 comprising the amino acid sequence of DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 49); an FR-L2 comprising the amino acid sequence of WYQQKPGQPPNLLIY (SEQ ID NO: 50); an FR-L3 comprising the amino acid sequence of GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 51); and/or an FR-L4 comprising the amino acid sequence of FGPGTKVEIK (SEQ ID NO: 52), or a combination of one or more of the above FRs and one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 49-52. In some instances, for example, the antibody further comprises an FR-L1 comprising the amino acid sequence of DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 49); an FR-L2 comprising the amino acid sequence of WYQQKPGQPPNLLIY (SEQ ID NO: 50); an FR-L3 comprising the amino acid sequence of GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 51); and an FR-L4 comprising the amino acid sequence of FGPGTKVEIK (SEQ ID NO: 52).

In some instances, the anti-TIGIT antagonist antibody further comprises at least one, two, three, or four of the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of XiVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 53), wherein Xi is E or Q; an FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 54); an FR-H3 comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 55); and/or an FR- H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 56), or a combination of one or more of the above FRs and one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 53- 56. The anti-TIGIT antagonist antibody may further include, for example, at least one, two, three, or four of the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of EVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 57); an FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 54); an FR-H3 comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 55); and/or an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 56), or a combination of one or more of the above FRs and one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 54-57. In some instances, the anti-TIGIT antagonist antibody includes an FR-H1 comprising the amino acid sequence of EVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 57); an FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 54); an FR-H3 comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 55); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 56). In another instance, for example, the anti-TIGIT antagonist antibody may further include at least one, two, three, or four of the following heavy chain variable region FRs: an FR-H1 comprising the amino acid sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 58); an FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 54); an FR-H3 comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 55); and/or an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 56), or a combination of one or more of the above FRs and one or more variants thereof having at least about 90% sequence identity (e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 54-56 and 58. In some instances, the anti-TIGIT antagonist antibody includes an FR-H1 comprising the amino acid sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 58); an FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 54); an FR-H3 comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 55); and an FR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 56).

In another aspect, an anti-TIGIT antagonist antibody is provided, wherein the antibody comprises a VH as in any of the instances provided above, and a VL as in any of the instances provided above, wherein one or both of the variable domain sequences include post-translational modifications.

In some instances, any one of the anti-TIGIT antagonist antibodies described above is capable of binding to rabbit TIGIT, in addition to human TIGIT. In some instances, any one of the anti-TIGIT antagonist antibodies described above is capable of binding to both human TIGIT and cynomolgus monkey (cyno) TIGIT. In some instances, any one of the anti-TIGIT antagonist antibodies described above is capable of binding to human TIGIT, cyno TIGIT, and rabbit TIGIT. In some instances, any one of the anti-TIGIT antagonist antibodies described above is capable of binding to human TIGIT, cyno TIGIT, and rabbit TIGIT, but not murine TIGIT.

In some instances, the anti-TIGIT antagonist antibody binds human TIGIT with a Ko of about 10 nM or lower and cyno TIGIT with a KD of about 10 nM or lower (e.g., binds human TIGIT with a KD of about 0.1 nM to about 1 nM and cyno TIGIT with a KD of about 0.5 nM to about 1 nM, e.g., binds human TIGIT with a KD of about 0.1 nM or lower and cyno TIGIT with a KD of about 0.5 nM or lower).

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

In some instances, the methods or uses described herein may include using or administering an isolated anti-TIGIT antagonist antibody that competes for binding to TIGIT with any of the anti-TIGIT antagonist antibodies described above. For example, the method may include administering an isolated anti-TIGIT antagonist antibody that competes for binding to TIGIT with an anti-TIGIT antagonist antibody having the following six HVRs: (a) an HVR-H1 comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 38); (b) an HVR-H2 comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 39); (c) an HVR-H3 comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 40); (d) an HVR-L1 comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 41), (e) an HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 42); and (f) an HVR-L3 comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 43). The methods described herein may also include administering an isolated anti-TIGIT antagonist antibody that binds to the same epitope as an anti-TIGIT antagonist antibody described above.

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

In other aspects, the anti-TIGIT antagonist antibody is an antibody that lacks Fc-mediated effector function (e.g., domvanalimab, BMS-986207, ASP8374, or COM902).

In some aspects, the anti-TIGIT antagonist antibody is an IgG class antibody. In some aspects, the anti-TIGIT antagonist antibody is an lgG1 class antibody, e.g., tiragolumab, vibostolimab, domvanalimab, BMS-986207, etigilimab, BGB-A1217, SGN-TGT, EQS084448 (EOS-448), TJ-T6, or AB308. In some aspects, the antibody is a human monoclonal full-length lgG1 class antibody comprising an Fc region.

In some aspects, the anti-TIGIT antagonist antibody is a human, monoclonal full-length lgG1 subclass antibody comprising a human lgG1 Fc region, a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 44, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 46.

In other aspects, the anti-TIGIT antagonist antibody is an lgG4 class antibody, e.g., ASP8374 or COM902.

In some embodiments, the anti-TIGIT antagonist antibody functions to inhibit TIGIT signaling. In some embodiments, the anti-TIGIT antagonist antibody inhibits the binding of TIGIT to its binding partners. Exemplary TIGIT binding partners include CD155 (PVR), CD112 (PVRL2 or Nectin-2), and CD113 (PVRL3 or Nectin-3). In some embodiments, the anti-TIGIT antagonist antibody is capable of inhibiting binding between TIGIT and CD155. In some embodiments, the anti-TIGIT antagonist antibody may inhibit binding between TIGIT and CD112. In some embodiments, the anti-TIGIT antagonist antibody inhibits binding between TIGIT and CD113. In some embodiments, the anti-TIGIT antagonist antibody inhibits TIGIT-mediated cellular signaling in immune cells. In some embodiments, the anti-TIGIT antagonist antibody inhibits TIGIT by depleting regulatory T cells (e.g., when engaging a FcyR).

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

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

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

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

VI. Pharmaceutical Compositions and Formulations

Also provided herein are pharmaceutical compositions and formulations comprising a bispecific antibody targeting PD-1 and LAG3 and, optionally, a pharmaceutically acceptable carrier. The disclosure also provides pharmaceutical compositions and formulations comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody (e.g., tiragolumab), and optionally, a pharmaceutically acceptable carrier. Pharmaceutical compositions and formulations of a bispecific antibody targeting PD-1 and LAG3 and/or other agents described herein (e.g., an anti-TIGIT antagonist antibody, e.g., tiragolumab) can be prepared by mixing the agent or agents having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (e.g., a bispecific antibody targeting PD-1 and LAG3 and/or an anti-TIGIT antagonist antibody (e.g., tiragolumab)) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (see, e.g., Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), e.g., in the form of lyophilized formulations or aqueous solutions.

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

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

Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171 ,586 and W02006/044908, the latter formulations including a histidine-acetate buffer.

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

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, for example, films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

VII. Articles of Manufacture or Kits

A. Kits comprising a bispecific antibody targeting PD-1 and LAG3

In another aspect, provided herein is an article of manufacture or a kit comprising a bispecific antibody targeting PD-1 and LAG3. In some instances, the article of manufacture or kit further comprises a package insert comprising instructions for using the bispecific antibody targeting PD-1 and LAG3 to treat or delay progression of a cancer (e.g., a UC, e.g., a locally advanced or metastatic UC) in a subject. Any of the bispecific antibodies targeting PD-1 and LAG3 described herein may be included in the article of manufacture or kits. In some aspects, the bispecific antibody is for use in combination with an anti-TIG IT antagonist antibody (e.g., tiragolumab).

In another embodiment of the invention, a kit is provided comprising a bispecific antibody targeting PD-1 and LAG3 for treating a subject having a cancer (e.g., a UC, e.g., a locally advanced or metastatic UC) according to any of the methods described herein. In some aspects, the bispecific antibody is for use in combination with an anti-TIGIT antagonist antibody (e.g., tiragolumab). In some instances, the kit further comprises the anti-TIGIT antagonist antibody (e.g., tiragolumab). In some instances, the article of manufacture or kit further comprises a package insert comprising instructions for using the bispecific antibody targeting PD-1 and LAG3 (e.g., in combination with the anti-TIGIT antagonist antibody (e.g., tiragolumab)) to treat or delay progression of a cancer (e.g., a UC, e.g., a locally advanced or metastatic UC) in a subject.

In another embodiment of the invention, a kit is provided comprising an anti-TIGIT antagonist antibody for treating a subject having a cancer (e.g., a UC, e.g., a locally advanced or metastatic UC) according to any of the methods described herein. In some aspects, the anti-TIGIT antagonist antibody is for use in combination with a bispecific antibody targeting PD-1 and LAG3. In some instances, the kit further comprises a bispecific antibody targeting PD-1 and LAG3. In some instances, the article of manufacture or kit further comprises a package insert comprising instructions for using the anti-TIGIT antagonist antibody in combination with the bispecific antibody targeting PD-1 and LAG3 to treat or delay progression of a cancer (e.g., a UC, e.g., a locally advanced or metastatic UC) in a subject.

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

Any of the bispecific antibodies targeting PD-1 and LAG3 and/or anti-TIGIT antagonist antibodies described herein may be included in the article of manufacture or kits. Any of the articles of manufacture or kits may include instructions to administer a bispecific antibody targeting PD-1 and LAG3 and/or an anti-TIGIT antagonist antibody to a subject in accordance with any of the methods described herein, e.g., any of the methods set forth in Section II above.

VIII. EXAMPLES

Example 1 : A Phase II, Randomized, Multicenter, Open Label, Controlled Study of RO7247669 Alone Or In Combination With Tiragolumab Versus Atezolizumab In Patients With Previously Untreated Locally Advanced Or Metastatic Urothelial Bladder Cancer Who Are Ineligible For Platinum-Containing Chemotherapy

A. Overview of Study Design

BO44157 is a Phase II, global, multicenter, open-label, three-arm, randomized, controlled study designed to evaluate the efficacy, safety, and pharmacokinetics of RO7247669 and

RO7247669 + tiragolumab compared with atezolizumab in patients with previously untreated, locally advanced urothelial bladder cancer (UC) or metastatic urothelial bladder cancer (mUC) who are ineligible to receive a platinum-containing chemotherapy.

The study enrolls approximately 240 participants at approximately 60 sites globally. Participants are randomized in a 1 :1 :1 ratio to receive one of the following:

• Arm A (control arm): Atezolizumab.

Participants receive atezolizumab on Day 1 of each 21 -day cycle (every 3 weeks (Q3W)) until disease progression or treatment discontinuation.

• Arm B (experimental arm): RO7247669.

Participants receive RO7247669 on Day 1 of each 21-day cycle (Q3W) until disease progression or treatment discontinuation.

• Arm C (experimental arm): RO7247669 + Tiragolumab.

Participants receive RO7247669 in combination with tiragolumab on Day 1 of each 21-day cycle (Q3W) until disease progression or treatment discontinuation.

The objectives and endpoints of the study are presented in Table 3. An overview of the study is presented in Fig. 1 .

Table 3. Objectives and Endpoints

The study consists of three phases: screening, treatment, and follow-up.

Screening Phase

After providing informed consent, participants are evaluated for study eligibility during a screening period of up to 28 days, as outlined in the schedule of activities. Participants who are determined to be eligible based on the screening assessments will be randomized to study treatment.

Participants who do not meet the criteria for participation in this study (screen failure) may qualify for one re-screening opportunity (for a total of two screenings per participant) at the investigator's discretion. Patients are required to re-sign the Informed Consent Form if they are re-screened. For participants who are re-screened, all eligibility criteria must be re-evaluated and the screening assessment should be repeated as applicable to meet eligibility criteria. The investigator will record reasons for screen failure in the screening log.

Treatment Phase

A total of approximately 240 participants are randomly assigned to each treatment arm in a 1 :1 :1 ratio, resulting in approximately 80 participants in each arm.

Randomization uses a permuted-block randomization method to ensure a balanced assignment to each treatment arm and will be stratified according to the following criteria:

• PD-L1 expression (positive vs. negative).

Positive PD-L1 expression status is defined as Tumor Area Positivity score (TAP) > 5% and negative PD-L1 status is defined as TAP < 5%. TAP is defined as the percentage of stained tumor and immune cell within the total tumor area. • Presence of liver metastases at baseline (yes vs. no).

• Eastern Cooperative Oncology Group Performance Status (0 or 1 vs. 2).

PD-L1 expression is determined by the investigational VENTANA PD-L1 (SP263) companion diagnostic (CDx) assay that is performed on leftover tumor samples.

Participants with liver metastases at baseline are capped at approximately 15% of the total planned enrollment. No crossover is allowed from the control arm to the experimental arms.

Patients are treated until unacceptable toxicity or loss of clinical benefit, as determined by the investigator after an integrated assessment of radiographic and biochemical data, local biopsy results (if available), and clinical status (e.g., symptomatic deterioration such as pain secondary to disease).

Safety Run-In Evaluation

No safety run-in phase is performed for the novel combination of RO7247669 and tiragolumab (Arm C) within this study protocol. A six-patient safety run-in phase for the same combination of RO7247669 and tiragolumab was completed on 6 May 2022 within Cohort 2 of Study BO43328. Study BO43328 is a Phase Ib/ll study evaluating the efficacy and safety of RO7247669 and tiragolumab in patients with Stage III (Cohortl) and Stage IV (Cohort 2) melanoma (MORPHEUS-Melanoma). The safety-run in from Study BO43328 identified no new safety signals and the safety events observed were in line with the known safety profile of the individual study treatments.

Assessments

Participants undergo tumor assessments at screening, every 6 weeks (±7 days) for the first 54 weeks following treatment initiation, and every 9 weeks thereafter, regardless of dose delays, until radiographic disease progression per Response Criteria in Solid Tumors, Version 1.1 (RECIST v1.1), withdrawal of consent, loss to follow-up, study termination, or death, whichever occurs first. Participants who are treated beyond disease progression per RECIST v1 .1 undergo tumor assessments every 6 weeks (±7 days) after initial documentation of progression, or more frequently if clinically indicated, regardless of time on study, until symptomatic deterioration attributed to disease progression, withdrawal of consent, loss to follow-up, study termination, or death, whichever occurs first. Participants who discontinue study treatment for reasons other than disease progression (e.g., toxicity) continue to undergo scheduled tumor assessments as if they were on the protocol schedule until disease progression per RECIST v1 .1 , withdrawal of consent, loss to follow-up, study termination, or death, whichever occurs first, regardless of whether the patient starts a new anti-cancer therapy.

Participants randomized into the study are asked to complete the patient-reported outcome questionnaire European Organisation for Research and Treatment of Cancer Item Library 187 to assess the treatment benefit of RO7247669 and RO7247669 in combination with tiragolumab compared to atezolizumab.

Safety assessments at study visits include the incidence, nature, and severity of adverse events, protocol-mandated vital signs, laboratory abnormalities, and other protocol-specified tests that are deemed critical to the safety evaluation of the study. The severity of adverse events is assessed by investigators according to the National Cancer Institute Common Terminology Criteria for Adverse Events, Version 5.0. The severity of cytokine release syndrome is determined according to the American Society for Transplantation and Cellular Therapy Consensus Grading Scale.

During the study, serum samples are collected to monitor RO7247669, tiragolumab and atezolizumab pharmacokinetic (PK) concentrations, and to detect the presence of antibodies to RO7247669, tiragolumab and atezolizumab. Participant samples, including serum, plasma, urine, and blood samples, are also collected for exploratory biomarker assessments. Fresh tumor tissue may be collected from participants who consent to undergo an optional biopsy, preferably 4 weeks (± 7 days) after treatment initiation.

Treatment Beyond Progression

During the study, participants who meet the criteria for disease progression as assessed by the investigator according to RECIST v1 .1 and show evidence of clinical benefit may continue treatment with atezolizumab, RO7247669, or tiragolumab at the investigator’s discretion, provided that participants meet all of the following criteria:

• Evidence of clinical benefit, as determined by the investigator following a review of all available data.

• Absence of symptoms and signs (including worsening of laboratory values (e.g., new or worsening hypercalcemia)) indicating unequivocal progression of disease.

• Absence of decline in Eastern Cooperative Oncology Group Performance Status that can be attributed to disease progression.

• Absence of tumor progression at critical anatomical sites (e.g., leptomeningeal disease) that cannot be managed by protocol-allowed medical interventions.

Investigator assessment of overall tumor response at all timepoints is based only on RECIST v1.1.

Follow-up Phase

All participants are followed-up for survival until death, loss to follow-up, participant withdrawal, or study termination.

B. Study Treatment

The investigational medicinal products for this study are atezolizumab, RO7247669, and tiragolumab.

Atezolizumab (Control Arm A)

Participants in the atezolizumab control arm receive treatment (1200 mg by IV infusion on Day 1 of every 21 -day cycle) until disease progression or unacceptable toxicity. Participants who meet the criteria for disease progression as assessed by the investigator according to RECIST v1 .1 and show evidence of clinical benefit may continue treatment with atezolizumab at the investigator’s discretion, provided that participants meet all the criteria listed in the Treatment Beyond Progression section above. Administration of atezolizumab is performed in a monitored setting where there is immediate access to trained personnel and adequate equipment and medicine to manage potentially serious reactions.

RO7247669 and Tiragolumab (Arms B and C)

Participants in the RO7247669 arm (Arm B) will receive treatment (600 mg by IV infusion on Day 1 of every 21-day cycle), while participants in the R07247669+tiragolumab arm (Arm C) receive treatment (RO7247669 600 mg by IV infusion and tiragolumab 600 mg by IV infusion on Day 1 of every 21-day cycle), until disease progression or unacceptable toxicity. Participants who meet the criteria for disease progression as assessed by the investigator according to RECIST v1 .1 and show evidence of clinical benefit may continue treatment with RO7247669 and/or tiragolumab at the investigator’s discretion, provided that participants meet all the criteria listed in the Treatment Beyond Progression section above.

On Day 1 of Cycle 1 , tiragolumab is administered at least 60 minutes after completion of the RO7247669 infusion. After the first infusion, if any subsequent infusion is tolerated without an infusion-related reaction, tiragolumab may be administered at least 30 minutes after the RO7247669 infusion.

Administration of RO7247669 and tiragolumab is performed in a monitored setting where there is immediate access to trained personnel and adequate equipment and medicine to manage potentially serious reaction.

Duration Of Participation

Treatment continues until disease progression per RECIST v1 .1 or for a total of 45 cycles. Participants are allowed to continue treatment with atezolizumab (Control Arm A), RO7247669 (Arm B) or RO7247669 and tiragolumab (Arm C), or beyond 45 cycles at the investigator’s discretion. The total duration of study participation for each individual is expected to range from 1 day to more than 30 months. Participants who have a confirmed complete response are allowed to discontinue treatment. Participants are allowed to continue treatment beyond disease progression if pre-specified criteria are met.

C. Background

Encouraging clinical data in the field of tumor immunotherapy have demonstrated that therapies focused on enhancing T-cell responses against cancer can result in a significant survival benefit 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).

In this study, the bispecific antibody (BsAb) RO7247669 as well as the combination of RO7247669 with tiragolumab are expected to stimulate the immune system through additional mechanisms from single agent atezolizumab with the aim of extending the benefit of atezolizumab to a larger population of patients with inoperable locally advanced urothelial carcinoma (UC) and mUC who are ineligible to receive any platinum-based chemotherapy. The PD-1/PD-L1 Pathway

The PD-1 /programmed death-ligand 1 (PD-L1) pathway serves as an immune checkpoint to temporarily dampen immune responses in states of chronic antigen stimulation, such as chronic infection or cancer. PD-1 is an inhibitory receptor that is expressed on activated and exhausted T cells, including tumor infiltrating CD8⁺ T cells that recognize mutated tumor antigens (neo-antigens). Binding of PD-L1 to PD-1 inhibits T-cell proliferation, activation, cytokine production, and cytolytic activity, leading to a functionally inactivated and exhausted T-cell state (Butte et al., Immunity, 27: 111-122, 2007; Yang et al., J. Immunol., 187: 1113-11 19, 2011). Therapeutic targeting of the PD-1/PD-L1 pathway to enhance anti tumor T-cell responses has been clinically validated across multiple solid tumors, as both a single agent and in combination with chemotherapy and other targeted agents.

The LAG3 Pathway

LAG3 is an immune checkpoint protein involved in the regulation of anti-tumor immunity and chronic infection. LAG3 is expressed on activated T cells, B cells, natural killer (NK) cells, and a subset of tolerogenic plasmacytoid dendritic cells, and constitutively on T-regulatory cells (Huard et al., Immunogenetics, 39: 213-217, 1994). Structurally similar to CD4, LAG3 is a member of the Ig superfamily and binds to major histocompatibility complex class II (MHC-II). The interaction of LAG3 with MHC-II inhibits T-cell proliferation, activation, cytolytic function, and proinflammatory cytokine production (Goldberg and Drake, Curr Top Microbiol Immunol, 344: 269-278, 2011).

Expression of LAG3 has been reported across various tumor types, including breast cancer, ovarian cancer, non-small cell lung cancer (NSCLC), melanoma, renal cell carcinoma, prostate cancer, hepatocellular carcinoma (HCC), and bladder cancer, and is associated with poor prognosis.

Targeting both the PD-1 and the LAG3 Pathway

Cancer immunotherapy (CIT) agents, particularly immune checkpoint inhibitors (CPIs), have had a significant impact on the treatment of patients with advanced malignancies in recent years. However, despite the remarkable clinical efficacy of these therapies, additional treatment options targeting immune checkpoints are needed, because the majority of patients eventually progress after an initial response or fail to respond to the PD-1/PD-L1 checkpoint blockade. This is believed to be mainly due to primary or secondary resistance mechanisms, immunosuppressive activity of myeloid-derived suppressor cells and/or T-regulatory cells (Sharma et al., Cell, 168: 707-723, 2017).

To overcome resistance mechanisms, additional treatment options and multiple combinations with anti-PD-L1 are being assessed. One potential reason for resistance to anti-PD-L1 therapy is the upregulation of alternative immune checkpoints with non-redundant regulatory functions (Sharma et al., Cell, 168: 707-723, 2017). LAG3 is one such alternative immune checkpoint.

LAG3 is frequently co-expressed with PD-1 on tumor-infiltrating lymphocytes (TILs), and dual blockade of PD-1 and LAG3 enhances CD8⁺ T-cell effector function and potentiates anti-tumor immunity in nonclinical models. Blockade of these two receptors in mice with colon, fibrosarcoma, or ovarian tumors resulted in tumor remission in approximately 80% of animals compared with 10% to 40% with blockade of either receptor using a single agent (Woo et al., Cancer Res, 72: 917-927, 2012; Huang et al., Oncotarget, 6: 27359-77, 2015). TILs from patients with ovarian cancer showed that antigen-specific CD8⁺ T cells co-expressing PD-1 and LAG3 exhibited greater impairment in their ability to respond to cognate antigen stimulation compared with CD8⁺ T cells that expressed one checkpoint molecule (Matsuzaki et al., Proc Natl Acad Sci USA, 107: 7875-80, 2010). In patients with NSCLC, overexpression of LAG3 on TILs correlated with PD-1/PD-L1 expression and was linked to higher risk of recurrence and poor survival outcomes (He et al., J Thorac Oncol, 12: 814-823, 2017). In addition, it was shown that in patients with metastatic melanoma and urothelial cancer treated with checkpoint immunotherapy, presence of PD1 + LAG3 + Ki67-expressing lymphocytes at baseline correlated with significantly worse OS as compared to patients whose lymphocytes at baseline did not express LAG3 and were Ki67 positive (Shen et al., Sci Transl Med, 13:eabf5107, 2021).

Clinical evaluation of anti-LAG3 agents, as a single-agent and in combination with other CPIs, is ongoing in several early phase studies in patients with advanced solid tumors (Long et al., Genes Cancer, 9: 176-189, 2018). Preliminary data demonstrate that anti-LAG3 therapy is well tolerated, both as a single agent and in combination with anti-PD-1 therapies, and the safety profiles are consistent with those of other CPIs ( Ascierto et aL, Ann Oncol, 28(Suppl 5):mdx440.011 , 2017; Hong et al., J Clin Oncol, 36: 3012, 2018; Stratton et aL, SITC Abstract P325, 2018). Notably, the combination of nivolumab (anti-PD-1) and relatlimab (anti-LAG3) showed a significantly improved progression-free survival (PFS) over nivolumab alone as first-line therapy in patients with melanoma in the Phase III Relativity-047 study (Tawbi et al., N Eng J Med, 386: 26-34, 2022).

Taken together, these data support the potential for LAG3 as a target for therapeutic intervention in urothelial cancer, and the potential synergistic effect of targeting both the PD-1/PD-L1 and the LAG3 pathways simultaneously in this indication.

Targeting the PD-1/LAG3 Pathways and the TIGIT Pathway

Durable clinical benefit is limited to a minority of patients treated with single-agent PD-L1/PD-1 inhibitors. Therapies targeting the mechanisms of resistance to anti-PD-L1/PD-1 therapies are needed to improve outcomes in patients with solid cancers. Resistance to PD-L1/PD-1 blockade may result in the expression of multiple co-inhibitory receptors on the surface of effector T cells including TIGIT and LAG3. Nonclinical tumor models have shown that TIGIT selectively suppressed the effector function of chronically stimulated CD8⁺ T cells, and that inhibiting both TIGIT and PD-L1/PD-1 resulted in superior efficacy compared with single-agent treatments (Johnston et aL, Cancer Cell, 26: 923-937, 2014). Similarly, in vivo proof-of-concept studies using different tumor mouse models have shown PD1-LAG3 BsAbs to be superior in controlling tumor growth and promoting tumor eradication when compared with anti-PD-1 antibodies as monotherapy.

Hence, targeting PD-1/LAG3 and TIGIT may enhance the efficacy of PD-L1/PD-1 blockade across different cancer types, including bladder cancer.

Benefit-Risk Assessment for RO7247669 + Tiragolumab

Tiragolumab is currently under investigation in multiple studies in both solid and hematological malignancies. Overall, tiragolumab in combination with atezolizumab has been well tolerated, adverse events have been manageable, and the safety profile seems to be consistent as reported across different solid tumor indications.

Early clinical experience with RO7247669 suggest a safety profile comparable to that of atezolizumab and other PD-1/PD-L1 blocking antibodies. The combination of nivolumab (anti-PD-1) and relatlimab (anti-LAG3) improved PFS over nivolumab alone as first-line therapy in melanoma patients in the Phase III Relativity-047 study (Tawbi et al., N Eng J Med, 386: 26-34, 2022), which supports the expected benefit from RO7247669 (anti-PD-1 /anti-LAG3 bispecific therapy) in combination with tiragolumab.

The combination of RO7247669 and tiragolumab is currently being clinically tested in Study BO43328, a Phase Ib/ll study evaluating the efficacy and safety of RO7247669 and tiragolumab in patients with Stage III (Cohortl) and Stage IV (Cohort 2) melanoma (MORPHEUS-Melanoma). A six-patient safety run-in phase for the combination of RO7247669 and tiragolumab was completed on 6 May 2022 within Cohort 2 of Study BO43328. This safety run-in identified no new safety signals and the safety events observed were in line with the known safety profile of the individual study treatments.

D. Rationale for Study Design

Rationale for Study Population

This study enrolls patients with advanced/unresectable or metastatic bladder cancer who are ineligible for any platinum-containing chemotherapy due to impaired functional status and/or the presence of comorbidities (e.g., renal failure, hearing loss, neuropathy). Treatment options for such patients are limited to single-agent systemic immunotherapy (pembrolizumab, atezolizumab), single-agent chemotherapy, and best supportive care. A recent real-world study evaluated the outcomes of platinum-ineligible patients with advanced bladder cancer treated with 1 L PD-1/PD-L1 inhibitors: although the ORR was comparable to efficacy observed in cisplatin-ineligible clinical trials, the median OS was only 8.5 months, highlighting the need for novel therapeutic options in this patient population (Pond et al., Clin Genitourin Cancer, 19: 425-433, 2021).

Notably, while the criteria describing medically frail patients in whom the use of cisplatin-based chemotherapy may be associated with unacceptable toxicity have been defined by a consensus working group (Gaisky et al., Lancet Oncol., 12: 211-214, 2011), no consensus yet exists in defining the criteria for patients who are ineligible to receive any form of platinum-based chemotherapy. In this study, this patient population is defined based on one of the following four criteria:

• ECOG Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and 30 mL/min/1 .73 m².

• ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1 .73 m² and 2 45 mL/min/1 .73 m².

• ECOG Performance Status of 0-2 with Grade (G) > 2 neuropathy.

• Patients for whom chemotherapy is not deemed appropriate.

In this study, the effect of RO7247669 and RO7247669 + tiragolumab is evaluated in the above-mentioned 1 L mUC platinum-ineligible population, regardless of PD-L1 expression. Rationale for Control Group

This study enrolls patients with previously untreated, locally advanced or mUC who are ineligible to receive a platinum-containing chemotherapy.

The current National Comprehensive Cancer Network (NCCN) treatment guidelines for patients with mUC in the first-line (1 L) setting who are ineligible to receive a platinum-based chemotherapy include atezolizumab, pembrolizumab, and certain non-platinum-based chemotherapies such as gemcitabine or participation in clinical trials of new or more tolerable therapies.

In this study, patients in the control arm will receive atezolizumab.

Justification for Dose and Schedule

The RO7247669 dose 600 mg IV every Q3W (600 mg on Day 1 of each 21 -day cycle) was selected on the basis of available clinical PK, efficacy, and safety data from the combined Phase la/Phase lb Study NP41300. In this study, MTD was not reached, and no DLTs were observed in dose escalation. Greater than 90% occupancy of peripheral PD-1 and LAG3 receptors on CD8⁺ cells was observed beginning at 50 mg of RO7247669 and remained sustained at all higher doses (unpublished Roche data on file). During the dose escalation phase of the Phase 1 study, responses were observed at 600 mg and 2100 mg. Further modelling of intratumoral PD-1 and LAG3 target engagement using estimated target properties was performed. A dose of 600 mg Q3Wwas selected to ensure the majority of patients have at least 90% PD-1 and LAG3 tumor receptor saturation by RO7247669, irrespective of intratumoral spatial heterogeneity and intersubject variability.

End of Study and Duration of Participation

A participant is considered to have completed the study if he or she has completed all phases of the study.

Treatment continues until disease progression per RECIST v1.1 or for a total of 45 cycles. Participants are allowed to continue treatment with atezolizumab (Control Arm A), RO7247669 (Arm B), or RO7247669 and tiragolumab (Arm C), or beyond 45 cycles at the investigator’s discretion. The total duration of study participation for each individual is expected to range from 1 day to more than 30 months. Participants who have a confirmed CR are allowed to discontinue treatment. Participants are allowed to continue treatment beyond disease progression if pre specified criteria are met.

The end of this study is defined as the date of the last visit of the last participant in the study/last scheduled procedure shown in the schedule of activities for the last participant in the study globally or the date at which the last data point required for statistical analysis (i.e., survival) or safety follow up is received from the last participant, whichever occurs later. The end of the study is expected to occur approximately 30 months after the last participant is enrolled. Example 2: Study Population

A. Inclusion Criteria

Potential participants are eligible to be included in the BO44157 study only if all of the following criteria apply:

• Age > 18 years.

• ECOG Performance Status of < 2.

• Histologically or cytologically documented locally advanced (T4b, any N; or any T, N2-3) or metastatic (M1 , Stage IV) cancer (also termed urothelial cell carcinoma) of the urothelium (including renal pelvis, ureters, urinary bladder, urethra). Patients with mixed histologies are required to have a dominant urothelial cell pattern. Locally advanced bladder cancer must be inoperable based on involvement of pelvic sidewall or adjacent viscera (clinical stage T4b) or bulky nodal metastasis (N2-N3).

• Considered to be ineligible ("unfit") to receive platinum-based chemotherapy as defined by one of the following criteria: o ECOG Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and < 30 mL/min/1 .73 m² o ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1 .73 m² and < 45 mL/min/1 .73 m². o ECOG Performance Status of 0-2 with G > 2 neuropathy.

• GFR is assessed by calculation through use of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation:

CKD-EPI = 142 x (serum creatinine/A)^B x 0.9938^A9® x (1.012 if female)

Where A and B are the following:

■ Female serum creatinine < 0.7: A = 0.7 and B = -0.241

■ Female serum creatinine > 0.7: A = 0.7 and B = -1.2

■ Male serum creatinine < 0.9: A = 0.9 and B = -0.302

■ Male serum creatinine > 0.9: A = 0.9 and B = -1 .2. o Patients for whom chemotherapy is not deemed appropriate

• No prior chemotherapy for inoperable locally advanced or metastatic or recurrent UC.

• For patients who received prior adjuvant/neoadjuvant chemotherapy or chemoradiation for UC, a treatment-free interval greaterthan 12 months between the last treatment administration and the date of recurrence is required in order to be considered treatment naive in the metastatic setting.

• Prior local intervesical chemotherapy or Bacillus Calmette-Guerin immunotherapy is allowed if completed at least 4 weeks prior to the initiation of study treatment.

• Measurable disease; at least one measurable lesion as defined by RECIST v1 .1 .

• Previously irradiated lesions should not be counted as target lesions unless there has been demonstrated progression in the lesion since radiotherapy and no other lesions are available for selection as target lesions.. • PD-L1 status is centrally assessed by IHC using the investigational VENTANA PD-L1 (SP263) CDx assay. Positive PD-L1 expression status is defined as TAP > 5% and negative PD-L1 status is defined as TAP < 5%. Patients with tumors that do not yield an evaluable PD-L1 status are not eligible.

• Life expectancy > 12 weeks.

• Adequate hematologic and end-organ function, defined by the following laboratory test results, obtained within 14 days prior to initiation of study treatment: o ANC > 1 .5 x 10⁹/L (> 1500/ .L) without granulocyte colony-stimulating factor support with the following exception: patients with benign ethnic neutropenia (BEN) and ANC > 1.3 x 10⁹/L (> 1300/ .L) are eligible. o Lymphocyte count > 0.5 x 10⁹/L (> 500/ .L). o Platelet count > 100 x 10⁹/L (> 100,000/ .L) without transfusion. o Hemoglobin > 90 g/L (> 9 g/dL). Patients may be transfused to meet this criterion. o Aspartate aminotransferase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) < 2.5 x upper limit of normal (ULN), with the following exceptions: Patients with documented liver metastases: AST and ALT < 5 x ULN; patients with documented liver or bone metastases: ALP < 5 x ULN. o Total bilirubin < 1 .5 x ULN with the following exception: patients with known Gilbert disease: total bilirubin < 3 x ULN. o Albumin > 25 g/L (> 2.5 g/dL) o For patients not receiving therapeutic anticoagulation: International Normalized Ratio (INR) and activated partial thromboplastin time (aPTT) < 1.5 x ULN.

• Negative HIV test at screening, with the following exception: individuals with a positive HIV test at screening are eligible provided they are stable on anti-retroviral therapy, have a CD4 count > 200/ .L, and have an undetectable viral load.

• Negative hepatitis B surface antigen (HBsAg) test at screening.

• Positive hepatitis B surface antibody (HBsAb) test at screening, or a negative HBsAb at screening accompanied by either of the following: negative hepatitis B core antibody (HBcAb) or positive HBcAb test followed by quantitative hepatitis B virus (HBV) DNA < 500 lU/mL.

• Negative hepatitis C virus (HCV) antibody test at screening, or a positive HCV antibody test followed by a negative HCV RNA test at screening.

• Adequate cardiovascular function: New York Heart Association (NYHA) Heart Failure Class < 2.

• Baseline-corrected QT (QTcF) interval < 480 ms. If the QTcF interval is longer than 480 ms but shorter than 500 ms, the participant may undergo a cardiac evaluation and be considered for treatment in case of no clinically significant findings.

• Resting systolic blood pressure < 150 mmHg and diastolic blood pressure 100 mmHg (average of > 3 readings on > 2 sessions with short break between sessions) (or no clinically significant hypertension).

• Resting heart rate between 45-100 bpm (or no clinically significant tachycardia). • Left ventricular ejection fraction (LVEF) > 50% assessed by either transthoracic echocardiogram (TTE) or multiple-gated acquisition (MUGA) scan (TTE preferred test) within 6 months prior to initiation of study treatment.

• Troponin T (TnT) or troponin I (Tnl) < 3 x institutional ULN. Participants with TnT or Tnl levels between > 1 and <3 x ULN should have a further TnT or Tnl reading within 3-6 hours and will be permitted to enter the study only if repeat levels remain < 3 x ULN and have not changed by > 20% compared to the first reading. These participants should also undergo a cardiac evaluation and consider consulting a cardiologist to confirm no clinically significant findings before they receive study treatment.

• For female participants of childbearing potential: agreement to remain abstinent (refrain from heterosexual intercourse) or use contraception and agreement to refrain from donating eggs.

• For male participants: agreement to remain abstinent (refrain from heterosexual intercourse) or use contraceptive methods, and agreement to refrain from donating sperm.

B. Exclusion Criteria

Potential participants are excluded from the BO44157 study if any of the following criteria apply:

• Pregnancy or breastfeeding, or intention of becoming pregnant during study treatment or within 5 months after the final dose of atezolizumab, 4 months after the final dose of RO7247669, or 90 days after the final dose of tiragolumab.

• GFR < 15 mL/min/1 .73 m² as calculated through use of the CKD-EPI equation or receiving dialysis

• Symptomatic, untreated, or actively progressing CNS metastases. Asymptomatic patients with treated CNS lesions are eligible, provided that all of the following criteria are met: Measurable disease, per RECIST v1 .1 , must be present outside the CNS; the patient has no history of intracranial hemorrhage or spinal cord hemorrhage; the patient has not undergone stereotactic radiotherapy within 7 days prior to initiation of study treatment, whole-brain radiotherapy within 14 days prior to initiation of study treatment, or neurosurgical resection within 28 days prior to initiation of study treatment; the patient has no ongoing requirement for corticosteroids as therapy for CNS disease; if the patient is receiving anti-convulsant therapy, the dose is considered stable; metastases are limited to the cerebellum or the supratentorial region (i.e., no metastases to the midbrain, pons, medulla, or spinal cord); no evidence of significant vasogenic edema; no evidence of interim progression between completion of CNS-directed therapy and initiation of study treatment. Asymptomatic patients with CNS metastases newly detected at screening are eligible for the study after receiving radiotherapy and/or surgery, with no need to repeat the screening brain scan.

• History of leptomeningeal disease.

• Uncontrolled tumor-related pain. Patients requiring pain medication must be on a stable regimen at study entry. Symptomatic lesions (e g., bone metastases or metastases causing nerve impingement) amenable to palliative radiotherapy should be treated prior to enrollment. Patients should be recovered from the effects of radiation. There is no required minimum recovery period. Asymptomatic metastatic lesions that would likely cause functional deficits or intractable pain with further growth (e.g., epidural metastasis that is not currently associated with spinal cord compression) should be considered for loco-regional therapy, if appropriate, prior to enrollment. • Uncontrolled pleural effusion, pericardial effusion, or ascites requiring recurrent drainage procedures (once monthly or more frequently). Patients with indwelling catheters (e.g., PLEURX®) are allowed.

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

• Active or history of autoimmune disease or immune deficiency, including, but not limited to, myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, anti-phospholipid antibody syndrome, Wegener granulomatosis, Sjogren syndrome, Guillain-Barre syndrome, or multiple sclerosis with the following exceptions: o Patients with a history of autoimmune-related hypothyroidism who are on thyroid-replacement hormone are eligible for the study. o Patients with controlled Type 1 diabetes mellitus who are on an insulin regimen are eligible for the study. o Patients with eczema, psoriasis, lichen simplex chronicus, or vitiligo with dermatologic manifestations only (e.g., patients with psoriatic arthritis are excluded) are eligible for the study provided all of following conditions are met: rash must cover < 10% of body surface area; disease is well controlled at baseline and requires only low-potency topical corticosteroids; there has been no occurrence of acute exacerbations of the underlying condition requiring psoralen plus ultraviolet A radiation, methotrexate, retinoids, biologic agents, oral calcineurin inhibitors, or high-potency or oral corticosteroids within the previous 12 months.

• History of idiopathic pulmonary fibrosis, organizing pneumonia (e.g., bronchiolitis obliterans), drug-induced pneumonitis, or idiopathic pneumonitis, or evidence of active pneumonitis on screening chest computed tomography (CT) scan. History of radiation pneumonitis in the radiation field (fibrosis) is permitted.

• Active tuberculosis (TB) (i.e., has signs and symptoms of TB).

• Active Epstein-Barr virus (EBV) infection or known or suspected chronic active EBV infection at screening.

• Significant cardiovascular/cerebrovascular disease within 3 months prior to initiation of study treatment, including any of the following: hypertensive crisis/encephalopathy; unstable angina; transient ischemic attack/stroke; congestive heart failure; serious cardiac arrhythmia requiring treatment (exceptions are atrial fibrillation, paroxysmal supraventricular tachycardia); history of thromboembolic events (such as myocardial infarction, stroke or pulmonary embolism).

• Major surgical procedure, other than for diagnosis, within 4 weeks prior to initiation of study treatment, or anticipation of need for a major surgical procedure during the study.

• History of another primary malignancy other than urothelial carcinoma within 5 years prior to Cycle 1 , Day 1 , with the exception of malignancies with a negligible risk of metastasis or death (e.g., 5-year OS rate > 90%), such as adequately treated carcinoma in situ of the cervix, non-melanoma skin carcinoma, localized prostate cancer, ductal carcinoma in situ, or Stage I uterine cancer with no plans for treatment intervention. Participants with localized prostate cancer (defined as Stage < pT2c, Gleason score < 7, and prostate-specific antigen (PSA) at prostate cancer diagnosis < 20 ng/mL) treated with curative intent and without PSA recurrence are eligible. Participants with preexisting low-risk prostate cancer (defined as Stage cT1/T2a, Gleason score < 6, and PSA < 10 ng/mL) who are treatment-naive and undergoing active surveillance are eligible.

• Severe infection within 4 weeks prior to initiation of study treatment, including, but not limited to, hospitalization for complications of infection, bacteremia, or severe pneumonia, or any active infection that could impact patient safety.

• Treatment with therapeutic oral or IV antibiotics within 2 weeks prior to initiation of study treatment. Patients receiving prophylactic antibiotics (e.g., to prevent a urinary tract infection or chronic obstructive pulmonary disease (COPD) exacerbation) are eligible for the study.

• Prior allogeneic stem cell or solid organ transplantation

• Any other disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding that contraindicates the use of an investigational drug, may affect the interpretation of the results, or may render the patient at high risk from treatment complications.

• Treatment with a live, attenuated vaccine within 4 weeks prior to initiation of study treatment, or anticipation of need for such a vaccine during treatment or within 5 months after the final dose of atezolizumab, 4 months after the final dose of RO7247669, or 90 days after the final dose of tiragolumab.

• Current treatment with anti-viral therapy for HBV

• Treatment with any approved anti-cancer therapy, including chemotherapy or hormonal therapy, within 3 weeks prior to initiation of study treatment; the following exceptions are allowed: palliative radiotherapy for bone metastases or soft tissue lesions should be completed > 7 days prior to baseline imaging; hormone-replacement therapy or oral contraceptives.

• Treatment with investigational therapy within 28 days prior to initiation of study treatment

• Prior treatment with CD137 agonists or immune checkpoint blockade therapies, including, but not limited to, anti-CTLA-4, anti-PD-1 , anti-PD-L1 , anti-TIGIT, and anti-LAG3 therapeutic antibodies or pathway-targeting agents.

• Treatment with systemic immunostimulatory agents (including, but not limited to, interferon and interleukin-2) within 4 weeks or 5 drug-elimination half-lives (whichever is longer) prior to initiation of study treatment.

• Treatment with systemic immunosuppressive medication (including, but not limited to, corticosteroids, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-tumor necrosis factor(TNF) agents) within 2 weeks prior to initiation of study treatment, or anticipation of need for systemic immunosuppressive medication during study treatment, with the following exceptions: patients who received acute, low-dose systemic immunosuppressant medication or a one-time pulse dose of systemic immunosuppressant medication (e.g., 48 hours of corticosteroids for a contrast allergy) are eligible for the study; patients who received mineralocorticoids (e.g., fludrocortisone), inhaled or low-dose corticosteroids for COPD or asthma, or low-dose corticosteroids for orthostatic hypotension or adrenal insufficiency are eligible for the study.

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

• Known hypersensitivity to Chinese hamster ovary cell products or to any component of the atezolizumab formulation. Example 3: Study Treatment

Table 4 provides a description of assigned study treatments for the BO44157 study.

Table 4. Study Treatment Description

A. Atezolizumab

Participants in the atezolizumab control arm receive treatment as outlined in Table 5 until disease progression or unacceptable toxicity. Participants who meet the criteria for disease progression as assessed by the investigator according to RECIST v1 .1 and show evidence of clinical benefit may continue treatment with atezolizumab at the investigator’s discretion.

Table 5. Treatment Regimen for Atezolizumab (Control Arm A)

Atezolizumab first and subsequent infusions are administered per the instructions outlined in Table 6.

Table 6. Administration of First and Subsequent Infusions of Atezolizumab

B. RO7247669 and Tiragolumab (Arms B and C)

Participants in the RO7247669 arm (Arm B) receive treatment as outlined in Table 7, while participants in the RO7247669 + tiragolumab arm (Arm C) receive treatment as outlined in Table 8, until disease progression or unacceptable toxicity. Participants who meet the criteria for disease progression as assessed by the investigator according to RECIST v1 .1 and show evidence of clinical benefit may continue treatment with RO7247669 and/or tiragolumab at the investigator’s discretion.

Table 7. Treatment Regimen for RO7247669 (Arm B)

Table 8. Treatment Regimen for RO7247669 + Tiragolumab (Arm C)

On Day 1 of Cycle 1 , tiragolumab is administered at least 60 minutes after completion of the RO7247669 infusion. After the first infusion, if any subsequent infusion is tolerated without an infusion-related reaction (IRR), tiragolumab may be administered at least 30 minutes after the RO7247669 infusion.

RO7247669 first and subsequent infusions are administered per the instructions outlined in Table 9.

Table 9. Administration of First and Subsequent Infusions of RO7247669

Tiragolumab first and subsequent infusions are administered per the instructions outlined in

Table 10.

Table 10. Administration of First and Subsequent Infusions of Tiragolumab

C. Concomitant Therapy

Permitted Therapy

In general, investigators may manage a participant's care (including preexisting conditions) through use of supportive therapies, as clinically indicated and per local standard practice, with the exception of prohibited therapies and taking into account cautionary therapies as described below. Participants who experience infusion-associated symptoms may be treated symptomatically with acetaminophen, ibuprofen, diphenhydramine, and/or Hz-receptor antagonists (e.g., famotidine, cimetidine), or equivalent medications per local standard practice. Serious infusion-associated events manifested by dyspnea, hypotension, wheezing, bronchospasm, tachycardia, reduced oxygen saturation, or respiratory distress are managed with supportive therapies as clinically indicated (e.g., supplemental oxygen and p2-adrenergic agonists).

Premedication with antihistamines, antipyretics, and/or analgesics may be administered at the discretion of the investigator.

All participants who experience diarrhea should be advised to drink liberal quantities of clear fluids. If sufficient oral fluid intake is not feasible, fluid and electrolytes should be substituted via IV infusion.

Use of the following concomitant therapies is permitted as described below:

• Oral contraceptives with a failure rate of < 1 % per year.

• Hormone-replacement therapy.

• Prophylactic or therapeutic anticoagulation therapy (such as warfarin at a stable dose or low-molecular-weight heparin).

• Vaccinations (such as influenza, COVID-19). Live, attenuated vaccines are not permitted.

• Megestrol acetate administered as an appetite stimulant.

• Mineralocorticoids (e.g., fludrocortisone).

• Inhaled or low-dose corticosteroids administered for COPD or asthma.

• Low-dose corticosteroids administered for orthostatic hypotension or adrenocortical insufficiency.

• Premedication with antihistamines, antipyretics, and/or analgesics may be administered for the second and subsequent RO7247669 and atezolizumab infusions only, at the discretion of the investigator.

• Palliative radiotherapy (e.g., treatment of known bony metastases or symptomatic relief of pain). Palliative radiotherapy is permitted provided it does not interfere with the assessment of tumor target lesions (e.g., the lesion to be irradiated must not be the only site of measurable disease). Treatment with atezolizumab, RO7247669, and tiragolumab may be continued during palliative radiotherapy.

• Local therapy (e.g., surgery, stereotactic radiosurgery, radiotherapy, radiofrequency ablation). Participants experiencing a mixed response requiring local therapy for control of three or fewer lesions may still be eligible to continue study treatment at the investigator’s discretion. Participants who receive local therapy directed at a target lesion are no longer be evaluable for radiographic response but remain evaluable for progression. Cautionary Therapy

Systemic corticosteroids, immunosuppressive medications, and TNF inhibitors may attenuate potential beneficial immunologic effects of treatment with atezolizumab, RO7247669, and tiragolumab. Therefore, in situations in which systemic corticosteroids, immunosuppressive medications, or TNF inhibitors would be routinely administered, alternatives, including antihistamines, should be considered. If the alternatives are not feasible, systemic corticosteroids, immunosuppressive medications, and TNF inhibitors may be administered at the discretion of the investigator.

Systemic corticosteroids or immunosuppressive medications are recommended, at the discretion of the investigator, for the treatment of specific adverse events when associated with RO7247669 and atezolizumab therapy.

Prohibited Therapy

Use of the following concomitant therapies is prohibited as described below:

• Investigational therapy within 28 days prior to initiation of study treatment and during study treatment.

• Concomitant therapy intended for the treatment of cancer (including, but not limited to, chemotherapy, hormonal therapy, immunotherapy, radiotherapy, and herbal therapy), whether health authority-approved or experimental, for various time periods prior to starting study treatment, depending on the agent (see above), and during study treatment, until disease progression is documented and the participant has discontinued study treatment, with the exception of palliative radiotherapy, radiotherapy to the brain, and local therapy under certain circumstances (see above).

• Live, attenuated vaccines within 4 weeks prior to initiation of study treatment, during RO7247669 and atezolizumab treatment, and for 4 months after the final dose RO7247669, 5 months after the final dose of atezolizumab, or 90 days after the final dose of tiragolumab.

• Systemic immunostimulatory agents (including, but not limited to, interferons and interleukin-2) within 4 weeks or 5 drug-elimination half-lives (whichever is longer) prior to initiation of study treatment and during study treatment because these agents could potentially increase the risk for autoimmune conditions when given in combination with RO7247669, atezolizumab or tiragolumab.

Example 4: Efficacy Assessments

Tumor and Response Evaluations

Participants in the the BO44157 study undergo tumor assessments at screening, every 6 weeks (± 7 days) for the first 54 weeks following treatment initiation, and every 9 weeks thereafter, regardless of dose delays, until radiographic disease progression per RECIST v1 .1 , withdrawal of consent, loss to follow-up, study termination, or death, whichever occurs first.

Participants who are treated beyond disease progression per RECIST v1 .1 undergo tumor assessments every 6 weeks (± 7 days) after initial documentation of progression, or more frequently if clinically indicated, regardless of time on study, until symptomatic deterioration attributed to disease progression, withdrawal of consent, loss to follow-up, study termination, or death, whichever occurs first. Participants who discontinue study treatment for reasons other than disease progression (e.g., toxicity) continue to undergo scheduled tumor assessments as if they were on the protocol schedule, even if they start a new anti-cancer therapy, until disease progression per RECIST v1.1 , withdrawal of consent, loss to follow-up, study termination, or death, whichever occurs first.

At the investigator's discretion, tumor assessments may be repeated at any time if progressive disease is suspected. All measurable and/or evaluable lesions are assessed and documented at screening. Tumor assessments performed as standard of care within 28 days prior to initiation of study treatment do not have to be repeated at screening, so long as they meet criteria outlined herein.

Response Evaluation

Objective response is determined by the investigator at specified timepoints according to RECIST v1.1. Assessments are performed by the same individual, if possible, to ensure internal consistency across visits.

Endpoints (e.g., ORR, PFS, DCR) are calculated programmatically on the basis of investigator assessment at each specified timepoint.

Clinical Outcome Assessments

Patient-reported outcome (PRO) instruments are completed to assess the treatment benefit of RO7247669 and RO7247669 in combination with tiragolumab compared to atezolizumab. In addition, PRO instruments enable the capture of each participant's direct experience with RO7247669 and RO7247669 in combination with tiragolumab.

PRO data are collected via European Organisation for Research and Treatment of Cancer (EORTC) item library 187 (EORTC IL187).

The EORTC IL187 is an item library, consisting of selected items or scales from the EORTC quality of life questionnaire (QLQ) C30 and EORTC QLQ BLM30 that was created specifically for this study from the EORTC Quality of Life Group Item Library. It consists of 33 questions that assess six symptoms (fatigue, nausea, vomiting, pain, insomnia, and appetite loss), five aspects of patient functioning (physical, role, emotional, social, and cognitive), and health-related quality of life (HRQoL) (global health status (GHS)/ quality of life (QoL)) from the EORTC QLQ C30, as well as urinary symptoms and problems (frequency, urgency, sleep interference, independence, incontinence, and pain) from the EORTC QLQ BLM30, all with a recall period of the previous week. The scoring for the EORTC IL187 follows that of the EORTC QLQ C30.

Example 5: Statistical Considerations

Statistical Hypotheses

The purpose of the BO44157 study is hypothesis generation regarding the effect of RO7247669 (Arm B) and RO7247669 in combination with tiragolumab (Arm C) on investigator-assessed confirmed objective response rate (ORR) relative to atezolizumab (Arm A). The null (HO) and alternative (H1) hypotheses regarding ORR in all randomized participants with measurable disease at baseline are tested independently for Arm C versus Arm A, and Arm B versus Arm A, each at a two-sided significance level of 0.1 , which can be phrased in terms of the population proportion rt between the experimental arm (7t_a) and the control arm (TIB):

Ho: 7^ - ?rp = 0 versus H1 : rra - <1,^ 0

No multiplicity corrections are planned for this study.

Sample Size Determination

A total of approximately 240 participants are enrolled in the study, and are expected to be randomized as 1 :1 :1 allocation ratio to each arm. The estimated sample size of 80 per arm is based on the comparison between the experimental arms (e.g., Arms B and C vs. the control arm (Arm A)) with regard to the primary endpoint of ORR with the following assumptions:

1 . A 15% improvement in ORR (i.e., AORR) in Arm B relative to Arm A with a 69% power and 10% alpha assuming a 20% ORR in Arm A

2. A 25% improvement in ORR (i.e., AORR) in Arm C relative to Arm A with 96% power and 10% alpha assuming a 20% ORR in Arm A

The study is not adequately powered to detect all potentially clinically meaningful differences in ORR, and thus, a statistically negative outcome in the primary endpoint does not necessarily rule out a clinically meaningful outcome. For instance, if the improvement of ORR is 10% assuming a 20% ORR in Arm A, such a study will have around 43% power with 10% alpha.

Analysis Sets

The participant analysis sets for the purposes of analyses are defined in Table 11 .

Table 11. Participant Analysis Sets

Statistical Analyses

All efficacy analyses are performed on the full analysis set (FAS), unless otherwise specified. Participants are analyzed according to the treatment assigned at randomization, regardless of whether they receive any assigned study drug.

All safety analyses are conducted on the safety analysis set (SAS), unless otherwise specified. Participants are analyzed according to the actual treatment they received, regardless of the initial treatment assignment at randomization according to the following: • Participants who receive atezolizumab only and do not receive either tiragolumab or RO7247669 are classified into Arm A.

• Participants who receive RO7247669 only and do not receive either atezolizumab or tiragolumab are classified into Arm B.

• Participants who receive any amount of tiragolumab are classified into Arm C.

Primary Endpoint

The primary endpoint is confirmed ORR according to RECIST v1.1 per investigator as defined in Table 3. The primary estimand corresponding to the primary endpoints is defined as:

• Population: Participants with previously untreated, locally advanced UC or mUC who are ineligible for platinum-containing chemotherapy, as defined by the study inclusion and exclusion criteria.

• Variables: Proportion of participants with a complete response (CR) or partial response (PR) on two consecutive occasions > 4 weeks apart after randomization, as determined by the investigator according to RECIST v1 .1 .

• Treatments:

Control Arm A: atezolizumab 1200 mg IV Q3W.

Experimental Arm B: RO7247669 600 mg IV Q3W.

Experimental Arm C: RO7247669 600 mg IV Q3W + tiragolumab 600 mg IV Q3W.

• Intercurrent events:

Early discontinuation from study treatment for any reason prior to a progression-free survival (PFS) event.

Initiation of another non-protocol anti-cancer therapy prior to a PFS event.

A treatment-policy strategy is planned for the primary estimand. This means that treatment effect of RO7247669 and RO7247669 in combination with tiragolumab versus atezolizumab on PFS is evaluated regardless of adherence to treatment and initiation of new non-protocol therapy.

• Population-level summary: difference in confirmed ORR.

The analysis population for confirmed ORR is the FAS with measurable disease at baseline. The 95% Cl for the differences in confirmed ORRs between two treatment arms (Arm B vs. Arm A, and Arm C vs. Arm A) is computed using the Newcombe method with continuity correction. The Cochran-Mantel-Haenszel test is used to compare the ORR between the two treatment arms, stratified according to the protocol-defined stratification factors. The 95% Cl of the confirmed ORR is calculated for each treatment arm using the Wilson score method.

The ORR primary analysis is expected to occur approximately 6 months after the last participant has been randomized. Secondary Endpoints

Progression-free survival (PFS) is defined as the time from randomization to the first occurrence of disease progression or death from any cause (whichever occurs first), as determined by the investigator according to RECIST v1 .1 . Data for participants who have not experienced disease progression or death are censored at the last tumor assessment date. Data for participants with no post-baseline tumor assessments are censored at the randomization date.

The PFS is compared between treatment arms with use of the stratified log-rank test at the two-sided level of significance. Hazard ratio is estimated using a stratified Cox regression model with the same stratification variables used for the stratified log-rank test. The 95% Cl is provided. Kaplan-Meier methodology is used to estimate the median PFS for each treatment arm, and Kaplan-Meier curves are produced. The Brookmeyer-Crowley methodology is used to construct the 95% Cl for the median PFS for each treatment arm.

Duration of response (DOR) is assessed in participants who have achieved a confirmed objective response, as determined by the investigator according to RECIST v1 .1 . DOR is defined as the time interval from the date of the first occurrence of a confirmed objective response until the first date of progressive disease, as determined by the investigator according to RECIST v1 .1 , or death from any cause, whichever occurs first. Participants who have not progressed and who have not died at the time of analysis are censored at the time of the last tumor assessment date.

Methods for comparison of DOR between treatment arms are the same as the methods for treatment comparison for PFS.

Overall survival (OS) is defined as the time between the date of randomization and death from any cause. Data for participants who are not reported as having died by the data cutoff date are censored at the date when they were last known to be alive. Data for participants who do not have postbaseline information are censored at the date of randomization.

Methods for comparison of OS between treatment arms are the same as the methods for treatment comparison for PFS.

The investigator-assessed PFS rates at 6 months and 12 months after randomization are estimated with use of the Kaplan-Meier methodology for each treatment arm, along with 95% Cis calculated with use of the standard error computed via the Greenwood method. The 95% Cl for the difference in PFS rates between the two treatment arms (Arm B vs. Arm A, and Arm C vs. Arm A) is estimated with use of the normal approximation method.

The OS rates at 6 months, 12 months, and 18 months after randomization are estimated with use of the Kaplan-Meier methodology for each treatment arm, along with 95% Cis calculated with use of the standard error computed via the Greenwood method. The 95% Cl for the difference in OS rates between the two treatment arms (Arm B vs. Arm A, and Arm C vs. Arm A) is estimated with use of the normal approximation method.

Disease control rate (DCR) is defined as the proportion of participants with a confirmed objective response or SD maintained for > 12 weeks, per RECIST v1 .1 . The analysis population for DCR is the FAS with measurable disease at baseline. The 95% Cl of DCR is calculated for each treatment arm using the Wilson score method. Similar analysis methods to those described for the PFS and OS analyses are performed for FAS with PD-L1 expression defined as TAP > 5% and TAP < 5%, as determined by the investigational VENTANA PD-L1 (SP263) CDx Assay. Table 12. Sequences

IX. Other Embodiments

Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

1. A method for treating a subject having a cancer (e.g., an urothelial bladder cancer), the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD-1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), wherein:

(i) the subject is ineligible for platinum-based chemotherapy; or (ii) the subject has not previously been treated with a cancer immunotherapy.

2. The method of paragraph 1 , wherein the bispecific antibody is administered as a monotherapy.

3. A method for treating a subject having a cancer (e.g., an urothelial bladder cancer), the method comprising administering to the subject one or more dosing cycles of (a) an anti-TIGIT antagonist antibody; and (b) a bispecific antibody targeting PD-1 and LAG3 comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein:

(i) the subject is ineligible for platinum-based chemotherapy; or

(ii) the subject has not previously been treated with a cancer immunotherapy.

4. The method of any one of paragraphs 1 -3, wherein the subject is ineligible for platinum-based chemotherapy.

5. The method of any one of paragraphs 1-3, wherein the subject has not previously been treated with a cancer immunotherapy.

6. The method of any one of paragraphs 1 -5, wherein the subject is ineligible for platinum-based chemotherapy and has not previously been treated with a cancer immunotherapy.

7. The method of any one of paragraphs 3-6, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody at a fixed dose of about 600 mg (e.g., at a fixed dose of 600 mg) every three weeks.

8. The method of any one of paragraphs 1-7, wherein the method comprises administering to the subject the bispecific antibody at a fixed dose of about 600 mg (e.g., at a fixed dose of 600 mg) every three weeks.

9. The method of any one of paragraphs 1-8, wherein the length of each of the one or more dosing cycles is about 21 days (e.g., is 21 days).

10. The method of any one of paragraphs 1-9, wherein the method comprises administering to the subject the bispecific antibody on or about Day 1 (e g., Day 1 ±1 day) of each of the one or more dosing cycles.

11 . The method of any one of paragraphs 3-10, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody on or about Day 1 (e.g., Day 1 ±1 day) of each of the one or more dosing cycles. 12. The method of any one of paragraphs 3-11 , wherein the method comprises administering to the subject the bispecific antibody before the anti-TI G IT antagonist antibody.

13. The method of paragraph 12, wherein the anti-TIGIT antagonist antibody is administered at least 60 minutes after the bispecific antibody in a first dosing cycle.

14. The method of paragraph 12 or 13, wherein the method comprises one or more additional dosing cycles, and the anti-TIGIT antagonist antibody is administered at least 30 minutes after the bispecific antibody in the one or more additional dosing cycles.

15. The method of any one of paragraphs 1-14, wherein the method comprises administering to the subject the bispecific antibody intravenously.

16. The method of any one of paragraphs 3-15, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody intravenously.

17. The method of any one of paragraphs 1-16, wherein the cancer (e.g., urothelial bladder cancer) is locally advanced or metastatic.

18. The method of paragraph 17, wherein:

(a) the clinical stage of the locally advanced cancer (e.g., urothelial bladder cancer) is (i) T4b, any N or (ii) any T, N2-3; or

(b) the clinical stage of the metastatic cancer (e.g., urothelial bladder cancer) is M1 and/or Stage IV.

19. The method of any one of paragraphs 1-18, wherein the cancer (e.g., urothelial bladder cancer) is a carcinoma.

20. The method of any one of paragraphs 1-19, wherein the subject is ineligible for platinum-based chemotherapy based on having:

(a) an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and < 30 mL/min/1.73 m²;

(b) an ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1 .73 m² and < 45 mL/min/1 .73 m²; or

(c) an ECOG Performance Status of 0-2 with Grade > 2 neuropathy.

21 . The method of paragraph 20, wherein the GFR is assessed by calculation through use of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

22. The method of any one of paragraphs 1-21 , wherein the subject has not previously been treated with a chemotherapy for locally advanced, metastatic, or recurrent cancer (e.g., urothelial bladder cancer). 23. The method of any one of paragraphs 1-22, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds to PD-1 comprising a heavy chain variable (VH) domain comprising:

(i) a hypervariable region H1 (HVR-H1) sequence comprising the amino acid sequence of SEQ ID NO: 1 ,

(ii) an HVR-H2 sequence comprising the amino acid sequence GGR, and

(iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; and a light chain variable (VL) domain comprising

(i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3;

(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: 4.

24. The method of paragraph 23, wherein the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG.

25. The method of paragraph 24, wherein the IgG Fc domain is an IgG 1 Fc domain or an lgG4 Fc domain.

26. The method of paragraph 24 or 25, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor.

27. The method of paragraph 26, wherein the Fc receptor is an Fey receptor.

28. The method of any one of paragraphs 23-27, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a second antigen-binding domain that specifically binds to LAG-3 comprising a VH domain comprising:

(i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7,

(ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and

(iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; and a VL domain comprising:

(i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10,

(ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and

(iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12.

29. The method of any one of paragraphs 23-28, wherein the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14. 30. The method of any one of paragraphs 23-29, wherein the bispecific antibody targeting PD-1 and LAG3 comprises:

(a) an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index); and/or

(b) an Fc domain comprising a modification promoting the association of the first and second subunit of the Fc domain.

31 . The method of any one of paragraphs 24-30, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index).

32. The method of any one of paragraphs 23-31 , wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first Fab fragment comprising said first antigen binding domain specifically binding to PD1 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and a second Fab fragment comprising the second antigen binding domain specifically binding to LAG3 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, and an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index).

33. The method of paragraph 32, wherein in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3 the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

34. The method of paragraph 33, wherein in the first Fab fragment the variable domains VL and VH are replaced by each other.

35. The method of any one of paragraphs 32-34, wherein in the constant domain CL of one of the Fab fragments the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

36. The method of paragraph 35, wherein in the constant domain CL of the second Fab fragment the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index). 37. The method of any one of paragraphs 23-36, wherein the bispecific antibody comprises a first heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15, a first light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16, a second heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17, and a second light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18.

38. The method of paragraph 37, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18.

39. The method of any one of paragraphs 3-38, wherein the anti-TIGIT antagonist antibody comprises the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 38); an HVR-H2 sequence comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 39); an HVR-H3 sequence comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 40); an HVR-L1 sequence comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 41); an HVR-L2 sequence comprising the amino acid sequence of WASTRES (SEQ ID NO: 42); and an HVR-L3 sequence comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 43).

40. The method of any one of paragraphs 3-39, wherein the anti-TIGIT antagonist antibody comprises:

(a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44 or 45;

(b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46; or

(c) a VH domain as in (a) and a VL domain as in (b).

41 . The method of any one of paragraphs 3-40, wherein the anti-TIGIT antagonist antibody is a monoclonal antibody, a human antibody, or a full-length antibody.

42. The method of any one of paragraphs 3-41 , wherein the anti-TIGIT antagonist antibody is a human, monoclonal full-length IgG 1 subclass antibody comprising a human lgG1 Fc region, a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 44, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 46.

43. The method of any one of paragraphs 3-42, wherein the anti-TIGIT antagonist antibody exhibits effector function. 44. The method of any one of paragraphs 3-43, wherein the anti-TIGIT antagonist antibody is tiragolumab.

45. The method of any one of paragraphs 3-41 , wherein the anti-TIGIT antagonist antibody is an antibody fragment that binds TIGIT selected from the group consisting of Fab, Fab’, Fab’-SH, Fv, single chain variable fragment (scFv), and (Fab’)2 fragments.

46. The method of any one of paragraphs 3-44, wherein the anti-TIGIT antagonist antibody is an IgG class antibody.

47. The method of paragraph 46, wherein the IgG class antibody is an lgG1 subclass antibody.

48. The method of any one of paragraphs 1-47, wherein a PD-L1 expression level of a tumor sample obtained from the subject has been determined.

49. The method of paragraph 48, wherein the PD-L1 expression level is a protein expression level, and the protein expression level has been determined by an immunohistochemical (IHC) assay comprising staining with an anti-PD-L1 antibody suitable for staining.

50. The method of paragraph 49, wherein the anti-PD-L1 antibody suitable for staining is the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8.

51 . The method of paragraph 49 or 50, wherein the protein expression level of PD-L1 is determined using a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, a Ventana SP142 IHC assay, or a pharmDx 28-8 IHC assay.

52. The method of any one of paragraphs 48-51 , wherein the tumor sample obtained from the subject has been determined to be PD-L1 -positive.

53. The method of any one of paragraphs 48-51 , wherein the tumor sample obtained from the subject has been determined to be PD-L1 -negative.

54. The method of any one of paragraphs 1-53, wherein the method results in an increase in objective response rate (ORR) in a population of subjects treated according to the method as compared to a reference ORR.

55. The method of paragraph 54, wherein the reference ORR is an ORR of a population of subjects who have received a control therapy.

56. The method of any one of paragraphs 1-55, wherein the method results in an increase in progression- free survival (PFS) as compared to a reference PFS. 57. The method of paragraph 56, wherein the reference PFS is a PFS of a population of subjects who have received a control therapy.

58. The method of any one of paragraphs 1-57, wherein the method results in an increase in overall survival (OS) as compared to a reference OS.

59. The method of paragraph 58, wherein the reference OS is an OS of a population of subjects who have received a control therapy.

60. The method of any one of paragraphs 1-59, wherein the method results in an increase in duration of response (DOR) as compared to a reference DOR.

61 . The method of paragraph 60, wherein the reference DOR is a DOR of a population of subjects who have received a control therapy.

62. The method of any one of paragraphs 1-61 , wherein the method results in:

(a) an increase in PFS rate at 6 months as compared to a reference PFS rate at 6 months; or

(b) an increase in PFS rate at 12 months as compared to a reference PFS rate at 12 months.

63. The method of paragraph 62, wherein the reference PFS rate is a PFS rate of a population of subjects who have received a control therapy.

64. The method of any one of paragraphs 1-63, wherein the method results in:

(a) an increase in OS rate at 6 months as compared to a reference OS rate at 6 months;

(b) an increase in OS rate at 12 months as compared to a reference OS rate at 12 months; or

(c) an increase in OS rate at 18 months as compared to a reference OS rate at 18 months.

65. The method of paragraph 64, wherein the reference OS rate is an OS rate of a population of subjects who have received a control therapy.

66. The method of any one of paragraphs 1-65, wherein the method results in an increase in disease control rate (DCR) as compared to a reference DCR.

67. The method of paragraph 66, wherein the reference DCR is a DCR of a population of subjects who have received a control therapy.

68. The method of any one of paragraphs 54-67, wherein the control therapy is a treatment comprising atezolizumab, and the control therapy does not comprise the bispecific antibody or an anti-TIGIT antagonist antibody. 69. A method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising:

(D a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy; or

(2)

(i) a first antigen-binding domain that specifically binds to PD-1 , comprising the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 1 , an HVR-H2 sequence comprising the amino acid sequence GGR, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; an HVR-L2 sequence comprising the amino acid sequence RSS, and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 4; and

(ii) a second antigen-binding domain that specifically binds to LAG3, comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

70. A method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising:

(1) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy; or

(2) (i) a first antigen-binding domain that specifically binds to PD-1 , comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 1 , an HVR-H2 sequence comprising the amino acid sequence GGR, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; an HVR-L2 sequence comprising the amino acid sequence RSS, and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 4; and

(ii) a second antigen-binding domain that specifically binds to LAG3, comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

71 . A method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of:

(1)

(a) an anti-TIGIT antagonist antibody; and

(b) a bispecific antibody targeting PD-1 and LAG3 comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy; or

(2)

(a) an anti-TIGIT antagonist antibody comprising the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 38; an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 39; an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 40; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 41 ; an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 42; and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 43; and

(b) a bispecific antibody targeting PD-1 and LAG3 comprising:

(i) a first antigen-binding domain that specifically binds to PD-1 , comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 1 , an HVR-H2 sequence comprising the amino acid sequence GGR, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; an HVR-L2 sequence comprising the amino acid sequence RSS, and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 4; and

(ii) a second antigen-binding domain that specifically binds to LAG3, comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

72. A method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of:

(1)

(a) an anti-TIGIT antagonist antibody; and

(b) a bispecific antibody targeting PD-1 and LAG3 comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy; or

(2)

(a) an anti-TIGIT antagonist antibody comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 38; an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 39; an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 40; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 41 ; an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 42; and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 43; and

(b) a bispecific antibody targeting PD-1 and LAG3 comprising:

(i) a first antigen-binding domain that specifically binds to PD-1 , comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 1 , an HVR-H2 sequence comprising the amino acid sequence GGR, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; an HVR-L2 sequence comprising the amino acid sequence RSS, and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 4; and

(ii) a second antigen-binding domain that specifically binds to LAG3, comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

73. The method of paragraph 71 or 72, wherein the anti-TIGIT antagonist antibody is tiragolumab.

74. The method of any one of paragraphs 1-73, wherein the subject is a human.

75. A bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a cancer (e.g., an urothelial bladder cancer), wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first antigenbinding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody, wherein:

(i) the subject is ineligible for platinum-based chemotherapy; or

(ii) the subject has not previously been treated with a cancer immunotherapy.

76. The bispecific antibody for use of paragraph 75, wherein the bispecific antibody is to be administered as a monotherapy.

77. A bispecific antibody targeting PD-1 and LAG3 and/or an anti-TIGIT antagonist antibody for use in a method of treating a subject having a cancer (e.g ., an urothelial bladder cancer), wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, 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 dosing regimen comprises administering to the subject one or more dosing cycles of (a) the anti-TIGIT antagonist antibody; and (b) the bispecific antibody, wherein: (i) the subject is ineligible for platinum-based chemotherapy; or

(ii) the subject has not previously been treated with a cancer immunotherapy.

78. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-77, wherein the subject is ineligible for platinum-based chemotherapy.

79. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-77, wherein the subject has not previously been treated with a cancer immunotherapy.

80. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-79, wherein the subject is ineligible for platinum-based chemotherapy and has not previously been treated with a cancer immunotherapy.

81 . The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-80, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody at a fixed dose of about 600 mg (e.g., at a fixed dose of 600 mg) every three weeks.

82. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-81 , wherein the method comprises administering to the subject the bispecific antibody at a fixed dose of about 600 mg (e.g., at a fixed dose of 600 mg) every three weeks.

83. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-82, wherein the length of each of the one or more dosing cycles is about 21 days (e.g., is 21 days).

84. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-83, wherein the method comprises administering to the subject the bispecific antibody on or about Day 1 (e.g., Day 1 ±1 day) of each of the one or more dosing cycles.

85. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-84, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody on or about Day 1 (e.g., Day 1 ±1 day) of each of the one or more dosing cycles.

86. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-85, wherein the method comprises administering to the subject the bispecific antibody before the anti-TIGIT antagonist antibody.

87. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 86, wherein the anti-TIGIT antagonist antibody is to be administered at least 60 minutes after the bispecific antibody in a first dosing cycle. 88. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 86 or 87, wherein the method comprises one or more additional dosing cycles, and the anti-TIGIT antagonist antibody is to be administered at least 30 minutes after the bispecific antibody in the one or more additional dosing cycles.

89. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-88, wherein the method comprises administering to the subject the bispecific antibody intravenously.

90. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-89, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody intravenously.

91 . The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-90, wherein the cancer (e.g., urothelial bladder cancer) is locally advanced or metastatic.

92. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 91 , wherein:

(a) the clinical stage of the locally advanced cancer (e.g., urothelial bladder cancer) is (i) T4b, any N or (ii) any T, N2-3; or

(b) the clinical stage of the metastatic cancer (e.g., urothelial bladder cancer) is M1 and/or Stage IV.

93. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-92, wherein the cancer (e.g., urothelial bladder cancer) is a carcinoma.

94. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-93, wherein the subject is ineligible for platinum-based chemotherapy based on having:

(a) an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and < 30 mL/min/1 .73 m²;

(b) an ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1 .73 m² and < 45 mL/min/1 .73 m²; or

(c) an ECOG Performance Status of 0-2 with Grade > 2 neuropathy.

95. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 94, wherein the GFR is assessed by calculation through use of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

96. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-95, wherein the subject has not previously been treated with a chemotherapy for locally advanced, metastatic, or recurrent cancer (e.g., urothelial bladder cancer). 97. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-96, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds to PD-1 comprising a heavy chain variable (VH) domain comprising:

(i) a hypervariable region H1 (HVR-H1) sequence comprising the amino acid sequence of SEQ ID NO: 1 ,

(ii) an HVR-H2 sequence comprising the amino acid sequence GGR, and

(iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; and a light chain variable (VL) domain comprising

(i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3;

(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: 4.

98. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 97, wherein the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG.

99. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 98, wherein the IgG Fc domain is an lgG1 Fc domain or an lgG4 Fc domain.

100. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 98 or 99, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor.

101. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 100, wherein the Fc receptor is an Fey receptor.

102. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 97-

101 , wherein the bispecific antibody targeting PD-1 and LAG3 comprises a second antigen-binding domain that specifically binds to LAG-3 comprising a VH domain comprising:

(i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7,

(ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and

(iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; and a VL domain comprising:

(i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10,

(ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and

(iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12.

103. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 97-

102, wherein the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14. 104. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 97-

103, wherein the bispecific antibody targeting PD-1 and LAG3 comprises:

(a) an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index); and/or

(b) an Fc domain comprising a modification promoting the association of the first and second subunit of the Fc domain.

105. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 98-

104, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index).

106. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 97-

105, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first Fab fragment comprising said first antigen binding domain specifically binding to PD1 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and a second Fab fragment comprising the second antigen binding domain specifically binding to LAG3 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, and an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index).

107. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 106, wherein in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3 the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

108. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 107, wherein in the first Fab fragment the variable domains VL and VH are replaced by each other.

109. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 106- 108, wherein in the constant domain CL of one of the Fab fragments the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

110. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 109, wherein in the constant domain CL of the second Fab fragment the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

111. The bispecific antibody and/or anti-TIG IT antagonist antibody for use of any one of paragraphs 97- 110, wherein the bispecific antibody comprises a first heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15, a first light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16, a second heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17, and a second light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18.

112. The bispecific antibody and/or a nti-TIG IT antagonist antibody for use of paragraph 111 , wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18.

113. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-

112, wherein the anti-TIGIT antagonist antibody comprises the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 38); an HVR-H2 sequence comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 39); an HVR-H3 sequence comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 40); an HVR-L1 sequence comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO:

41); an HVR-L2 sequence comprising the amino acid sequence of WASTRES (SEQ ID NO: 42); and an HVR-L3 sequence comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 43).

114. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-

113, wherein the anti-TIGIT antagonist antibody comprises:

(a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44 or 45;

(b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46; or

(c) a VH domain as in (a) and a VL domain as in (b).

115. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-

114, wherein the anti-TIGIT antagonist antibody is a monoclonal antibody, a human antibody, or a full- length antibody. 116. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-

115, wherein the anti-TIGIT antagonist antibody is a human, monoclonal full-length lgG1 subclass antibody comprising a human IgG 1 Fc region, a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 44, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 46.

117. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-

116, wherein the anti-TIGIT antagonist antibody exhibits effector function.

118. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77-

117, wherein the anti-TIGIT antagonist antibody is tiragolumab.

119. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77- 115, wherein the anti-TIGIT antagonist antibody is an antibody fragment that binds TIGIT selected from the group consisting of Fab, Fab’, Fab’-SH, Fv, single chain variable fragment (scFv), and (Fab’)2 fragments.

120. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 77- 119, wherein the anti-TIGIT antagonist antibody is an IgG class antibody.

121 . The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 120, wherein the IgG class antibody is an IgG 1 subclass antibody.

122. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75- 121 , wherein a PD-L1 expression level of a tumor sample obtained from the subject has been determined.

123. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 122, wherein the PD-L1 expression level is a protein expression level, and the protein expression level has been determined by an immunohistochemical (IHC) assay comprising staining with an anti-PD-L1 antibody suitable for staining.

124. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 123, wherein the anti-PD-L1 antibody suitable for staining is the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8.

125. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 123 or 124, wherein the protein expression level of PD-L1 is determined using a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, a Ventana SP142 IHC assay, or a pharmDx 28-8 IHC assay.

126. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 122- 125, wherein the tumor sample obtained from the subject has been determined to be PD-L1 -positive. 127. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 122- 125, wherein the tumor sample obtained from the subject has been determined to be PD-L1 -negative.

128. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75- 127, wherein the method results in an increase in objective response rate (ORR) in a population of subjects treated according to the method as compared to a reference ORR.

129. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 128, wherein the reference ORR is an ORR of a population of subjects who have received a control therapy.

130. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75- 129, wherein the method results in an increase in progression-free survival (PFS) as compared to a reference PFS.

131. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 130, wherein the reference PFS is a PFS of a population of subjects who have received a control therapy.

132. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75- 131 , wherein the method results in an increase in overall survival (OS) as compared to a reference OS.

133. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 132, wherein the reference OS is an OS of a population of subjects who have received a control therapy.

134. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75- 133, wherein the method results in an increase in duration of response (DOR) as compared to a reference DOR.

135. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 134, wherein the reference DOR is a DOR of a population of subjects who have received a control therapy.

136. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75- 135, wherein the method results in:

(a) an increase in PFS rate at 6 months as compared to a reference PFS rate at 6 months; or

(b) an increase in PFS rate at 12 months as compared to a reference PFS rate at 12 months.

137. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 136, wherein the reference PFS rate is a PFS rate of a population of subjects who have received a control therapy.

138. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75- 137, wherein the method results in:

(a) an increase in OS rate at 6 months as compared to a reference OS rate at 6 months;

(b) an increase in OS rate at 12 months as compared to a reference OS rate at 12 months; or

(c) an increase in OS rate at 18 months as compared to a reference OS rate at 18 months. 139. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 138, wherein the reference OS rate is an OS rate of a population of subjects who have received a control therapy.

140. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 75-

139, wherein the method results in an increase in disease control rate (DOR) as compared to a reference DCR.

141 . The bispecific antibody and/or anti-TIGIT antagonist antibody for use of paragraph 140, wherein the reference DCR is a DCR of a population of subjects who have received a control therapy.

142. The bispecific antibody and/or anti-TIGIT antagonist antibody for use of any one of paragraphs 129- 141 , wherein the control therapy is a treatment comprising atezolizumab, and the control therapy does not comprise the bispecific antibody or an anti-TIGIT antagonist antibody.

143. A bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a cancer (e.g., a locally advanced or metastatic urothelial bladder cancer), wherein the method comprises:

(1) a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody; the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and the subject is ineligible for platinum-based chemotherapy; or

(2)

(i) a first antigen-binding domain that specifically binds to PD-1 , comprising the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 1 , an HVR-H2 sequence comprising the amino acid sequence GGR, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; an HVR-L2 sequence comprising the amino acid sequence RSS, and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 4; and

(ii) a second antigen-binding domain that specifically binds to LAG3, comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

144. A bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a locally advanced or metastatic urothelial bladder cancer, wherein the method comprises:

(1) a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody; the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and the subject has not previously been treated with a cancer immunotherapy; or

(2)

(i) a first antigen-binding domain that specifically binds to PD-1 , comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 1 , an HVR-H2 sequence comprising the amino acid sequence GGR, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; an HVR-L2 sequence comprising the amino acid sequence RSS, and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 4; and

(ii) a second antigen-binding domain that specifically binds to LAG3, comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

145. A bispecific antibody targeting PD-1 and LAG3 and/or an anti-TIGIT antagonist antibody for use in a method of treating a subject having a cancer (e.g., a locally advanced or metastatic urothelial bladder cancer), wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of: (1)

(a) the anti-TIGIT antagonist antibody; and

(b) the bispecific antibody; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy; or

(2)

(a) an anti-TIGIT antagonist antibody comprising the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 38; an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 39; an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 40; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 41 ; an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 42; and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 43; and

(b) a bispecific antibody targeting PD-1 and LAG3 comprising:

(i) a first antigen-binding domain that specifically binds to PD-1 , comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 1 , an HVR-H2 sequence comprising the amino acid sequence GGR, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; an HVR-L2 sequence comprising the amino acid sequence RSS, and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 4; and

(ii) a second antigen-binding domain that specifically binds to LAG3, comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

146. A bispecific antibody targeting PD-1 and LAG3 and/or an anti-TIGIT antagonist antibody for use in a method of treating a subject having a cancer (e.g., a locally advanced or metastatic urothelial bladder cancer), wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of:

(1)

(a) the anti-TIGIT antagonist antibody; and

(b) the bispecific antibody;

Wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy; or

(2)

(a) an anti-TIGIT antagonist antibody comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 38; an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 39; an HVR-H3 sequence comprising the amino acid sequence of SEQ ID NO: 40; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 41 ; an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 42; and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 43; and

(b) a bispecific antibody targeting PD-1 and LAG3 comprising:

(i) a first antigen-binding domain that specifically binds to PD-1 , comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 1 , an HVR-H2 sequence comprising the amino acid sequence GGR, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3; an HVR-L2 sequence comprising the amino acid sequence RSS, and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 4; and

(ii) a second antigen-binding domain that specifically binds to LAG3, comprising the following HVRs: an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10, an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

147. The bispecific antibody for use of paragraph 145 or 146, wherein the anti-TIGIT antagonist antibody is tiragolumab.

148. The bispecific antibody for use of any one of paragraphs 75-147, wherein the subject is a human. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.

Claims

WHAT IS CLAIMED IS:

1 . A method for treating a subject having an urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising a first antigen-binding domain that specifically binds to programmed cell death protein 1 (PD- 1) and a second antigen-binding domain that specifically binds to lymphocyte activation gene 3 (LAG3), wherein:

(i) the subject is ineligible for platinum-based chemotherapy; or

(ii) the subject has not previously been treated with a cancer immunotherapy.

2. The method of claim 1 , wherein the bispecific antibody is administered as a monotherapy.

3. A method for treating a subject having an urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of (a) an anti-TIGIT antagonist antibody; and (b) a bispecific antibody targeting PD-1 and LAG3 comprising a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, wherein:

(i) the subject is ineligible for platinum-based chemotherapy; or

(ii) the subject has not previously been treated with a cancer immunotherapy.

4. The method of any one of claims 1-3, wherein the subject is ineligible for platinum-based chemotherapy.

5. The method of any one of claims 1-3, wherein the subject has not previously been treated with a cancer immunotherapy.

6. The method of any one of claims 1-5, wherein the subject is ineligible for platinum-based chemotherapy and has not previously been treated with a cancer immunotherapy.

7. The method of any one of claims 3-6, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody at a fixed dose of 600 mg every three weeks.

8. The method of any one of claims 1-7, wherein the method comprises administering to the subject the bispecific antibody at a fixed dose of 600 mg every three weeks.

9. The method of any one of claims 1 -8, wherein the length of each of the one or more dosing cycles is 21 days.

10. The method of any one of claims 1-9, wherein the method comprises administering to the subject the bispecific antibody on Day 1 of each of the one or more dosing cycles.

11 . The method of any one of claims 3-10, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody on Day 1 of each of the one or more dosing cycles.

12. The method of any one of claims 3-11 , wherein the method comprises administering to the subject the bispecific antibody before the anti-TIGIT antagonist antibody.

13. The method of claim 12, wherein the anti-TIGIT antagonist antibody is administered at least 60 minutes after the bispecific antibody in a first dosing cycle.

14. The method of claim 12 or 13, wherein the method comprises one or more additional dosing cycles, and the anti-TIGIT antagonist antibody is administered at least 30 minutes after the bispecific antibody in the one or more additional dosing cycles.

15. The method of any one of claims 1-14, wherein the method comprises administering to the subject the bispecific antibody intravenously.

16. The method of any one of claims 3-15, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody intravenously.

17. The method of any one of claims 1-16, wherein the urothelial bladder cancer is locally advanced or metastatic.

18. The method of claim 17, wherein:

(a) the clinical stage of the locally advanced urothelial bladder cancer is (i) T4b, any N or (ii) any

T, N2-3; or

(b) the clinical stage of the metastatic urothelial bladder cancer is M1 and/or Stage IV.

19. The method of any one of claims 1-18, wherein the urothelial bladder cancer is a carcinoma.

20. The method of any one of claims 1-19, wherein the subject is ineligible for platinum-based chemotherapy based on having:

(a) an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and < 30 mL/min/1.73 m²;

(b) an ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1 .73 m² and < 45 mL/min/1 .73 m²; or

(c) an ECOG Performance Status of 0-2 with Grade > 2 neuropathy.

21. The method of claim 20, wherein the GFR is assessed by calculation through use of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

22. The method of any one of claims 1 -21 , wherein the subject has not previously been treated with a chemotherapy for locally advanced, metastatic, or recurrent urothelial bladder cancer.

23. The method of any one of claims 1-22, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds to PD-1 comprising a heavy chain variable (VH) domain comprising:

(i) a hypervariable region H1 (HVR-H1) sequence comprising the amino acid sequence of SEQ ID NO: 1 ,

(ii) an HVR-H2 sequence comprising the amino acid sequence GGR, and

(iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; and a light chain variable (VL) domain comprising

(i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3;

(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: 4.

24. The method of claim 23, wherein the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG.

25. The method of claim 24, wherein the IgG Fc domain is an IgG 1 Fc domain or an lgG4 Fc domain.

26. The method of claim 24 or 25, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor.

27. The method of claim 26, wherein the Fc receptor is an Fey receptor.

28. The method of any one of claims 23-27, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a second antigen-binding domain that specifically binds to LAG-3 comprising a VH domain comprising:

(i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7,

(ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and

(iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; and a VL domain comprising:

(i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10,

(ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and

(iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12.

29. The method of any one of claims 23-28, wherein the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14.

30. The method of any one of claims 23-29, wherein the bispecific antibody targeting PD-1 and LAG3 comprises:

(a) an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index); and/or

(b) an Fc domain comprising a modification promoting the association of the first and second subunit of the Fc domain.

31 . The method of any one of claims 24-30, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index).

32. The method of any one of claims 23-31 , wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first Fab fragment comprising said first antigen binding domain specifically binding to PD1 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and a second Fab fragment comprising the second antigen binding domain specifically binding to LAG3 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, and an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index).

33. The method of claim 32, wherein in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3 the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

34. The method of claim 33, wherein in the first Fab fragment the variable domains VL and VH are replaced by each other.

35. The method of any one of claims 32-34, wherein in the constant domain CL of one of the Fab fragments the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

36. The method of claim 35, wherein in the constant domain CL of the second Fab fragment the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

37. The method of any one of claims 23-36, wherein the bispecific antibody comprises a first heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15, a first light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16, a second heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17, and a second light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18.

38. The method of claim 37, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18.

39. The method of any one of claims 3-38, wherein the anti-TIGIT antagonist antibody comprises the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 38); an HVR-H2 sequence comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 39); an HVR-H3 sequence comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 40); an HVR-L1 sequence comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 41); an HVR-L2 sequence comprising the amino acid sequence of WASTRES (SEQ ID NO: 42); and an HVR-L3 sequence comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 43).

40. The method of any one of claims 3-39, wherein the anti-TIGIT antagonist antibody comprises:

(a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44 or 45;

(b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46; or

(c) a VH domain as in (a) and a VL domain as in (b).

41 . The method of any one of claims 3-40, wherein the anti-TIGIT antagonist antibody is a monoclonal antibody, a human antibody, or a full-length antibody.

42. The method of any one of claims 3-41 , wherein the anti-TIGIT antagonist antibody is a human, monoclonal full-length IgG 1 subclass antibody comprising a human lgG1 Fc region, a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 44, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 46.

43. The method of any one of claims 3-42, wherein the anti-TIGIT antagonist antibody exhibits effector function.

44. The method of any one of claims 3-43, wherein the anti-TIGIT antagonist antibody is tiragolumab.

45. The method of any one of claims 3-41 , wherein the anti-TIGIT antagonist antibody is an antibody fragment that binds TIGIT selected from the group consisting of Fab, Fab’, Fab’-SH, Fv, single chain variable fragment (scFv), and (Fab’)2 fragments.

46. The method of any one of claims 3-44, wherein the anti-TIGIT antagonist antibody is an IgG class antibody.

47. The method of claim 46, wherein the IgG class antibody is an lgG1 subclass antibody.

48. The method of any one of claims 1-47, wherein a PD-L1 expression level of a tumor sample obtained from the subject has been determined.

49. The method of claim 48, wherein the PD-L1 expression level is a protein expression level, and the protein expression level has been determined by an immunohistochemical (IHC) assay comprising staining with an anti-PD-L1 antibody suitable for staining.

50. The method of claim 49, wherein the anti-PD-L1 antibody suitable for staining is the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8.

51 . The method of claim 49 or 50, wherein the protein expression level of PD-L1 is determined using a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, a Ventana SP142 IHC assay, or a pharmDx 28-8 IHC assay.

52. The method of any one of claims 48-51 , wherein the tumor sample obtained from the subject has been determined to be PD-L1 -positive.

53. The method of any one of claims 48-51 , wherein the tumor sample obtained from the subject has been determined to be PD-L1 -negative.

54. The method of any one of claims 1-53, wherein the method results in an increase in objective response rate (ORR) in a population of subjects treated according to the method as compared to a reference ORR.

55. The method of claim 54, wherein the reference ORR is an ORR of a population of subjects who have received a control therapy.

56. The method of any one of claims 1-55, wherein the method results in an increase in progression- free survival (PFS) as compared to a reference PFS.

57. The method of claim 56, wherein the reference PFS is a PFS of a population of subjects who have received a control therapy.

58. The method of any one of claims 1-57, wherein the method results in an increase in overall survival (OS) as compared to a reference OS.

59. The method of claim 58, wherein the reference OS is an OS of a population of subjects who have received a control therapy.

60. The method of any one of claims 1 -59, wherein the method results in an increase in duration of response (DOR) as compared to a reference DOR.

61 . The method of claim 60, wherein the reference DOR is a DOR of a population of subjects who have received a control therapy.

62. The method of any one of claims 1 -61 , wherein the method results in:

(c) an increase in PFS rate at 6 months as compared to a reference PFS rate at 6 months; or

(d) an increase in PFS rate at 12 months as compared to a reference PFS rate at 12 months.

63. The method of claim 62, wherein the reference PFS rate is a PFS rate of a population of subjects who have received a control therapy.

64. The method of any one of claims 1-63, wherein the method results in:

(d) an increase in OS rate at 6 months as compared to a reference OS rate at 6 months;

(e) an increase in OS rate at 12 months as compared to a reference OS rate at 12 months; or

(f) an increase in OS rate at 18 months as compared to a reference OS rate at 18 months.

65. The method of claim 64, wherein the reference OS rate is an OS rate of a population of subjects who have received a control therapy.

66. The method of any one of claims 1 -65, wherein the method results in an increase in disease control rate (DCR) as compared to a reference DCR.

67. The method of claim 66, wherein the reference DCR is a DCR of a population of subjects who have received a control therapy.

68. The method of any one of claims 54-67, wherein the control therapy is a treatment comprising atezolizumab, and the control therapy does not comprise the bispecific antibody or an anti-TIGIT antagonist antibody.

69. A method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

70. A method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of a bispecific antibody targeting PD-1 and LAG3 comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

71 . A method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of:

(a) an anti-TIGIT antagonist antibody; and

(b) a bispecific antibody targeting PD-1 and LAG3 comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

72. A method for treating a subject having a locally advanced or metastatic urothelial bladder cancer, the method comprising administering to the subject one or more dosing cycles of:

(a) an anti-TIGIT antagonist antibody; and

(b) a bispecific antibody targeting PD-1 and LAG3 comprising: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

73. The method of claim 71 or 72, wherein the anti-TIGIT antagonist antibody is tiragolumab.

74. The method of any one of claims 1-73, wherein the subject is a human.

75. A bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having an urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to PD-1 and a second antigen-binding domain that specifically binds to LAG3, and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody, wherein:

(i) the subject is ineligible for platinum-based chemotherapy; or

(ii) the subject has not previously been treated with a cancer immunotherapy.

76. The bispecific antibody for use of claim 75, wherein the bispecific antibody is to be administered as a monotherapy.

77. A bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having an urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, 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 dosing regimen comprises administering to the subject one or more dosing cycles of (a) the anti-TIGIT antagonist antibody; and (b) the bispecific antibody, wherein:

(i) the subject is ineligible for platinum-based chemotherapy; or

(ii) the subject has not previously been treated with a cancer immunotherapy.

78. The bispecific antibody for use of any one of claims 75-77, wherein the subject is ineligible for platinum-based chemotherapy.

79. The bispecific antibody for use of any one of claims 75-77, wherein the subject has not previously been treated with a cancer immunotherapy.

80. The bispecific antibody for use of any one of claims 75-79, wherein the subject is ineligible for platinum-based chemotherapy and has not previously been treated with a cancer immunotherapy.

81 . The bispecific antibody for use of any one of claims 77-80, wherein the method comprises administering to the subject the anti-TIG IT antagonist antibody at a fixed dose of 600 mg every three weeks.

82. The bispecific antibody for use of any one of claims 75-81 , wherein the method comprises administering to the subject the bispecific antibody at a fixed dose of 600 mg every three weeks.

83. The bispecific antibody for use of any one of claims 75-82, wherein the length of each of the one or more dosing cycles is 21 days.

84. The bispecific antibody for use of any one of claims 75-83, wherein the method comprises administering to the subject the bispecific antibody on Day 1 of each of the one or more dosing cycles.

85. The bispecific antibody for use of any one of claims 77-84, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody on Day 1 of each of the one or more dosing cycles.

86. The bispecific antibody for use of any one of claims 77-85, wherein the method comprises administering to the subject the bispecific antibody before the anti-TIGIT antagonist antibody.

87. The bispecific antibody for use of claim 86, wherein the anti-TIGIT antagonist antibody is to be administered at least 60 minutes after the bispecific antibody in a first dosing cycle.

88. The bispecific antibody for use of claim 86 or 87, wherein the method comprises one or more additional dosing cycles, and the anti-TIGIT antagonist antibody is to be administered at least 30 minutes after the bispecific antibody in the one or more additional dosing cycles.

89. The bispecific antibody for use of any one of claims 75-88, wherein the method comprises administering to the subject the bispecific antibody intravenously.

90. The bispecific antibody for use of any one of claims 77-89, wherein the method comprises administering to the subject the anti-TIGIT antagonist antibody intravenously.

91 . The bispecific antibody for use of any one of claims 75-90, wherein the urothelial bladder cancer is locally advanced or metastatic.

92. The bispecific antibody for use of claim 91 , wherein: (a) the clinical stage of the locally advanced urothelial bladder cancer is (i) T4b, any N or (ii) any

T, N2-3; or

(b) the clinical stage of the metastatic urothelial bladder cancer is M1 and/or Stage IV.

93. The bispecific antibody for use of any one of claims 75-92, wherein the urothelial bladder cancer is a carcinoma.

94. The bispecific antibody for use of any one of claims 75-93, wherein the subject is ineligible for platinum-based chemotherapy based on having:

(a) an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 with baseline glomerular filtration rate (GFR) > 15 mL/min/1 .73 m² and < 30 mL/min/1 .73 m²;

(b) an ECOG Performance Status of 1 or 2 with baseline GFR > 15 mL/min/1 .73 m² and < 45 mL/min/1 .73 m²; or

(c) an ECOG Performance Status of 0-2 with Grade > 2 neuropathy.

95. The bispecific antibody for use of claim 94, wherein the GFR is assessed by calculation through use of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.

96. The bispecific antibody for use of any one of claims 75-95, wherein the subject has not previously been treated with a chemotherapy for locally advanced, metastatic, or recurrent urothelial bladder cancer.

97. The bispecific antibody for use of any one of claims 75-96, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first antigen-binding domain that specifically binds to PD-1 comprising a heavy chain variable (VH) domain comprising:

(i) a hypervariable region H1 (HVR-H1) sequence comprising the amino acid sequence of SEQ ID NO: 1 ,

(ii) an HVR-H2 sequence comprising the amino acid sequence GGR, and

(iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 2; and a light chain variable (VL) domain comprising

(i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 3;

(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: 4.

98. The bispecific antibody for use of claim 97, wherein the bispecific antibody targeting PD-1 and LAG3 comprises an Fc domain that is an IgG.

99. The bispecific antibody for use of claim 98, wherein the IgG Fc domain is an IgG 1 Fc domain or an lgG4 Fc domain.

100. The bispecific antibody for use of claim 98 or 99, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor.

101. The bispecific antibody for use of claim 100, wherein the Fc receptor is an Fey receptor.

102. The bispecific antibody for use of any one of claims 97-101 , wherein the bispecific antibody targeting PD-1 and LAG3 comprises a second antigen-binding domain that specifically binds to LAG-3 comprising a VH domain comprising:

(i) an HVR-H1 sequence comprising the amino acid sequence of SEQ ID NO: 7,

(ii) an HVR-H2 sequence comprising the amino acid sequence of SEQ ID NO: 8, and

(iii) an HVR-H3 sequence comprising an amino acid sequence of SEQ ID NO: 9; and a VL domain comprising:

(i) an HVR-L1 sequence comprising the amino acid sequence of SEQ ID NO: 10,

(ii) an HVR-L2 sequence comprising the amino acid sequence of SEQ ID NO: 11 , and

(iii) an HVR-L3 sequence comprising the amino acid sequence of SEQ ID NO: 12.

103. The bispecific antibody for use of any one of claims 97-102, wherein the first antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14.

104. The bispecific antibody for use of any one of claims 97-103, wherein the bispecific antibody targeting PD-1 and LAG3 comprises:

(a) an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index); and/or

(b) an Fc domain comprising a modification promoting the association of the first and second subunit of the Fc domain.

105. The bispecific antibody for use of any one of claims 98-104, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index).

106. The bispecific antibody for use of any one of claims 97-105, wherein the bispecific antibody targeting PD-1 and LAG3 comprises a first Fab fragment comprising said first antigen binding domain specifically binding to PD1 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 5 and a VL domain comprising the amino acid sequence of SEQ ID NO: 6, and a second Fab fragment comprising the second antigen binding domain specifically binding to LAG3 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, and an Fc domain of human lgG1 subclass with the amino acid mutations L234A, L235A, and P329G (numbering according to Kabat EU index).

107. The bispecific antibody for use of claim 106, wherein in one of the Fab fragments of the bispecific antibody targeting PD-1 and LAG3 the variable domains VL and VH are replaced by each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

108. The bispecific antibody for use of claim 107, wherein in the first Fab fragment the variable domains VL and VH are replaced by each other.

109. The bispecific antibody for use of any one of claims 106-108, wherein in the constant domain CL of one of the Fab fragments the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

110. The bispecific antibody for use of claim 109, wherein in the constant domain CL of the second Fab fragment the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (numbering according to Kabat EU Index), and in the constant domain CH1 the amino acids at positions 147 and 213 are substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

111. The bispecific antibody for use of any one of claims 97-110, wherein the bispecific antibody comprises a first heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15, a first light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16, a second heavy chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17, and a second light chain comprising an amino acid sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18.

112. The bispecific antibody for use of claim 1 11 , wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18.

113. The bispecific antibody for use of any one of claims 77-112, wherein the anti-TIGIT antagonist antibody comprises the following hypervariable regions (HVRs): an HVR-H1 sequence comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 38); an HVR-H2 sequence comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 39); an HVR-H3 sequence comprising the amino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 40); an HVR-L1 sequence comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 41); an HVR-L2 sequence comprising the amino acid sequence of WASTRES (SEQ ID NO: 42); and an HVR-L3 sequence comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 43).

114. The bispecific antibody for use of any one of claims 77-113, wherein the anti-TIGIT antagonist antibody comprises:

(a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44 or 45;

(b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46; or

(c) a VH domain as in (a) and a VL domain as in (b).

115. The bispecific antibody for use of any one of claims 77-114, wherein the anti-TIGIT antagonist antibody is a monoclonal antibody, a human antibody, or a full-length antibody.

116. The bispecific antibody for use of any one of claims 77-115, wherein the anti-TIGIT antagonist antibody is a human, monoclonal full-length lgG1 subclass antibody comprising a human IgG 1 Fc region, a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 44, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 46.

117. The bispecific antibody for use of any one of claims 77-116, wherein the anti-TIGIT antagonist antibody exhibits effector function.

118. The bispecific antibody for use of any one of claims 77-117, wherein the anti-TIGIT antagonist antibody is tiragolumab.

119. The bispecific antibody for use of any one of claims 77-115, wherein the anti-TIGIT antagonist antibody is an antibody fragment that binds TIGIT selected from the group consisting of Fab, Fab’, Fab’- SH, Fv, single chain variable fragment (scFv), and (Fab’)2 fragments.

120. The bispecific antibody for use of any one of claims 77-119, wherein the anti-TIGIT antagonist antibody is an IgG class antibody.

121. The bispecific antibody for use of claim 120, wherein the IgG class antibody is an lgG1 subclass antibody.

122. The bispecific antibody for use of any one of claims 75-121 , wherein a PD-L1 expression level of a tumor sample obtained from the subject has been determined.

123. The bispecific antibody for use of claim 122, wherein the PD-L1 expression level is a protein expression level, and the protein expression level has been determined by an immunohistochemical (IHC) assay comprising staining with an anti-PD-L1 antibody suitable for staining.

124. The bispecific antibody for use of claim 123, wherein the anti-PD-L1 antibody suitable for staining is the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8.

125. The bispecific antibody for use of claim 123 or 124, wherein the protein expression level of PD- L1 is determined using a Ventana SP263 IHC assay, a pharmDx 22C3 IHC assay, a Ventana SP142 IHC assay, or a pharmDx 28-8 IHC assay.

126. The bispecific antibody for use of any one of claims 122-125, wherein the tumor sample obtained from the subject has been determined to be PD-L1 -positive.

127. The bispecific antibody for use of any one of claims 122-125, wherein the tumor sample obtained from the subject has been determined to be PD-L1 -negative.

128. The bispecific antibody for use of any one of claims 75-127, wherein the method results in an increase in objective response rate (ORR) in a population of subjects treated according to the method as compared to a reference ORR.

129. The bispecific antibody for use of claim 128, wherein the reference ORR is an ORR of a population of subjects who have received a control therapy.

130. The bispecific antibody for use of any one of claims 75-129, wherein the method results in an increase in progression-free survival (PFS) as compared to a reference PFS.

131. The bispecific antibody for use of claim 130, wherein the reference PFS is a PFS of a population of subjects who have received a control therapy.

132. The bispecific antibody for use of any one of claims 75-131 , wherein the method results in an increase in overall survival (OS) as compared to a reference OS.

133. The bispecific antibody for use of claim 132, wherein the reference OS is an OS of a population of subjects who have received a control therapy.

134. The bispecific antibody for use of any one of claims 75-133, wherein the method results in an increase in duration of response (DOR) as compared to a reference DOR.

135. The bispecific antibody for use of claim 134, wherein the reference DOR is a DOR of a population of subjects who have received a control therapy.

136. The bispecific antibody for use of any one of claims 75-135, wherein the method results in:

(c) an increase in PFS rate at 6 months as compared to a reference PFS rate at 6 months; or

(d) an increase in PFS rate at 12 months as compared to a reference PFS rate at 1 months.

137. The bispecific antibody for use of claim 136, wherein the reference PFS rate is a PFS rate of a population of subjects who have received a control therapy.

138. The bispecific antibody for use of any one of claims 75-137, wherein the method results in:

(d) an increase in OS rate at 6 months as compared to a reference OS rate at 6 months;

(e) an increase in OS rate at 12 months as compared to a reference OS rate at 12 months; or

(f) an increase in OS rate at 18 months as compared to a reference OS rate at 18 months.

139. The bispecific antibody for use of claim 138, wherein the reference OS rate is an OS rate of a population of subjects who have received a control therapy.

140. The bispecific antibody for use of any one of claims 75-139, wherein the method results in an increase in disease control rate (DCR) as compared to a reference DCR.

141. The bispecific antibody for use of claim 140, wherein the reference DCR is a DCR of a population of subjects who have received a control therapy.

142. The bispecific antibody for use of any one of claims 129-141 , wherein the control therapy is a treatment comprising atezolizumab, and the control therapy does not comprise the bispecific antibody or an anti-TIGIT antagonist antibody.

143. A bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a locally advanced or metastatic urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody; the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and the subject is ineligible for platinum-based chemotherapy.

144. A bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a locally advanced or metastatic urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; wherein the dosing regimen comprises administering to the subject one or more dosing cycles of the bispecific antibody; the bispecific antibody is administered at a fixed dose of 600 mg every three weeks, and the subject has not previously been treated with a cancer immunotherapy.

145. A bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a locally advanced or metastatic urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of:

(a) the anti-TIGIT antagonist antibody; and

(b) the bispecific antibody; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject is ineligible for platinum-based chemotherapy.

146. A bispecific antibody targeting PD-1 and LAG3 for use in a method of treating a subject having a locally advanced or metastatic urothelial bladder cancer, wherein the method comprises a dosing regimen comprising a bispecific antibody targeting PD-1 and LAG3 and an anti-TIGIT antagonist antibody, wherein the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a first light chain comprising the amino acid sequence of SEQ ID NO: 16, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a second light chain comprising the amino acid sequence of SEQ ID NO: 18; and wherein the dosing regimen comprises administering to the subject one or more dosing cycles of:

(a) the anti-TIGIT antagonist antibody; and

(b) the bispecific antibody; wherein the anti-TIGIT antagonist antibody and the bispecific antibody are each administered at a fixed dose of 600 mg every three weeks, and wherein the subject has not previously been treated with a cancer immunotherapy.

147. The bispecific antibody for use of claim 145 or 146, wherein the anti-TIGIT antagonist antibody is tiragolumab.

148. The bispecific antibody for use of any one of claims 75-147, wherein the subject is a human.