# US 11,306,154 B2 · Methods of treating cancer by administering antigen-binding molecules comprising a TNF family ligand trimer

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- Number: US 11,306,154 B2
- Document ID: US11306154B2
- Status: Granted
- Kind: B2
- Application number: 16/522,391
- Filed: 2019-07-25
- Published: 2020-08-06
- Granted: 2022-04-19
- Earliest claimed: 2014-11-14
- Assignees: Hoffmann-La Roche Inc.
- Applicants: Hoffmann-La Roche Inc.
- Inventors: Maria Amann; Peter Bruenker; Christina Claus; Claudia Ferrara Koller; Sandra Grau-Richards; Christian Klein; Viktor Levitski; Ekkehard Moessner; Joerg Thomas Regula; Pablo Umana
- Web page: https://patentgrep.com/patent/US11306154B2

## CPC

- C07K 16/40: against enzymes
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61P 35/00: Antineoplastic agents
- C07K 14/70575: {NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525)}
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2815: {against CD8}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C07K 16/3007: {Carcino-embryonic Antigens}
- C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
- C07K 2317/35: Valency
- C07K 2317/522: CH1 domain
- C07K 2317/524: CH2 domain
- C07K 2317/526: CH3 domain
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/60: characterized by non-natural combinations of immunoglobulin fragments
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The invention relates to novel TNF family ligand trimer-containing antigen binding molecules comprising (a) at least one moiety capable of specific binding to a target cell antigen and (b) a first and a second polypeptide that are linked to each other by a disulfide bond, characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof.

## Claims

### Claim 1

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising:
(a) at least one antigen-binding domain comprising a light chain variable region (VL) and a heavy chain variable region (VH) capable of specific binding to a target cell antigen, wherein the target cell antigen is Fibroblast Activation Protein (FAP),
(b) a first polypeptide and a second polypeptide that are linked to each other by a disulfide bond, wherein the first polypeptide comprises two ectodomains of a TNF family ligand, or fragments thereof, that are connected to each other by a peptide linker and the second polypeptide comprises only one ectodomain of the TNF family ligand, or fragment thereof,
wherein the ectodomain of the TNF family ligand, or fragment thereof, comprises the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:96, SEQ ID NO: 373, SEQ ID NO:374, and SEQ ID NO:375, and
(c) an Fc domain composed of a first subunit and a second subunit, wherein the first subunit and the second subunit are capable of stable association with each other,
wherein the TNF family ligand is 4-1BBL, and wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 3 (depends on 1)

The method of claim 1, wherein
(i) the first polypeptide comprises a heavy chain constant 1 (CH1) domain or a light chain constant (CL) domain and the second polypeptide comprises a CL domain or a CH1 domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 domain and the CL domain, wherein the first polypeptide comprises two ectodomains of the TNF family ligand, or fragments thereof, that are connected to each other and to the CH1 domain or the CL domain of the first polypeptide by a peptide linker, and wherein the second polypeptide comprises only one ectodomain of the TNF family ligand, or fragment thereof, connected to the CL domain or the CH1 domain of the second polypeptide by a peptide linker,
(ii) the first polypeptide comprises a heavy chain constant 3 (CH3) domain and the second polypeptide comprises a CH3 domain, wherein the first polypeptide comprises two ectodomains of the TNF family ligand, or fragments thereof, that are connected to each other and to the C-terminus of the CH3 domain of the first polypeptide by a peptide linker, and wherein the second polypeptide comprises only one ectodomain of the TNF family ligand, or fragment thereof, connected to C-terminus of the CH3 domain of the second polypeptide by a peptide linker, or
(iii) the first polypeptide comprises a heavy chain variable region-light chain constant domain (VH-CL) or a light chain variable region-heavy chain constant 1 domain (VL-CH1) and the second polypeptide comprises a VL-CH1 domain or a VH-CL domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 domain and the CL domain, wherein the first polypeptide comprises two ectodomains of the TNF family ligand, or fragments thereof, that are connected to each other and to the VH or the VL of the first polypeptide by a peptide linker, and wherein the second polypeptide comprises only one ectodomain of the TNF family ligand, or fragment thereof, connected to the VL or the VH of the second polypeptide by a peptide linker.

### Claim 4 (depends on 1)

The method of claim 1, wherein the TNF family ligand costimulates human T-cell activation.

### Claim 5 (depends on 1)

The method of claim 1, wherein the ectodomain of the TNF family ligand, or fragment thereof, comprises the amino acid sequence of SEQ ID NO:1.

### Claim 6 (depends on 1)

The method of claim 1, wherein the ectodomain of the TNF family ligand, or fragment thereof, comprises the amino acid sequence of SEQ ID NO:96.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99 and the second polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:96, SEQ ID NO:3, and SEQ ID NO:4.

### Claim 8 (depends on 1)

The method of claim 1, wherein the antigen-binding domain capable of specific binding to Fibroblast Activation Protein (FAP) is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, a single domain antibody, an aVH, and a scaffold antigen-binding protein.

### Claim 9 (depends on 1)

The method of claim 1, wherein the TNF family ligand trimer-containing antigen-binding molecule comprises one antigen-binding domain capable of specific binding to Fibroblast Activation Protein (FAP).

### Claim 10 (depends on 1)

The method of claim 1, wherein the antigen-binding domain capable of specific binding to Fibroblast Activation Protein (FAP) is a Fab molecule.

### Claim 11 (depends on 1)

The method of claim 1, wherein the Fc domain is an IgG domain.

### Claim 12 (depends on 1)

The method of claim 1, wherein the Fc domain is an IgG1 Fc domain comprising amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering) of the IgG heavy chain.

### Claim 13 (depends on 1)

The method of claim 1, wherein the Fc domain is an IgG1 Fc domain or an IgG4 Fc domain.

### Claim 14 (depends on 1)

The method of claim 1, wherein the TNF family ligand trimer-containing antigen-binding molecule activates the NFκB signaling pathway.

### Claim 15 (depends on 1)

The method of claim 1, wherein the VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:100, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:101, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:102, and the VL comprises (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:103, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:104, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:105.

### Claim 16 (depends on 1)

The method of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO:16 and the VL comprises the amino acid sequence of SEQ ID NO:17 or wherein the VH comprises the amino acid sequence of SEQ ID NO:106 and the VL comprises the amino acid sequence of SEQ ID NO:107.

### Claim 17 (depends on 1)

The method of claim 1, the first polypeptide comprises a CH3 domain and the second polypeptide comprises a CH3 domain, wherein the first polypeptide comprises two ectodomains of the TNF family ligand, or fragments thereof, that are connected to each other and to the C-terminus of the CH3 domain by a peptide linker, and wherein the second polypeptide comprises only one ectodomain of the TNF family ligand, or fragment thereof, connected to the C-terminus of the CH3 domain of the second polypeptide by a peptide linker.

### Claim 18 (depends on 17)

The method of claim 17, wherein the TNF family ligand trimer-containing antigen-binding molecule comprises two antigen-binding domains capable of specific binding to Fibroblast Activation Protein (FAP).

### Claim 19 (depends on 1)

The method of claim 1, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.

### Claim 20

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising:
(a) a first heavy chain and a first light chain, wherein the first heavy chain and the first light chain taken together comprise a Fab molecule capable of specific binding to a target cell antigen, wherein the target cell antigen is Fibroblast Activation Protein (FAP), and a first polypeptide comprising two ectodomains of a TNF family ligand or fragments thereof connected to each other by a first peptide linker and fused at its C-terminus by a second peptide linker to a second heavy chain, and a second polypeptide comprising only one ectodomain of the TNF family ligand or fragment thereof fused at its C-terminus by a third peptide linker to a second light chain, wherein the TNF family ligand is 4-1BBL, or
(b) a first heavy chain and a first light chain, wherein the first heavy chain and the first light chain taken together comprise a Fab molecule capable of specific binding to a target cell antigen, wherein the target cell antigen is FAP, and a first polypeptide comprising two ectodomains of a TNF family ligand or fragments thereof connected to each other by a first peptide linker and fused at its C-terminus by a second peptide linker to a second light chain, and a second polypeptide comprising only one ectodomain of the TNF family ligand or fragment thereof fused at its C-terminus by a third peptide linker to a second heavy chain, wherein the TNF family ligand is 4-1BBL,
wherein the ectodomain of the TNF family ligand, or fragment thereof, comprises the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:96, SEQ ID NO: 373, SEQ ID NO:374, and SEQ ID NO:375,
wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 21 (depends on 20)

The method of claim 20, wherein the TNF family ligand trimer-containing antigen-binding molecule activates the NFκB signaling pathway.

### Claim 22 (depends on 20)

The method of claim 20, wherein the first polypeptide comprising two ectodomains of the TNF family ligand, or fragments thereof, connected to each other by the first peptide linker is fused at its C-terminus by the second peptide linker to a CH1 domain that is part of a heavy chain, and the second polypeptide comprising only one ectodomain of the TNF family ligand, or fragment thereof, is fused at its C-terminus by the third peptide linker to a CL domain that is part of a light chain.

### Claim 23 (depends on 22)

The method of claim 22, wherein in the CL domain connected to the second polypeptide comprising only one ectodomain of the TNF family ligand, or fragment thereof, the amino acid at position 123 (light chain EU numbering) has been substituted by arginine (R) and the amino acid at position 124 (light chain EU numbering) has been substituted by lysine (K), and wherein in the CH1 domain connected to the first polypeptide comprising two ectodomains of the TNF family ligand, or fragments thereof, the amino acids at position 147 (heavy chain EU numbering) and at position 213 (heavy chain EU numbering) have been substituted by glutamic acid (E).

### Claim 24 (depends on 20)

The method of claim 20, wherein the first polypeptide comprising two ectodomains of the TNF family ligand, or fragments thereof, connected to each other by the first peptide linker is fused at its C-terminus by the second peptide linker to a CL domain that is part of a heavy chain, and the second polypeptide comprising only one ectodomain of the TNF family ligand, or fragment thereof, is fused at its C-terminus by the third peptide linker to a CH1 domain that is part of a light chain.

### Claim 25 (depends on 24)

The method of claim 24, wherein in the CL domain connected to the first polypeptide comprising two ectodomains of the TNF family ligand, or fragments thereof, the amino acid at position 123 (light chain EU numbering) has been substituted by arginine (R) and the amino acid at position 124 (light chain EU numbering) has been substituted by lysine (K), and wherein in the CH1 domain connected to the second polypeptide comprising only ectodomain of the TNF family ligand, or fragment thereof, the amino acids at position 147 (heavy chain EU numbering) and at position 213 (heavy chain EU numbering) have been substituted by glutamic acid (E).

### Claim 26 (depends on 20)

The method of claim 20, wherein the first polypeptide comprising two ectodomains of the TNF family ligand, or fragments thereof, connected to each other by the first peptide linker is fused at its C-terminus by the second peptide linker to a VH that is part of a heavy chain, and the second polypeptide comprising only one ectodomain of the TNF family ligand, or fragment thereof, is fused at its C-terminus by the third peptide linker to a VL that is part of a light chain.

### Claim 27 (depends on 20)

The method of claim 20, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 28 (depends on 20)

The method of claim 20, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.

### Claim 29

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising:
(a) a first heavy chain and a first light chain, wherein the first heavy chain and the first light chain taken together comprise a Fab molecule capable of specific binding to a target cell antigen, wherein the target cell antigen is Fibroblast Activation Protein (FAP),
(b) a second heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99; and
(c) a second light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:96, SEQ ID NO:3, and SEQ ID NO:4,
wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 30 (depends on 29)

The method of claim 29, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 31 (depends on 29)

The method of claim 29, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.

### Claim 32

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising:
(a) a first heavy chain comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:16 and a first light chain comprising a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:17 or a first heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO:106 and a first light chain comprising a VL comprising the amino acid sequence of SEQ ID NO:107;
(b) a second heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, and SEQ ID NO:173; and
(c) a second light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, and SEQ ID NO:174,
wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 33 (depends on 32)

The method of claim 32, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 34 (depends on 32)

The method of claim 32, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.

### Claim 35

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising:
(a) a first heavy chain comprising the amino acid sequence of SEQ ID NO:121, a second heavy chain comprising the amino acid sequence of SEQ ID NO:122, and two light chains each comprising the amino acid sequence of SEQ ID NO:19,
(b) a first heavy chain comprising the amino acid sequence of SEQ ID NO:123, a second heavy chain comprising the amino acid sequence of SEQ ID NO:124, and two light chains each comprising the amino acid sequence of SEQ ID NO:125, or
(c) a first heavy chain comprising the amino acid sequence of SEQ ID NO:126, a second heavy chain comprising the amino acid sequence of SEQ ID NO:127, and two light chains each comprising the amino acid sequence of SEQ ID NO:125,
wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 36 (depends on 35)

The method of claim 35, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 37 (depends on 35)

The method of claim 35, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.

### Claim 38

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising:
(a) an antigen-binding domain capable of specific binding to Fibroblast Activation Protein (FAP), comprising a heavy chain variable region (VH) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:100, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:101, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:102, and a light chain variable region (VL) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:103, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:104, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:105;
(b) a first polypeptide and a second polypeptide that are linked to each other by a disulfide bond, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:97, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:96, and
(c) an Fc domain composed of a first subunit and a second subunit, wherein the first subunit and the second subunit are capable of stable association with each other,
wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 39 (depends on 38)

The method of claim 38, wherein the Fc domain is an IgG1 Fc domain comprising amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering) of the IgG heavy chain.

### Claim 40 (depends on 38)

The method of claim 38, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 41 (depends on 38)

The method of claim 38, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.

### Claim 42

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising:
(a) an antigen-binding domain capable of specific binding to Fibroblast Activation Protein (FAP) comprising a first heavy chain comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:106, and a first light chain comprising a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:107,
(b) a second heavy chain comprising the amino acid sequence of SEQ ID NO:97,
(c) a second light chain comprising the amino acid sequence of SEQ ID NO:96, and
(d) an Fc domain composed of a first subunit and a second subunit, wherein the first subunit and the second subunit are capable of stable association with each other,
wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 43 (depends on 42)

The method of claim 42, wherein the Fc domain is an IgG1 Fc domain comprising amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering) of the IgG heavy chain.

### Claim 44 (depends on 42)

The method of claim 42, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 45 (depends on 42)

The method of claim 42, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.

### Claim 46

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising:
(a) an antigen-binding domain capable of specific binding to Fibroblast Activation Protein (FAP) comprising a first heavy chain comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:106, and a first light chain comprising a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:107,
(b) a second heavy chain comprising the amino acid sequence of SEQ ID NO:119,
(c) a second light chain comprising the amino acid sequence of SEQ ID NO:120, and
(d) an Fc domain composed of a first subunit and a second subunit, wherein the first subunit and the second subunit are capable of stable association with each other,
wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 47 (depends on 46)

The method of claim 46, wherein the Fc domain is an IgG1 Fc domain comprising amino acid substitutions at positions 234 and 235 (EU numbering) and/or 329 (EU numbering) of the IgG heavy chain.

### Claim 48 (depends on 46)

The method of claim 46, wherein in the CL domain of the second heavy chain, the amino acid at position 123 (light chain EU numbering) has been substituted by arginine (R) and the amino acid at position 124 (light chain EU numbering) has been substituted by lysine (K), and wherein in the CH1 domain of the second light chain, the amino acids at position 147 (heavy chain EU numbering) and at position 213 (heavy chain EU numbering) have been substituted by glutamic acid (E).

### Claim 49 (depends on 46)

The method of claim 46, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 50 (depends on 46)

The method of claim 46, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.

### Claim 51

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising:
(a) a first heavy chain comprising the amino acid sequence of SEQ ID NO:164, a first light chain comprising the amino acid sequence of SEQ ID NO:125, a second heavy chain comprising the amino acid sequence of SEQ ID NO:115, and a second light chain comprising the amino acid sequence of SEQ ID NO:116;
(b) a first heavy chain comprising the amino acid sequence of SEQ ID NO:164, a first light chain comprising the amino acid sequence of SEQ ID NO:125, a second heavy chain comprising the amino acid sequence of SEQ ID NO:117, and a second light chain comprising the amino acid sequence of SEQ ID NO:118;
(c) two light chains each comprising the amino acid sequence of SEQ ID NO:125, a first heavy chain comprising the amino acid sequence of SEQ ID NO:123, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:124;
(d) a first heavy chain comprising the amino acid sequence of SEQ ID NO:164, a first light chain comprising the amino acid sequence of SEQ ID NO:125, a second heavy chain comprising the amino acid sequence of SEQ ID NO:119, and a second light chain comprising the amino acid sequence of SEQ ID NO:120;
(e) a first heavy chain comprising the amino acid sequence of SEQ ID NO:164, a first light chain comprising the amino acid sequence of SEQ ID NO:125, a second heavy chain comprising the amino acid sequence of SEQ ID NO:173, and a second light chain comprising the amino acid sequence of SEQ ID NO:174; or
(f) two light chains each comprising the amino acid sequence of SEQ ID NO:125, a first heavy chain comprising the amino acid sequence of SEQ ID NO:126, and a second heavy chain comprising the amino acid sequence of SEQ ID NO:127,
wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 52 (depends on 51)

The method of claim 51, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 53 (depends on 51)

The method of claim 51, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.

### Claim 54

A method of treating a cancer in an individual, the method comprising administering to said individual a therapeutically effective amount of a composition comprising a TNF family ligand trimer-containing antigen-binding molecule, said molecule comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO:164, a first light chain comprising the amino acid sequence of SEQ ID NO:125, a second heavy chain comprising the amino acid sequence of SEQ ID NO:119, and a second light chain comprising the amino acid sequence of SEQ ID NO:120, wherein the composition comprising the TNF family ligand trimer-containing antigen-binding molecule is in a pharmaceutically acceptable form.

### Claim 55 (depends on 54)

The method of claim 54, wherein the cancer is a solid tumor, bladder cancer, renal cell carcinoma, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma, or acute lymphoblastic leukemia.

### Claim 56 (depends on 54)

The method of claim 54, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC), or mesothelioma.