Bispecific antigen binding molecules targeting ox40 and fap

Abstract

This invention relates to novel bispecific antigen-binding molecules comprising at least two antigen-binding domains that specifically bind to OX40 and a specific antigen-binding domain that specifically binds to fibroblast activation protein (FAP), and to methods for producing such molecules and methods for using them.

Description

[Technical Field]

[0001] This invention relates to novel bispecific antigen-binding molecules comprising at least two antigen-binding domains specifically binding to OX40 and an antigen-binding domain specifically binding to fibroblast activation protein (FAP), and an Fc domain, specifically comprising one or more amino acid-substituted Fc domains that reduce binding to Fc receptors and / or effector function. Other embodiments of the invention are methods for producing such molecules and methods for using such molecules. [Previous Technology]

[0002] Despite the emergence of several new agents offering survival benefits to patients, cancer remains one of the leading causes of death worldwide. Despite advances in treatment options, the prognosis for patients with advanced cancer remains poor. Many cancer indications have poor prognoses, and the management of most advanced solid tumors remains challenging due to high rates of tumor recurrence or distant metastasis. Patients with advanced solid tumors have clearly benefited from checkpoint inhibitor (CPI) therapy. Even after treatment interruption, it can prolong overall survival in a subgroup of patients, possibly through the generation of a memory immune response. While this therapy has transformed the treatment landscape for many cancer types, unfortunately, 60-80% of patients with metastatic disease do not derive long-term benefit from this type of cancer immunotherapy. Therefore, there is an urgent medical need to continuously develop new and optimal therapies that can be added to existing treatments to prolong the survival of cancer patients without causing unacceptable toxicity.

[0003] The immunosuppressive microenvironment in some tumors exhibits high levels of co-inhibitory signals, such as PD-L1, but lacks adequate expression of the OX40 ligand. OX40 (CD134; TNFRSF4) is a member of the tumor necrosis factor (TNF) receptor superfamily, and is transiently expressed by T cells after binding to the T cell receptor (TCR). OX40 binding bidirectionally regulates the interaction between T cells and OX40L+ antigen-presenting cells (e.g., B cells, dendritic cells (DCs), monocytes). In the case of TCR binding, OX40 primarily provides co-stimulatory signals to CD4+ effector T cells and also to CD8+ effector T cells, thereby enhancing proliferation, survival, and effector functions (e.g., cytokine secretion). Conversely, OX40 signaling leads to the suppression and loss of regulatory T cell function. OX40 agonism counteracts TGF-β effects (e.g., inhibits FoxP3 induction) and reduces IL-10 secretion. In murine tumor models, agonist anti-OX40 antibodies conjugated to OX40 can promote anti-tumor T-cell responses, tumor shrinkage, and reproducible distant effects. Monotherapy with OX40 agonists typically has low efficacy, but potent anti-tumor effects are achieved when combined with immunogenic therapies (chemotherapy, radiation, and vaccination), checkpoint inhibitors (PD-1, CTLA-4), and other co-stimulatory agonists (such as 4-1BB, ICOS, or GITR).

[0004] Fibroblast activation protein-α (FAP) is a serine protease that is highly expressed on the cell surface of cancer-associated stromal cells in >90% of human epithelial malignancies, on reticular fibroblasts located in the T-cell promoter region of lymph nodes, and can also be found on activated fibroblasts in normal tissues. The high prevalence of FAP in various cancer indications makes it a potential target for drugs that accumulate in the tumor environment.

[0005] One approach to specifically restore OX40 co-stimulation in the tumor microenvironment is through bispecific antibodies comprising at least one antigen-binding domain targeting fibroblast activation protein (FAP) in the tumor stroma and at least one antigen-binding domain targeting OX40. For example, such bispecific antibodies have been described in WO 2017 / 055398 A2 and WO 2017 / 060144 A1. Cross-linking and surface immobilization of these bispecific molecules via cell surface FAP will generate a highly potentiating stroma against OX40-positive T cells, in which case it supports NFκB-mediated effector function and can substitute for OX40 ligand linkage. High FAP expression has been reported in numerous human oncology indications, both on tumor cells themselves and in immunosuppressive cancer-associated fibroblasts (CAF). Therefore, the industry needs improved OX40 bispecific antibodies targeting FAP that have excellent pharmacological properties, such as better shelf life, lower immunogenicity, and fewer nonspecific interactions, such as allergic reactions or uncontrolled cytokine release. [Summary of the Invention]

[0007] This invention relates to novel bispecific antigen-binding molecules with improved properties that specifically bind to OX40 and fibroblast activation protein (FAP), wherein OX40 co-stimulation is provided via cross-linking of FAP expressed on tumor stromal cells and potentially also via FAP expressed intermediately in secondary lymphoid tissues. Therefore, the antigen-binding molecules of this invention not only effectively trigger OX40 at the desired site but are also highly selective, while overcoming the need for FcγR cross-linking, thereby reducing side effects. These novel bispecific antigen-binding molecules are characterized by comprising a novel FAP antigen-binding domain fused to the C-terminus of the Fc domain and improved pharmacokinetic properties.

[0008] The bispecific antigen-binding molecules of the present invention combine at least two antigen-binding domains capable of specifically binding to the co-stimulatory TNF receptor family member OX40 with an antigen-binding domain targeting fibroblast activation protein (FAP), the FAP comprising the novel murine anti-human FAP pure line 212 and its humanized variants. These bispecific antigen-binding molecules are advantageous as OX40 agonists because they bind to FAP with high affinity, thus better activating co-stimulatory OX40 receptors near tumor sites expressing FAP. These molecules are further designed to have favorable pharmacokinetic characteristics to optimize the treatment regimen, thereby achieving an enhanced balance between safety and efficacy.

[0009] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) at least two antigen-binding domains specifically binding to OX40, and (b) an antigen-binding domain specifically binding to fibroblast activation protein (FAP), comprising: a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, and (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:5. (vi) the amino acid sequence of NO:7, and (vi) CDR-L3, which contains the amino acid sequence of SEQ ID NO:8; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0010] In one state, the Fc region contains one or more amino acid substitutions that reduce the binding affinity of the antibody to the Fc receptor and / or the effector function.

[0011] In another embodiment, a bispecific antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to the FAP comprises: a heavy chain variable region (VHFAP) containing an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9; and a light chain variable region (VLFAP) containing an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:10. In one embodiment, the antigen-binding domain capable of specifically binding to the FAP comprises: a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:9; and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:10.

[0012] In another state, the antigen-binding domain that can specifically bind to the FAP includes: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20; and a light chain variable region (VLFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. In one state, the antigen-binding domains that can specifically bind to FAP include (a) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:15 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:21, (b) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:16 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:21, (c) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:16 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:22, or (d) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:19 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:25. Specifically, the antigen-binding domain that can specifically bind to FAP includes (a) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:15 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:21.

[0013] In one state, the antigen-binding domain of OX40 can specifically bind to the amino acid sequence containing SEQ ID NO:1 or a polypeptide composed of therefrom.

[0014] In another state, a bispecific antigen-binding molecule is provided, wherein the antigen-binding domains capable of specifically binding to OX40 each comprise (a) a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:27, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:28, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:29; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:30, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:31, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:32, or (b) a heavy chain variable region (VHOX40) comprising (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:35, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:36, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:37; and a light chain variable region (VLOX40) comprising (iv) CDR-L1, comprising the amino acid sequence of SEQ ID NO:38, (v) CDR-L2, comprising the amino acid sequence of SEQ ID NO:39, and (vi) CDR-L3, comprising the amino acid sequence of SEQ ID NO:40, or (c) a heavy chain variable region (VHOX40) comprising (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:43, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:44, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:37. The amino acid sequence of SEQ ID NO:45; and the light chain variable region (VLOX40), which comprises (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:46, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:47, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:48, or (d) the heavy chain variable region (VHOX40), which comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:51, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:52, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:53;And a light chain variable region (VLOX40), which comprises (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:54, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:55, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:56.

[0015] In one state, the antigen-binding domains (each) capable of specifically binding to OX40 include a heavy chain variable region (VHOX40), which includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:27, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:28, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:29; and a light chain variable region (VLOX40), which includes (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:30, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:31, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:32. In another state, the antigen-binding domain that specifically binds to OX40 includes: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:35, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:36, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:37; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:38, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:39, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:40. In another state, the antigen-binding domain that specifically binds to OX40 includes: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:43, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:44, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:45; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:46, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:47, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:48.In another state, the antigen-binding domain that specifically binds to OX40 includes: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:51, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:52, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:53; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:54, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:55, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:56.

[0016] In one state, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domains that specifically bind to OX40 each comprise (i) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34, or (ii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:41 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:42, or (iii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:49 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:50, or (iv) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:57 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:57. The light chain variable region (VLOX40) of the amino acid sequence NO:58.

[0017] In one embodiment, a bispecific antigen-binding molecule as defined above is provided, wherein each antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34. In another embodiment, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:41 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:42. In another embodiment, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VHOX40) containing the amino acid sequence of SEQ ID NO:49 and a light chain variable region (VLOX40) containing the amino acid sequence of SEQ ID NO:50. In yet another embodiment, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VHOX40) containing the amino acid sequence of SEQ ID NO:57 and a light chain variable region (VLOX40) containing the amino acid sequence of SEQ ID NO:58.

[0018] In a specific state, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domains (each) capable of specifically binding to OX40 include a heavy chain variable region (VHOX40) containing the amino acid sequence of SEQ ID NO:41 and a light chain variable region (VLOX40) containing the amino acid sequence of SEQ ID NO:42.

[0019] In another embodiment, a bispecific antigen-binding molecule as defined above is provided, capable of specifically binding to the antigen-binding domain of OX40 comprising (i) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:59 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34, or (ii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:60 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34, or (iii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:61 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34.

[0020] In one state, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:59 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34. In another state, the antigen-binding domain specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:60 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34. In yet another state, the antigen-binding domain specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:61 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34.

[0021] In another embodiment, the bispecific antigen-binding molecule is a humanized or chimeric antibody, specifically a humanized antibody. In another embodiment, the bispecific antigen-binding molecule comprises an IgG Fc region, specifically an IgG1 Fc region or an IgG4 Fc region. Specifically, the Fc region comprises one or more amino acid substitutions that reduce the binding affinity of the antibody to the Fc receptor and / or effector function. In one particular embodiment, a bispecific antigen-binding molecule is provided, wherein the Fc region is a human IgG1 subclass Fc region with amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

[0022] In another state, a bispecific antigen-binding molecule as defined above is provided, wherein, according to the ridge-pore method, the first subunit of the Fc region contains a ridge and the second subunit of the Fc region contains a pore. Specifically, a bispecific antigen-binding molecule is provided wherein the first subunit of the Fc region contains amino acid substitutions S354C and T366W (numbered according to the Kabat EU index) and the second subunit of the Fc region contains amino acid substitutions Y349C, T366S, and Y407V (numbered according to the Kabat EU index). In yet another state, the Fc region is of murine origin, and the first subunit of the Fc region contains amino acid substitutions K392D and K409D (numbered according to the Kabat EU index) and the second subunit of the Fc region contains amino acid substitutions E356K and D399K (numbered according to the Kabat EU index). More specifically, a bispecific antigen-binding molecule is provided, wherein the first subunit of the Fc region contains amino acid substitutions S354C and T366W (numbered according to the Kabat EU index) and the second subunit of the Fc region contains amino acid substitutions Y349C, T366S and Y407V (numbered according to the Kabat EU index).

[0023] In another embodiment, a bispecific antigen-binding molecule is provided, wherein the bispecific antigen-binding molecule comprises (a) at least two Fab fragments capable of specifically binding to OX40, each linked to the N-terminus of a subunit of the Fc region, and (b) a cross-Fab fragment capable of specifically binding to FAP, fused to the C-terminus of a subunit of the Fc region, and (c) an Fc region composed of a first subunit and a second subunit capable of stable association.

[0024] Therefore, a bispecific antigen-binding molecule is provided, which provides bivalent binding to OX40 and monovalent binding to FAP (2+1 format). Specifically, the bispecific antigen-binding molecule comprises a cross-fab fragment capable of specifically binding to FAP, wherein the VH-Cκ chain of the cross-fab fragment capable of specifically binding to FAP is fused to the C-terminus of a subunit of the Fc region. In a particular state, the VH-Cκ chain of the cross-fab fragment capable of specifically binding to FAP is fused to the C-terminus of the first subunit of the Fc region containing amino acid substitutions S354C and T366W (numbered according to the Kabat EU index), i.e., fused to the bulge chain.

[0025] In one state, a bispecific antigen-binding molecule is provided, wherein the bispecific antigen-binding molecule comprises: (aa) a first Fab fragment capable of specifically binding to OX40, (ab) a second Fab fragment capable of specifically binding to OX40, (b) a cross-Fab fragment capable of specifically binding to FAP, which is fused to the C-terminus of a subunit of the Fc region, and (c) an Fc region composed of a first subunit and a second subunit capable of stable association, wherein the first Fab fragment (aa) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the first subunit and the second Fab fragment (ab) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the second subunit. Therefore, a bispecific antigen-binding molecule is provided that provides bivalent binding to OX40 and monovalent binding to FAP (2+1 format).

[0026] In another embodiment, a bispecific antigen-binding molecule is provided, wherein the bispecific antigen-binding molecule comprises: (aa) a first Fab fragment capable of specifically binding to OX40, (ab) a second Fab fragment capable of specifically binding to OX40, (ac) a third Fab fragment capable of specifically binding to OX40, (b) a cross-Fab fragment capable of specifically binding to FAP, which is fused to the C-terminus of a subunit of the Fc region, and (c) an Fc region composed of a first subunit and a second subunit capable of stable association, wherein the second Fab fragment (ab) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the first Fab fragment (aa), the VH-CH1 chain of the first Fab fragment (aa) is fused at its C-terminus to the N-terminus of the first subunit, and the third Fab fragment (ac) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit.

[0027] Therefore, a bispecific antigen-binding molecule is provided, which provides trivalent binding to OX40 and monovalent binding to FAP (3+1 format).

[0028] In one state, a bispecific antigen-binding molecule is provided, wherein the bispecific antigen-binding molecule comprises: (aa) a first Fab fragment capable of specifically binding to OX40, (ab) a second Fab fragment capable of specifically binding to OX40, (ac) a third Fab fragment capable of specifically binding to OX40, (b) a cross-Fab fragment capable of specifically binding to FAP, which is fused to the C-terminus of a subunit of the Fc region, and (c) an Fc region composed of a first subunit and a second subunit capable of stable association, wherein the second Fab fragment (ab) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the first Fab fragment (aa), and the VH-CH1 chain of the first Fab fragment (aa) further comprises amino acid substitutions Y349C, T366S and Y407V (according to Kabat) at its C-terminus and in the Fc region. The third Fab fragment (ac) is fused at the N-terminus of the second subunit (numbered according to the Kabat EU index) and at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit containing amino acid-substituted S354C and T366W (numbered according to the Kabat EU index). Specifically, the VH-Cκ chain capable of specifically binding to the cross-fab fragment of FAP is fused at the C-terminus of the first subunit containing amino acid-substituted S354C and T366W (numbered according to the Kabat EU index) in the Fc region.

[0029] In one state, a bispecific antigen-binding molecule is provided, comprising (a) two heavy chains, one heavy chain comprising two VH-CH1 chains, which may be linked to each other by a peptide linker, and an Fc subunit, which are capable of specifically binding to the Fab fragment of OX40, and the other heavy chain comprising a VH-CH1 chain and an Fc subunit, which are capable of specifically binding to the Fab fragment of OX40; (b) three light chains, each light chain comprising a VL and Cκ domains, which are capable of specifically binding to the Fab fragment of OX40; and (c) a cross-Fab fragment comprising a VL-CH1 light chain and a VH-Cκ chain, which are capable of specifically binding to FAP, wherein the VH-Cκ chain is optionally linked to the C-terminus of one of the two heavy chains in (a) by a peptide linker.

[0030] In one state sample, a bispecific antigen-binding molecule is provided, wherein the bispecific antigen-binding molecule comprises: (aa) specifically binding to a first Fab fragment of OX40, (ab) specifically binding to a second Fab fragment of OX40, (ac) specifically binding to a third Fab fragment of OX40, and (ad) specifically binding to a fourth Fab fragment of OX40. (b) a cross-Fab fragment capable of specifically binding to FAP, which fuses with the C-terminus of a subunit of the Fc region, and (c) an Fc region composed of a first subunit and a second subunit capable of stable association, wherein the second Fab fragment (ab) fuses with the N-terminus of the VH-CH1 chain of the first Fab fragment (aa) at the C-terminus of the VH-CH1 chain, the VH-CH1 chain of the first Fab fragment (aa) fuses with the N-terminus of the first subunit at its C-terminus, and the fourth Fab fragment (ad) fuses with the N-terminus of the VH-CH1 chain of the third Fab fragment (ac) at the C-terminus of the VH-CH1 chain, the VH-CH1 chain of the third Fab fragment (ac) fuses with the N-terminus of the second subunit at its C-terminus.

[0031] Therefore, a bispecific antigen-binding molecule is provided, which provides tetravalent binding to OX40 and monovalent binding to FAP (4+1 format). In a specific state, the VH-Cκ chain capable of specifically binding to the cross-fab fragment of FAP is fused to the C-terminus of the first subunit containing amino acid substitutions S354C and T366W (numbered according to the Kabat EU index) in the Fc region.

[0032] In one embodiment, a bispecific antigen-binding molecule is provided, wherein each of the two heavy chains comprises two VH-CH1 chains, which are optionally fused together by a peptide linker, capable of specifically binding to a Fab fragment of OX40. Therefore, in one embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two heavy chains, each heavy chain comprising two VH-CH1 chains, which are optionally linked together by a peptide linker, capable of specifically binding to a Fab fragment of OX40, and an Fc subunit; (b) four light chains, each light chain comprising VL and Cκ domains capable of specifically binding to a Fab fragment of OX40; and (c) a cross-Fab fragment comprising a VL-CH1 light chain and a VH-Cκ chain capable of specifically binding to FAP, wherein the VH-Cκ chain is optionally linked to the C-terminus of one of the two heavy chains in (a) by a peptide linker.

[0033] According to another aspect of the invention, an isolated nucleic acid is provided that encodes a bispecific antigen-binding molecule as described above. The invention further provides a vector, specifically an expression vector, comprising the isolated nucleic acid of the invention, and a host cell comprising the isolated nucleic acid or the expression vector of the invention. In some aspects, the host cell is a eukaryotic cell, specifically a mammalian cell. In another aspect, a method for generating a bispecific antigen-binding molecule as described above is provided, comprising culturing the host cell as described above under conditions suitable for expressing the bispecific antigen-binding molecule and isolating the bispecific antigen-binding molecule. The invention also covers bispecific antigen-binding molecules that specifically bind to OX40 and FAP generated by the method of the invention.

[0034] The present invention further provides a pharmaceutical composition comprising a bispecific antigen-binding molecule as described above and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises an additional therapeutic agent.

[0035] The present invention also covers bispecific antigen-binding molecules or antibodies as described above, or pharmaceutical compositions containing such bispecific antigen-binding molecules, which are used as pharmaceutical agents.

[0036] In one embodiment, a bispecific antigen-binding molecule as described above or a pharmaceutical composition of the present invention is provided for (i) inducing immune stimulation, (ii) stimulating tumor-specific T cell responses, (iii) inducing apoptosis of tumor cells, (iv) treating cancer, (v) delaying cancer progression, (vi) prolonging the survival of patients with cancer, and (vii) treating infections.

[0037] In one specific embodiment, a bispecific antigen-binding molecule or a pharmaceutical composition of the present invention as described above is provided for the treatment of cancer. In another specific embodiment, the present invention provides a bispecific antigen-binding molecule for the treatment of cancer as described above, wherein the bispecific antigen-binding molecule is for combined administration with a chemotherapy agent, radiation, and / or other agents for cancer immunotherapy. In one embodiment, a bispecific activating CD28 antigen-binding molecule or a pharmaceutical composition for the treatment of cancer is provided, wherein the bispecific activating CD28 antigen-binding molecule is for combined administration with a T-cell activating anti-CD3 bispecific antibody (e.g., an anti-CEA / anti-CD3 bispecific antibody). In another embodiment, the bispecific antigen-binding molecule, as described herein, is used to treat cancer, wherein the bispecific antigen-binding molecule is administered in combination with an agent that blocks PD-L1 / PD-1 interaction (such as a PD-L1 antibody, for example atezolizumab, or a PD-1 antibody, for example nivolumab or pembrolizumab). In yet another embodiment, the bispecific antigen-binding molecule as described above or the pharmaceutical composition of the present invention is provided for upregulating or prolonging cytotoxic T cell activity.

[0038] In another embodiment, the present invention provides a method for inhibiting the growth of tumor cells in an individual, comprising administering to the individual an effective amount of a bispecific antigen-binding molecule as described above or a pharmaceutical composition of the present invention to inhibit the growth of such tumor cells. In another embodiment, the present invention provides a method for treating or delaying cancer in an individual, comprising administering to the individual an effective amount of a bispecific antigen-binding molecule as described above or a pharmaceutical composition of the present invention.

[0039] Also provided are uses of the bispecific antigen-binding molecule as described above for manufacturing medicaments for treating diseases in individuals in need, particularly for manufacturing medicaments for treating cancer, and methods for treating individual diseases comprising administering to the individual a therapeutically effective amount of a composition containing the bispecific antigen-binding molecule of the present invention in a pharmaceutically acceptable form. In a particular embodiment, the disease is cancer. In any of the above embodiments, the system is mammal, specifically human.

Implementation Method

[0040] Definition

[0041] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa.

[0042] As used herein, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, antibody fragments, and scaffold antigen-binding proteins.

[0043] As used herein, the terms "antigen-binding domain capable of specifically binding to a target cell antigen" or "part of a protein capable of specifically binding to a target cell antigen" refer to a polypeptide molecule that specifically binds to an antigenic determinant. In one state, the antigen-binding domain can activate signal transduction via its target cell antigen. In a particular state, the antigen-binding domain can guide its attached entity (e.g., an OX40 activating antibody) to a target site, such as to a specific type of tumor cell or tumor stroma carrying an antigenic determinant. Antigen-binding domains capable of specifically binding to target cell antigens include antibodies and fragments thereof as further defined herein. Additionally, antigen-binding domains capable of specifically binding to target cell antigens include scaffold antigen-binding proteins as further defined herein, such as binding domains based on designed repeating sequence proteins or designed repeating domains (e.g., see WO 2002 / 020565). Specifically, an antigen-binding domain capable of specifically binding to a target cell antigen is an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP).

[0044] Regarding antibodies or fragments thereof, the term "antigen-binding domain capable of specifically binding to a target cell antigen" refers to a molecular portion comprising a region that specifically binds to some or all of the antigen and is complementary to it. For example, an antigen-binding domain capable of specifically binding to an antigen may be provided, for instance, by one or more antibody variable domains (also called antibody variable regions). Specifically, an antigen-binding domain capable of specifically binding to an antigen comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In a particular state, the "antigen-binding domain capable of specifically binding to a target cell antigen" is a Fab fragment or a cross-Fab fragment.

[0045] The term “antibody” is used in the broadest sense herein and covers a variety of antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0046] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homologous antibodies, that is, individual antibodies constituting that population are identical and / or bind to the same antigenic determinant, excluding possible variant antibodies, such as those containing natural mutations or generated during the production of a monoclonal antibody formulation, which are typically present in very small amounts. In contrast to multiclonal antibody formulations, which typically include different antibodies targeting different determinants (antigenic determinants), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0047] As used herein, a "monospecific" antibody means an antibody having one or more binding sites, each binding to the same antigenic determinant of the same antigen. The term "bispecific" means that an antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each specific to a different antigenic determinant. In some embodiments, a bispecific antigen-binding molecule is capable of simultaneously binding to two antigenic determinants, specifically two antigenic determinants expressed on two different cells. Bispecific antigen-binding molecules as described herein can also form part of a multispecific antibody.

[0048] As used in this application, the term "valence" indicates the presence of a specified number of binding sites specific to a particular antigenic determinant in an antigen-binding molecule that is specific to a particular antigenic determinant. Therefore, the terms "bivalent," "trivalent," "tetravalent," and "hexavalent" indicate the presence of two, three, four, and six binding sites specific to a particular antigenic determinant, respectively, in an antigen-binding molecule. In a particular embodiment of the present invention, the bispecific antigen-binding molecule of the present invention may be monovalent for a particular antigenic determinant, meaning it has only one binding site for that antigenic determinant, or it may be divalent or tetravalent for a particular antigenic determinant, meaning it has two or four binding sites for that antigenic determinant, respectively.

[0049] The terms "full-length antibody," "intact antibody," and "all antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of natural antibodies. "Natural antibody" refers to a natural immunoglobulin molecule with a different structure. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody can be assigned to one of five types, namely α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM). Some of these can be further subdivided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Based on the amino acid sequence of the antibody's constant structural domain, its light chain can be assigned to one of two types, namely kappa (κ) and lambda (λ).

[0050] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include (but are not limited to) Fv, Fab, Fab', Fab'-SH, F(ab')2; bivalent antibodies, trivalent antibodies, tetravalent antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); 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, for example, Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding antigenic determinant residues and having an extended in vivo half-life, see U.S. Patent No. 5,869,046. Bivalent antibody systems may be bivalent or bispecific antibody fragments with two antigen-binding sites, 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). Trivalent and tetravalent antibodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibody systems comprise antibody fragments containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, a single-domain anti-system is used for human single-domain antibodies (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1). As described herein, antibody fragments can be prepared using various techniques, including (but not limited to) proteolytic digestion of intact antibodies and production via recombinant host cells (e.g., E. coli or bacteriophages).

[0051] Papain digestion of an intact antibody produces two identical antigen-binding fragments (referred to as "Fab" fragments), each containing a variable domain of the heavy chain and a variable domain of the light chain, as well as a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term "Fab fragment" refers to an antibody fragment comprising: a light chain fragment (containing the VL domain and constant domain of the light chain (CL)) and a VH domain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is a Fab' fragment with a free thiol group at the cysteine ​​residue of the constant domain. Pepsin treatment produces an F(ab')2 fragment (two Fab fragments) having two antigen-binding sites and a portion of the Fc region. According to the invention, the term "Fab fragment" also includes a "cross-Fab fragment" (or crossover Fab fragment) as defined below.

[0052] The terms "cross-Fab fragment," "xFab fragment," or "crossover Fab fragment" refer to Fab fragments in which the variable or constant regions of the heavy and light chains are exchanged. A cross-Fab molecule may have two different chain compositions and is included in the bispecific antibody of the present invention: on the one hand, the variable regions of the Fab heavy and light chains are exchanged, i.e., the cross-Fab molecule comprises a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1) and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). This cross-Fab molecule is also called cross-Fab (VLVH). On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the cross-Fab molecule comprises a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL) and a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1). This cross-Fab molecule is also called cross-Fab (CLCH1).

[0053] A "single-chain Fab fragment" or "scFab" is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; and wherein the linker system has at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat number) to form interchain disulfide bonds.

[0054] A "cross-linked single-chain Fab fragment" or "x-scFab" is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL together form a polypeptide that specifically binds to an antigen binding site of an antigen and wherein the linker system has at least 30 amino acids. Furthermore, the x-scFab molecules can be further stabilized by inserting cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering) to form interchain disulfide bonds.

[0055] A "single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an antibody, linked by a short linker peptide having 10 to 25 amino acids. This linker is typically rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, this protein retains the specificity of the original antibody. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Additionally, antibody fragments may contain single-chain polypeptides with VH domain characteristics (i.e., capable of assembling with the VL domain) or VL domain characteristics (i.e., capable of assembling with the VH domain), which can form functional antigen-binding sites and thereby provide the antigen-binding properties of the full-length antibody.

[0056] "Scaffold antigen-binding protein" is known in this art. For example, fibronectin and engineered ankyrin repeat protein (DARPin) have been used as alternative scaffolds for the antigen-binding domain, see Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009), and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In one embodiment of the present invention, the scaffold antigen-binding protein is selected from the group consisting of: CTLA-4 (Evibody), lipocalin, etc. (Anticalin), protein A-derived molecules (such as the Z domain of protein A, affibody), A domain (high-affinity polymer (Avimer) / maxibody)), serum transferrin (trans-body); engineered ankyrin repeat protein (DARPin), variable domains of antibody light or heavy chains (single-domain antibody, sdAb), variable domains of antibody heavy chains (nanoantibodies, VH), VNAR fragments, fibronectin (AdNectin)), C-type lectin domains (tetranectin); variable domains of neoantigen receptor β-endoramiase (VNAR fragments), human γ-crystal proteins or ubiquitin (avidin molecules); Kunitz-type domains of human protease inhibitors, microbodies (such as proteins from the knottin family), peptide aptamers, and fibronectin (AdNectin). CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is a CD28 family receptor primarily expressed on CD4+ T cells. Its extracellular domain possesses a variable domain-like Ig fold. The loop corresponding to the antibody CDR can be heterologously substituted to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as erve bodies (e.g., US7166697B1). Erve bodies and antibodies (e.g., domain-specific antibodies) have separable variable regions of approximately the same size. For further details, see Journal of Immunological Methods 248 (1-2), 31-45 (2001).Lipid carriers are a family of extracellular proteins that transport small hydrophobic molecules, such as steroids, bilins, rhodopsins, and lipids. They possess a rigid β-sheet secondary structure with multiple loops at the open end of the cone structure, which can be engineered to bind to different target antigens. Anticarriers are between 160 and 180 amino acids in size and are derived from lipid carriers. For further details, see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B1, and US20070224633. The affinity system is derived from the scaffold of protein A from Staphylococcus aureus, which can be engineered to bind to antigens. This domain consists of a triple-helix bundle with approximately 58 amino acids. Libraries have been generated by randomizing surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP 1641818A1. High-affinity polymers are multidomain proteins derived from the A-domain scaffold family. The native domains, containing approximately 35 amino acids, are bound by defined disulfide bonds. Diversity is generated by reorganizing the natural variations exhibited by the A-domain family. For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind to different target antigens by inserting peptide sequences into a permissive surface loop. Examples of engineered transferrin scaffolds include transotropic transferrin. For further details, see J. Biol. Chem 274, 24066-24073 (1999). Designed ankyrin repeat proteins (DARPin) are derived from ankyrin, a family of proteins that mediate the integration of membrane proteins into the cytoskeleton. A single ankyrin repeat consists of a 33-residue motif composed of two α-helices and one β-turn. Each repeat can be engineered to bind to different target antigens by randomizing the residues in the first α-helix and β-turn. The binding interface can be increased by increasing the number of modules (affinity maturation method). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003), J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.A single-domain antibody system is an antibody fragment composed of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of an antibody heavy chain from camels (nanoantibody or VHH fragment). Additionally, the term single-domain antibody includes autonomous human heavy chain variable domains (VH) or VNAR fragments derived from sharks. Fibronectin can be engineered to bind to the scaffold of the antigen. Adenatin is composed of a backbone of the native amino acid sequence of the 10th domain of 15 repeating units of human type III fibronectin (FN3). The three loops at one end of the beta-sandwich can be engineered to enable adenatin to specifically recognize the therapeutic target of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764, and US6818418B1. Peptide aptamer systems are combinatorial recognition molecules composed of a constant scaffold protein, typically thioredoxin (TrxA), containing a restrictive variable peptide ring inserted at the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from natural microproteins of 25 to 50 amino acids in length, containing 3 to 4 cysteine ​​bridges. Examples of microproteins include KalataBI, conotoxin, and knotting agents. Microproteins can be engineered to include a ring of up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knotting agent domains, see WO2008098796.

[0057] "Antigen-binding molecules that bind to the same antigenic determinant" (as a reference molecule) refers to antigen-binding molecules that block the binding of the reference molecule to its antigen by 50% or more in competitive analysis, and conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen by 50% or more in competitive analysis. "Antigen-binding molecules that do not bind to the same antigenic determinant" (as a reference molecule) refers to antigen-binding molecules that do not block the binding of the reference molecule to its antigen by 50% or more in competitive analysis, and conversely, the reference molecule does not block the binding of the antigen-binding molecule to its antigen by 50% or more in competitive analysis.

[0058] The terms "antigen-binding domain" or "antigen-binding site" refer to a portion of an antigen-binding molecule that contains a region specifically binding to and complementary to some or all of the antigen. In the case of a large antigen, the antigen-binding molecule may bind only to a specific portion of the antigen, which is called an antigenic determinant. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain includes a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain.

[0059] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "antigenic determinant" and refers to a site on a polypeptide macromolecule where the antigen-binding portion binds to form an antigen-binding portion-antigen complex (e.g., an adjacent extension of an amino acid or a conformational configuration composed of different regions of non-adjacent amino acids). Useful antigenic determinants may be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or located in the extracellular matrix (ECM). Unless otherwise indicated, proteins that can be used as antigens herein may be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. In the context of reference to a specific protein herein, the term covers "full-length," unprocessed protein, and any form of protein processed from cells. The term also covers natural variants of proteins, such as splice variants or allelic variants.

[0060] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in this technique (e.g., surface plasma resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, as measured by, for example, SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the binding of the antigen-binding molecule to the antigen. In some embodiments, the molecules bound to the antigen have a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM or ≤ 0.001 nM (e.g., 10⁻⁸ M or less, e.g., 10⁻⁸ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M).

[0061] "Affinity" or "binding affinity" refers to the sum strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its bound ligand (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y is typically expressed by the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constants (kdissociation and kassociation, respectively). Therefore, equivalent affinity can contain different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by commonly used methods known in this art (including those described herein). A specific method for measuring affinity is surface plasma resonance (SPR).

[0062] "Affinity maturation" antibody system refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) that improve the antibody's affinity for the antigen compared to a parent antibody that does not have such alterations.

[0063] As used herein, "target cell antigen" refers to an antigenic determinant presented on the surface of target cells, specifically target cells in a tumor (such as cancer cells or tumor stromal cells). Therefore, target cell antigens are tumor-associated antigens. Specifically, tumor target cell antigens are fibroblast activation proteins (FAP).

[0064] Unless otherwise indicated, the term "fibroblast activation protein (FAP)" (also known as prolyl endopeptidase FAP or Seprase (EC 3.4.21)) means any natural FAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term covers "full-length," untreated FAP, and any form of FAP processed from cells. The term also covers natural variants of FAP, such as splice variants or allelic variants. In one embodiment, the antigen-binding molecule of the present invention is capable of specifically binding to human, mouse, and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 149, SEQ ID NO:2) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to position 760. The amino acid sequence of the His-tagged human FAP ECD is shown in SEQ ID NO: 62. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 126, SEQ ID NO:63) or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to position 761. SEQ ID NO:64 shows the amino acid sequence of the His-tagged mouse FAP ECD. SEQ ID NO:65 shows the amino acids of the His-tagged cynomolgus monkey FAP ECD. Preferably, the anti-FAP binding molecule of the present invention binds to the extracellular domain of FAP.

[0065] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that participates in the binding of the antigen-binding molecule to the antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0066] As used herein, the term "hypervariant region" or "HVR" refers to each region in the variable domain of an antibody that is sequence-hypervariant and determines antigen-binding specificity, such as the "complementarity-determining region" ("CDR"). Typically, an antibody contains six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs in this article include: (a) hypervariable rings located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and (b) CDRs located 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) antigenic contacts, which appear 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)).

[0067] Unless otherwise indicated, the CDR is determined according to Kabat et al., above. Those skilled in this art will understand that the CDR name may also be determined according to Chothia, above, McCallum, above, or any other scientifically accepted naming system.

[0068] "Frame" or "FR" refers to the variable domain residues other than the complementarity-determining region (CDR). The variable domain FR is usually composed of four FR domains: FR1, FR2, FR3 and FR4. Therefore, the CDR and FR sequences usually 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.

[0069] The term "chimeric" antibody system refers to an antibody in which one part of the heavy chain and / or light chain is derived from a specific source or species, while the remaining part of the heavy chain and / or light chain is derived from a different source or species.

[0070] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0071] A “humanized” antibody system refers to a chimeric antibody comprising amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise at least one, and typically two, variable domains, wherein all or substantially all of the CDRs correspond to the corresponding CDRs of the non-human antibody, and all or substantially all of the FRs correspond to the corresponding FRs of the human antibody. The humanized antibody may, where appropriate, comprise at least a portion of the antibody constant region derived from the human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. Other forms of “humanized antibodies” as covered by this invention are those in which the constant region has been further modified or altered relative to the constant region of the original antibody to produce the properties according to the invention, particularly concerning C1q binding and / or Fc receptor (FcR) binding.

[0072] The term "CH1 domain" refers to the antibody heavy chain polypeptide portion extending approximately from EU position 118 to EU position 215 (according to Kabat's EU numbering system). In one state, the CH1 domain has the following amino acid sequence: ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 166). Typically, this is followed by a segment having the amino acid sequence EPKSC (SEQ ID NO: 163) to link the CH1 domain to the hinge region. The inventors have found that a CH1 domain not fused to the hinge region can lead to a reaction with pre-existing antibodies (ADA) in the human body, which are absent if variants EPKSCD (SEQ ID NO: 164) or EPKSCS (SEQ ID NO: 165) are present. For example, CH1 domains with free C-termini can be found in cross-fab fragments.

[0073] The term "hinge region" refers to a portion of an antibody heavy chain polypeptide that binds the CH1 and CH2 domains in the wild-type antibody heavy chain, for example, from approximately position 216 to approximately position 230 according to the Kabat EU numbering system, or from approximately position 226 to approximately position 230 according to the Kabat EU numbering system. Hinge regions of other IgG subclasses can be determined by aligning the hinge region with the cysteine ​​residues of the IgG1 subclass sequence. Hinge regions are typically dimer molecules composed of two polypeptides having the same amino acid sequence. Hinge regions typically contain up to 25 amino acid residues and are flexible, allowing the associated target binding site to move independently. Hinge regions can be subdivided into three domains: upper, middle, and lower hinge domains (e.g., see Roux et al., J. Immunol. 161 (1998) 4083). In one state, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 160), where X is S or P. In another state, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO: 161), where X is S or P. In yet another state, the hinge region has the amino acid sequence CPXCP (SEQ ID NO: 162), where X is S or P.

[0074] The terms "Fc domain" or "Fc region" are used herein to define a C-terminal region in an antibody heavy chain that contains at least a portion of a constant region. This term includes both native and variant Fc regions. The IgG Fc region comprises the IgG CH2 and IgG CH3 domains. The "CH2 domain" of the human IgG Fc region typically extends from an amino acid residue at approximately position 231 to an amino acid residue at approximately position 340 (according to Kabat's EU numbering system). In one state, the CH2 domain has the following amino acid sequence: APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 153). The CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact natural Fc region. It is presumed that the carbohydrates can replace the domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, the carbohydrate chain is linked to the CH2 domain. The CH2 domain as used herein can be the natural sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the C-terminus extension of the CH2 domain in the Fc region (i.e., from amino acid residue approximately 341 to amino acid residue approximately 447 according to the EU numbering system of Kabat for IgG). In one state, the CH3 domain has the following amino acid sequence: GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 154). The CH3 region herein can be a native CH3 domain or a variant CH3 domain (e.g., a CH3 domain with a "protrusion" ("ridge") introduced in one chain and a corresponding "cavity" ("hole") introduced in the other chain; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two distinct antibody heavy chains as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present.Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0075] The term "wild-type Fc domain" refers to an amino acid sequence that is identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include the natural human IgG1 Fc region (non-A and A allotypes), the natural human IgG2 Fc region, the natural human IgG3 Fc region, and the natural human IgG4 Fc region, as well as their natural variants. Wild-type Fc regions are represented by SEQ ID NO: 155 (IgG1, Caucasian allotype), SEQ ID NO: 156 (IgG1, African American allotype), SEQ ID NO: 157 (IgG2), SEQ ID NO: 158 (IgG3), and SEQ ID NO: 159 (IgG4). The term "variant (human) Fc domain" refers to an amino acid sequence that differs from the amino acid sequence of the "wild-type" (human) Fc domain due to at least one "amino acid mutation". In a single-state sample, the variant Fc region has at least one amino acid mutation compared to the native Fc region, for example, about 1 to about 10 amino acid mutations, and in a single-state sample, the native Fc region has about 1 to about 5 amino acid mutations. In a single-state sample, the (variant) Fc region has at least about 95% homology with the wild-type Fc region.

[0076] The "protrusion-cavity" technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a protrusion ("protrusion") at the interface of a first polypeptide and a corresponding cavity ("cavity") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing the small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). Protrusions and cavities can be generated, for example, by site-specific mutagenesis altering the nucleic acid encoding the polypeptide or by peptide synthesis. In a specific embodiment, the bulge modification includes an amino acid substitution of T366W in one of the two subunits of the Fc domain, and the cavitation modification includes an amino acid substitution of T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit of the Fc domain containing the bulge modification further includes an amino acid substitution of S354C, and the subunit of the Fc domain containing the cavitation modification further includes an amino acid substitution of Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits in the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0077] "Region equivalent to the Fc region of immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of immunoglobulin, as well as variants with altered abilities to produce substitutions, additions, or deletions that do not substantially reduce the function of immunoglobulin-mediated effects (such as antibody-dependent cytotoxicity). For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the Fc region of immunoglobulin without substantially losing biological function. Such variants may be selected according to general rules known in this art to minimize the effect on activity (e.g., see Bowie, JU et al., Science 247:1306-10 (1990)).

[0078] The term "effective function" refers to biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, antigen uptake by antigen-presenting cells mediated by immune complexes, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0079] Fc receptor binding-dependent effector function is mediated by the interaction between the antibody Fc region and the Fc receptor (FcR), which is a specialized cell surface receptor on hematopoietic cells. The Fc receptor belongs to the immunoglobulin superfamily and has been shown to mediate both: the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g., tumor cells) coated with corresponding antibodies via antibody-dependent cell-mediated cytotoxicity (ADCC) (see, for example, Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined based on their specificity to immunoglobulin isotypes: the Fc receptor for IgG antibodies is called FcγR. Fc receptor binding is described in, for example, Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ et al., Immunomethods 4 (1994) 25-34; de Haas, M. et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE et al., Ann. Hematol. 76 (1998) 231-248.

[0080] Cross-linking of the IgG antibody Fc receptor (FcγR) triggers a variety of effector functions, including phagocytosis, antibody-dependent cytotoxicity and release of inflammatory mediators, as well as the clearance of immune complexes and regulation of antibody production. In humans, three classes of FcγRs have been characterized: - FcγRI (CD64), which binds monomeric IgG with high affinity and is found on macrophages, monocytes, neutrophils, and eosinophils. Modification of the IgG Fc region at at least one of the amino acid residues E233-G236, P238, D265, N297, A327, and P329 (according to Kabat's EU index number) reduces binding to FcγRI. Replacing IgG2 residues at positions 233-236 with IgG1 and IgG4 residues reduces binding to FcγRI by 10³-fold and eliminates the response of human monocytes to antibody-sensitized erythrocytes (Armour, KL et al., Eur. J. Immunol. 29 (1999) 2613-2624). FcγRII (CD32) binds complexed IgG with low to medium affinity and is widely expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to activate the killing process. FcγRIIB appears to play a role in inhibitory processes and is found on B cells, macrophages, mast cells, and eosinophils. On B cells, it appears to inhibit further immunoglobulin production and, for example, isotype switching of IgE classes. On macrophages, FcγRIIB inhibits phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B form can help inhibit cell activation by binding IgE to its individual receptor. For example, reduced binding to FcγRIIA has been found for antibodies containing mutated Fc regions of IgG at at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (according to Kabat's EU index number). - FcγRIII (CD16) binds IgG with medium to low affinity and exists in two forms. FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. FcγRIIIB is highly expressed on neutrophils.For example, antibodies containing mutations in the Fc region of IgG at at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (according to Kabat's EU index number) have been found to have reduced binding to FcγRIIIA.

[0081] The method for locating the binding site against the Fc receptor on human IgG1, the mutation site mentioned above, and measuring the binding with FcγRI and FcγRIIA is described in Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604.

[0082] The term "ADCC" or "antibody-dependent cytotoxicity" refers to the function mediated by Fc receptor binding and refers to the lysis of target cells by antibodies in the presence of effector cells, as reported herein. The ability of antibodies to induce the initial steps of ADCC is studied by measuring the binding of antibodies to Fcγ receptor-expressing cells (such as recombinant cells expressing FcγRI and / or FcγRIIA or NK cells (basically expressing FcγRIIIA)). Specifically, the binding to FcγR on NK cells is measured.

[0083] "Activated Fc receptors" are Fc receptors that trigger signal transduction events upon binding to the Fc region of an antibody. These events stimulate the cellular effector functions of the receptor. Activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activated Fc receptor system is human FcγRIIIa (see UniProt accession number P08637, p. 141).

[0084] Unless otherwise indicated, the term "OX40" as used herein means any natural OX40 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," untreated OX40, and any form of OX40 processed from cells. The term also encompasses natural variants of OX40, such as splice variants or allelic variants. An exemplary human OX40 amino acid sequence is shown in SEQ ID NO: 1 (Uniprot P43489, version 112), and an exemplary mouse OX40 amino acid sequence is shown in SEQ ID NO: 66 (Uniprot P47741, version 101).

[0085] As used herein, the term "OX40 activator" includes any portion that has a activating effect on the OX40 / OX40L interaction. In this context, OX40 preferably refers to human OX40, and therefore, an OX40 activator is preferably a activator of human OX40. Typically, this portion will be an activating OX40 antibody or antibody fragment, specifically a Fab fragment.

[0086] The terms "anti-OX40 antibody," "anti-OX40," "OX40 antibody," and "antibody specifically binding to OX40" refer to antibodies capable of binding to OX40 with sufficient affinity, thereby enabling the antibody to be used as a diagnostic and / or therapeutic agent targeting OX40. In one embodiment, as measured by flow cytometry (FACS), the degree of binding of the anti-OX40 antibody to unrelated non-OX40 proteins is less than about 10% of the binding of the antibody to OX40. In some embodiments, the dissociation constant (KD) of the antibody bound to OX40 is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10⁻⁶ M or less, e.g., 10⁻⁶⁸ M to 10⁻¹³ M, e.g., 10⁻⁸ M to 10⁻¹⁰ M).

[0087] The term "peptide linker" refers to a peptide containing one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are those known in the art or described herein. Suitable non-immunogenic linker peptide systems (e.g.) (G4S)n, (SG4)n, or G4(SG4)n peptide linkers, wherein "n" is generally a value between 1 and 10, generally between 2 and 4, and specifically 2. That is, such peptides are selected from the group consisting of: GGGGS (SEQ ID NO:67), GGGGSGGGGS (SEQ ID NO:68), SGGGGSGGGG (SEQ ID NO:69), and GGGGSGGGGSGGG (SEQ ID NO:70), but also include the sequences GSPGSSSSGS (SEQ ID NO:71), (G4S)3 (SEQ ID NO:72), (G4S)4 (SEQ ID NO:73), GSGSGSGS (SEQ ID NO:74), GSGSGNGS (SEQ ID NO:75), GGSGSGSG (SEQ ID NO:76), GGSGSG (SEQ ID NO:77), GGSG (SEQ ID NO:78), GGSG (SEQ ID NO:79), GGSG (SEQ ID NO:70), GGSG ... (SEQ ID NO:78), GGSGNGSG (SEQ ID NO:79), GGNGSGSG (SEQ ID NO:80), and GGNGSG (SEQ ID NO:81). Of particular interest are (G4S) (SEQ ID NO:67), (G4S)2 or GGGGSGGGGGS (SEQ ID NO:68), (G4S)3 (SEQ ID NO:72), and (G4S)4 (SEQ ID NO:73).

[0088] As used in this application, the term "amino acid" refers to the group of naturally occurring carboxylated α-amino acids, including alanine (three-letter code: ala, single-letter code: A), arginine (arg, R), aspartic acid (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamic acid (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0089] "Fusion" or "linkage" means that components (such as antibody heavy chains and Fab fragments) are directly linked by peptide bonds or linked through one or more peptide linkers.

[0090] The "percentage of amino acid sequence identity (%)" for a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be achieved in various ways known to those skilled in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, SAWI, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this document, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and its source code, along with user files, has been submitted to the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use with UNIX operating systems (including digital UNIX version 4.0D). All sequence comparison parameters are set by the ALIGN-2 program and will not be changed. When using ALIGN-2 for amino acid sequence comparison, the amino acid sequence similarity percentage (APS) of a given amino acid sequence A relative to, with, or against a given amino acid sequence B (or, as can be expressed as a given amino acid sequence A having or containing a certain APS of APS relative to, with, or against a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y, where X is the number of amino acid residues assessed as a consistent match by the sequence alignment program ALIGN-2 in the procedural alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the APS of A relative to B will not be equal to the APS of B relative to A. Unless otherwise explicitly stated, all amino acid sequence similarity percentage values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraphs.

[0091] In some embodiments, amino acid sequence variants of the bispecific antigen-binding molecules provided herein are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of antigen-binding molecules containing TNF ligand trimers. Amino acid sequence variants of such molecules can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen-binding molecule containing TNF ligand trimers or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the antibody amino acid sequence. Any combination of deletion, insertion, and substitution can be performed to obtain the final construct, provided that the final construct has the desired properties (e.g., antigen binding). Sites of interest for substitution mutagenesis include HVR and frame (FR). Conservative substitutions are provided in Table B entitled “Preferred Substitutions” and are further described below with reference to amino acid side chain categories (1) to (6). Amino acid substitutions can be introduced into the molecule of interest, and the product can be screened for desired activities, such as (e.g.) preserved / improved antigen binding, reduced immunogenicity, or modified ADCC or CDC. Table A Original residues Exemplary substitution Better alternative Ala (A) Val; Leu; Ile Val Arg (R) Lys;Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu;Asn Glu Cys (C) Ser;Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leucine Leu Leu (L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg;Gln;Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leucine Leu

[0092] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residual groups that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0093] Non-conservative substitution requires that members of one of these categories be exchanged for members of another category.

[0094] The term "amino acid sequence variant" includes a substantial variant in which an amino acid is substituted at one or more hypervariable residues of a parent antigen-binding molecule (e.g., humanized or human antibody). Typically, variants selected for further research are chosen to alter (e.g., modify) certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antigen-binding molecule and / or substantially retain certain biological properties of the parent antigen-binding molecule. An illustrative substitution variant system affinity maturation antibody can be conveniently generated (e.g., using phage display-based affinity maturation techniques, such as those described herein). In short, one or more HVR residues are mutated, and the variant antigen-binding molecule is displayed on a phage and screened for specific biological activities (e.g., binding affinity). In some embodiments, substitution, insertion, or deletion may occur within one or more HVRs, as long as such alterations do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conserved alterations to HVR that do not substantially reduce binding affinity can be made (e.g., conserved substitutions as presented herein). A useful method for identifying targetable mutagenic residues or regions in an antibody is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or target residue groups (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) have been identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Other substitutions can be introduced at amino acid sites that exhibit functional sensitivity to the initial substitution. Alternatively, the crystal structure of the antigen-antigen binding molecule complex can be used to identify the contact points between the antibody and the antigen. These contact residues and adjacent residues can be targeted as substitution candidates or eliminated. Variants can be screened to determine whether they contain the desired properties.

[0095] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions (ranging in length from one residue to a polypeptide containing hundreds or more residues), as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the bispecific antigen-binding molecule of the present invention having an N-terminal methionine residue. Other insertion variants of the molecule include fusions with the N-terminus or C-terminus of the polypeptide, which prolong the serum half-life of the bispecific antigen-binding molecule.

[0096] In some embodiments, the bispecific antigen-binding molecules provided herein are modified to increase or decrease the degree of antibody glycosylation. Glycosylation variants of the molecule can be readily obtained by altering the amino acid sequence to create or remove one or more glycosylation sites. If the antigen-binding molecule contains an Fc region, the carbohydrate linked to it can be modified. Natural antibodies produced by mammalian cells typically contain branched, dibranched oligosaccharides, which are typically linked to the CH2 domain of the Fc region via N-bonds to Asn297. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc linked to the "stem" of the dibranched oligosaccharide structure. In some embodiments, the oligosaccharides in antigen-binding molecules containing TNF family ligand trimers can be modified to produce variants with certain modified properties. In one embodiment, a variant of the bispecific antigen-binding molecule or antibody of the present invention is provided, wherein such variant has a carbohydrate structure lacking (directly or indirectly) fucosylation linked to the Fc region. These fucosylated variants may possess modified ADCC functionality, see, for example, U.S. Patent Publication No. 2003 / 0157108 (Presta, L.) or No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). In another embodiment, a variant of the bispecific antigen-binding molecule or antibody of the present invention is provided having bisected oligosaccharides, for example, wherein the bisected oligosaccharide linked to the Fc region is bisected by GlcNAc. These variants may exhibit reduced fucosylation and / or improved ADCC function, see, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. These antibody variants may have improved CDC function and are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0097] In certain forms, it is desirable to generate cysteine-engineered variants of the bispecific antigen-binding molecule of the present invention, such as "thio-MAb", wherein one or more residues of the molecule are substituted with cysteine ​​residues. In certain forms, the substituted residues appear at accessible sites of the molecule. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to bind the antibody to other parts (such as drug portions or linker-drug portions) to produce an immunoconjugate. In certain forms, one or more of the following residues may be substituted with cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-engineered antigen-binding molecules can be generated as described, for example, in U.S. Patent No. 7,521,541.

[0098] The terms "nucleic acid" or "polynucleotide" include any compound and / or substance comprising a nucleotide polymer. Each nucleotide consists of: a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by a base sequence, where these bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), specifically messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Additionally, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain natural or non-natural nucleotides. Examples of non-natural nucleotides include modified nucleotide bases having derivative sugar or phosphate ester backbones or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for directly expressing the antibodies of the present invention in vitro and / or in vivo (e.g., in a host or patient). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA carrier and / or the expression of the encoded molecule, enabling the mRNA to be injected into an individual to generate antibodies in vivo (e.g., see Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0099] "Isolated" nucleic acids refer to nucleic acid molecules that have been isolated from components in their natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells, which normally contain such nucleic acid molecules, but which are located outside chromosomes or at chromosome locations different from their natural chromosome locations.

[0100] "Isolated nucleic acid encoding a bispecific antigen-binding molecule or antibody" refers to one or more nucleic acid molecules encoding a heavy chain and a light chain (or fragments thereof), including such nucleic acid molecules in a single vector or in a single vector, and such nucleic acid molecules present at one or more locations in a host cell.

[0101] For a nucleic acid or polynucleotide having a nucleotide sequence that is at least (e.g.) 95% "identical" to the reference nucleotide sequence of the present invention, the nucleotide sequence of the polynucleotide is expected to be identical to the reference sequence, except that the polynucleotide sequence may include up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. Such changes to the reference sequence may occur at the 5' or 3' end of the reference nucleotide sequence or at any position between those ends, individually scattered between residues in the reference sequence or in the form of one or more adjacent groups within the reference sequence. In practice, known computer programs (such as those discussed above for polypeptides, e.g., ALIGN-2) can be used to conventionally determine whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the present invention.

[0102] The term "expression cassette" refers to a recombinant or synthetically produced polynucleotide having a series of designated nucleic acid elements that allow transcription of specific nucleic acids in target cells. Recombinant expression cassettes can be incorporated into plastids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes the nucleic acid sequence to be transcribed and sequences such as a promoter. In some embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding a bispecific antigen-binding molecule or a fragment thereof of the present invention.

[0103] The terms "vector" or "expression vector" are synonymous with "expression construct" and refer to a DNA molecule used to introduce a specific gene operatively associated with a target cell and guide its expression. This term includes vectors that have a self-replicating nucleic acid structure and vectors incorporated into the host cell genome to which they are introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows transcription of large amounts of stable mRNA. After the expression vector is located inside the target cell, the cell's transcription and / or translation apparatus produces a ribonucleic acid molecule or protein encoded by the gene. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding a bispecific antigen-binding molecule or a fragment thereof of the present invention.

[0104] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformed cells" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. The nucleic acid content of progeny cells may not be completely identical to that of parent cells and may contain mutations. This article includes mutant progeny that have the same function or biological activity as those screened or selected from the original transformed cells.

[0105] The host cell line is any type of cell system that can be used to generate the bispecific antigen-binding molecule of the present invention. Host cells include cultured cells, such as mammalian cultured cells, such as CHO cells, BHK cells, NSO cells, SP2 / O cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells and plant cells (to name just a few), and also includes cells contained in genetically modified animals, genetically modified plants or cultured plant or animal tissues.

[0106] The “effective amount” of an agent refers to the amount required to produce physiological changes in the cells or tissues to which the agent is administered.

[0107] The "therapeutic effective amount" of an agent (e.g., a pharmaceutical composition) means the amount that effectively achieves the desired therapeutic or preventive effect at the required dose and within the required time period. For example, a therapeutically effective amount of an agent eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0108] "Individual" or "subject" refers to mammals. Mammals include (but are not limited to) domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats). In particular, "individual" or "subject" refers to humans.

[0109] The terms "pharmaceutical composition" or "pharmaceutical formulation" refer to preparations that are in a form that allows the biological activity of the active ingredient contained therein to be effective and that do not contain any other components that would have unacceptable toxicity to an individual to whom the pharmaceutical composition will be administered.

[0110] "Pharmaceutical-acceptable carriers" refers to components in a pharmaceutical composition or formulation other than the active ingredient that are non-toxic to individuals. Pharmaceutical-acceptable carriers include (but are not limited to) buffers, excipients, stabilizers, or preservatives.

[0111] The term "packaging insert" is used to refer to the instruction leaflet that is usually included in the commercial packaging of a therapeutic product, which contains information about indications, usage, dosage, administration, combination therapy, contraindications and / or warnings about the use of such therapeutic products.

[0112] As used herein, "treatment" (and its grammatical variations, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of disease in the treated individual, and may be implemented for prevention or during the clinicopathological course of disease. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological outcome of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the molecules of the present invention are used to delay disease development or slow disease progression.

[0113] As used herein, the term "cancer" refers to proliferative diseases such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung cancer (NSCL), bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, epidermal or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, and Hodgkin's disease. Diseases, including esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, bile duct cancer, central nervous system (CNS) lesions, spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma, including refractory forms of any of the above cancers or combinations of one or more of the above cancers.

[0114] As used herein, the term "chemotherapy agent" refers to a compound that can be used to treat cancer. In one state, the chemotherapy agent is an antimetabolite. In one state, the antimetabolite is selected from the group consisting of: aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine, capecitabine, cytarabine, fluorouracil, floxuridine, and gemcitabine. In a specific state, the antimetabolite is capecitabine or gemcitabine. In another state, the antimetabolite is fluorouracil. In one state, the chemotherapeutic agent is an agent that affects microtubule formation. In another state, the agent affecting microtubule formation is selected from the group consisting of: paclitaxel, docetaxel, vincristine, vinblastine, vindesine, vinorelbin, taxotere, etoposide, and teniposide. In another state, the chemotherapeutic agent is an alkylating agent, such as cyclophosphamide. In one state, the chemotherapeutic agent is a cytotoxic antibiotic, such as a topoisomerase II inhibitor. In another state, the topoisomerase II inhibitor is doxorubicin. The bispecific antibody of this invention...

[0115] This invention provides a novel bispecific antigen-binding molecule comprising a novel anti-FAP antibody (pure line 212). The bispecific antigen-binding molecule comprising this novel anti-FAP antibody has particularly advantageous properties such as: produceability, stability, binding affinity, biological activity, targeting efficiency, reduced internalization, excellent pharmacokinetic (PK) properties (such as improved clearance), reduced toxicity, a wider range of doses that can be administered to patients, and thereby potentially enhanced efficacy. Furthermore, depending on the OX40 antibody included, the bispecific antigen-binding molecule has been prepared in an advantageous format. Exemplary bispecific antigen-binding molecules

[0116] In one embodiment, the present invention provides a bispecific antigen-binding molecule characterized by targeted agonist binding to OX40. Specifically, the bispecific antigen-binding molecule is an OX40 agonist targeting FAP. In another specific embodiment, the bispecific antigen-binding molecule of the present invention comprises an Fc region consisting of a first subunit and a second subunit capable of stable association, the Fc region containing a mutation that reduces effector function. Using an Fc region containing a mutation that reduces or eliminates effector function will prevent nonspecific agonist effects caused by Fc receptor crosslinking and will prevent ADCC in OX40+ cells. The bispecific antigen-binding molecules described herein have advantages over conventional antibodies that can specifically bind to OX40 because these molecules selectively induce an immune response at target cells (typically near the tumor, i.e., in the tumor stroma).

[0117] Therefore, these bispecific antigen-binding molecules are characterized by their FAP-targeting activating effect on OX40. In the presence of FAP-expressing cells, the bispecific antigen-binding molecules can induce NFκB activation in human OX40-positive NFκB reporter cells.

[0118] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) at least two antigen-binding domains specifically binding to OX40, and (b) an antigen-binding domain specifically binding to fibroblast activation protein (FAP), comprising: a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing an amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing an amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, and (v) CDR-L2 ...3 containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and (vi) the amino acid sequence of NO:7, and (vi) CDR-L3, which contains the amino acid sequence of SEQ ID NO:8; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0119] In one state, the antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP) comprises: a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing an amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NO:4, and (iii) CDR-H3 containing an amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, (v) CDR-L2 containing an amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3 containing an amino acid sequence of SEQ ID NO:8.

[0120] In one state, the Fc region contains one or more amino acid substitutions that reduce the binding affinity of the antibody to the Fc receptor and / or the effector function.

[0121] In another embodiment, a bispecific antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to the FAP comprises: a heavy chain variable region (VHFAP) containing an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9; and a light chain variable region (VLFAP) containing an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:10. In one embodiment, the antigen-binding domain capable of specifically binding to the FAP comprises: a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:9; and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:10.

[0122] In another state, the antigen-binding domain that can specifically bind to the FAP includes: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20; and a light chain variable region (VLFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. In one state, the antigen-binding domains that can specifically bind to FAP include (a) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:15 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:21, (b) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:16 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:21, (c) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:16 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:22, or (d) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:19 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:25. Specifically, the antigen-binding domain that can specifically bind to FAP includes (a) a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO:15 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO:21.

[0123] In one state, the antigen-binding domain of OX40 can specifically bind to the amino acid sequence containing SEQ ID NO:1 or a polypeptide composed of therefrom.

[0124] In another state, a bispecific antigen-binding molecule is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises (a) a heavy chain variable region (VHOX40), which comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:27, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:28, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:29; and a light chain variable region (VLOX40), which comprises (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:30, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:31, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:32, or (b) a heavy chain variable region (VHOX40), which comprises (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:35, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:36, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:37; and a light chain variable region (VLOX40) comprising (iv) CDR-L1, comprising the amino acid sequence of SEQ ID NO:38, (v) CDR-L2, comprising the amino acid sequence of SEQ ID NO:39, and (vi) CDR-L3, comprising the amino acid sequence of SEQ ID NO:40, or (c) a heavy chain variable region (VHOX40) comprising (i) CDR-H1, comprising the amino acid sequence of SEQ ID NO:43, (ii) CDR-H2, comprising the amino acid sequence of SEQ ID NO:44, and (iii) CDR-H3, comprising the amino acid sequence of SEQ ID NO:37. The amino acid sequence of SEQ ID NO:45; and the light chain variable region (VLOX40), which comprises (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:46, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:47, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:48, or (d) the heavy chain variable region (VHOX40), which comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:51, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:52, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:53;And a light chain variable region (VLOX40), which comprises (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:54, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:55, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:56.

[0125] In one state, the antigen-binding domain that can specifically bind to OX40 includes: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:27, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:28, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:29; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:30, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:31, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:32. In another state, the antigen-binding domain that specifically binds to OX40 includes: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:35, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:36, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:37; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:38, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:39, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:40. In another state, the antigen-binding domain that specifically binds to OX40 includes: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:43, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:44, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:45; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:46, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:47, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:48.In another state, the antigen-binding domain that specifically binds to OX40 includes: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:51, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:52, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:53; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:54, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:55, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:56. In a specific state, the antigen-binding domain capable of specifically binding to OX40 comprises: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:35, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:36, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:37; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:38, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:39, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:40.

[0126] In one state, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises (i) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34, or (ii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:41 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:42, or (iii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:49 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:50, or (iv) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:57 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:57. The light chain variable region (VLOX40) of the amino acid sequence NO:58.

[0127] In one embodiment, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:33 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34. In another embodiment, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:41 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:42. In another embodiment, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain specifically binding to OX40 comprises a heavy chain variable region (VHOX40) containing the amino acid sequence of SEQ ID NO:49 and a light chain variable region (VLOX40) containing the amino acid sequence of SEQ ID NO:50. In yet another embodiment, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain specifically binding to OX40 comprises a heavy chain variable region (VHOX40) containing the amino acid sequence of SEQ ID NO:57 and a light chain variable region (VLOX40) containing the amino acid sequence of SEQ ID NO:58. In a particular embodiment, each of the antigen-binding domains specifically binding to OX40 comprises a heavy chain variable region (VHOX40) containing the amino acid sequence of SEQ ID NO:41 and a light chain variable region (VLOX40) containing the amino acid sequence of SEQ ID NO:42.

[0128] In another state, a bispecific antigen-binding molecule as defined above is provided, capable of specifically binding to the antigen-binding domain of OX40 comprising (i) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:59 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34, or (ii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:60 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34, or (iii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:61 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34.

[0129] In one state, a bispecific antigen-binding molecule as defined above is provided, wherein the antigen-binding domain specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:59 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34. In another state, the antigen-binding domain specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:60 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34. In yet another state, the antigen-binding domain specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:61 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34. In a specific state, each antigen-binding domain that specifically binds to OX40 comprises a heavy chain variable region (VHOX40) containing the amino acid sequence of SEQ ID NO: 60 and a light chain variable region (VLOX40) containing the amino acid sequence of SEQ ID NO: 34. A bispecific antigen-binding molecule that binds to OX40 and FAP.

[0130] In another state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:27, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:28, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:29; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:30, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:31, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:32. (b) An antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), comprising: a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing the amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing the amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:5. (c) The amino acid sequence of NO:8; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0131] In another state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61; and a light chain variable region (VLOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:34; (b) at least one antigen-binding domain specifically binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20; and a light chain variable region (VLF ... The amino acid sequences of the group consisting of NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0132] In a specific state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically capable of binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61; and a light chain variable region (VLOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:34; (b) at least one antigen-binding domain specifically capable of binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15; and a light chain variable region (VLFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21; and (c) an Fc region consisting of a first subunit and a second subunit capable of stable association.

[0133] In another state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:35, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:36, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:37; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:38, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:39, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:40. (b) An antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), comprising: a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing the amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing the amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:5. (c) The amino acid sequence of NO:8; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0134] In another state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41; and a light chain variable region (VLOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:42; (b) at least one antigen-binding domain specifically binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20; and a light chain variable region (VLFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24. (c) The amino acid sequence of the group consisting of NO:24, SEQ ID NO:25 and SEQ ID NO:26; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0135] In a specific state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically capable of binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41; and a light chain variable region (VLOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:42; (b) at least one antigen-binding domain specifically capable of binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15; and a light chain variable region (VLFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21; and (c) an Fc region consisting of a first subunit and a second subunit capable of stable association.

[0136] In another state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:43, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:44, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:45; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:46, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:47, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:48. (b) An antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), comprising: a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing the amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing the amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:5. (c) The amino acid sequence of NO:8; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0137] In another state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:49; and a light chain variable region (VLOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:50; (b) at least one antigen-binding domain specifically binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20; and a light chain variable region (VLFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24. (c) The amino acid sequence of the group consisting of NO:24, SEQ ID NO:25 and SEQ ID NO:26; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0138] In a specific state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically capable of binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:49; and a light chain variable region (VLOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:50; (b) at least one antigen-binding domain specifically capable of binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15; and a light chain variable region (VLFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21; and (c) an Fc region consisting of a first subunit and a second subunit capable of stable association.

[0139] In another state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:51, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO:52, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:53; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO:54, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:55, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:56. (b) An antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), comprising: a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing the amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing the amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:5. (c) The amino acid sequence of NO:8; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0140] In another state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:57; and a light chain variable region (VLOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:58; (b) at least one antigen-binding domain specifically binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20; and a light chain variable region (VLFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24. (c) The amino acid sequence of the group consisting of NO:24, SEQ ID NO:25 and SEQ ID NO:26; and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

[0141] In a specific state, a bispecific antigen-binding molecule is provided, comprising (a) at least two antigen-binding domains specifically capable of binding to OX40, comprising: a heavy chain variable region (VHOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:57; and a light chain variable region (VLOX40) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:58; (b) at least one antigen-binding domain specifically capable of binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15; and a light chain variable region (VLFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21; and (c) an Fc region consisting of a first subunit and a second subunit capable of stable association. For bispecific antigen-binding molecules that are divalent to OX40 and monovalent to FAP (2+1 format)

[0142] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two Fab fragments specifically binding to OX40, and (b) a cross-Fab fragment specifically binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing the amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing the amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3 contains the amino acid sequence of SEQ ID NO:8; and (c) an Fc domain consisting of a first subunit and a second subunit capable of stable association.

[0143] Therefore, a bispecific antigen-binding molecule is provided, wherein the bispecific antigen-binding molecule is bivalently bound to OX40 and monovalently bound to FAP.

[0144] In one state, a bispecific antigen-binding molecule is provided, comprising (a) two heavy chains, each heavy chain comprising VH and CH1 domains and an Fc subunit specifically binding to the Fab fragment of OX40; (b) two light chains, each light chain comprising VL and CL domains specifically binding to the Fab fragment of OX40; and (c) a cross-fab fragment specifically binding to FAP, comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain is attached to the C-terminus of one of the two heavy chains in (a). In one state, the VH-CL (VH-Cκ) chain is attached to the C-terminus of the Fc ridge heavy chain. In one state, the VH-Cκ chain is attached to the C-terminus of the Fc ridge heavy chain comprising amino acid-substituted S354C and T366W (numbered according to the Kabat EU index).

[0145] In another state, a bispecific antigen-binding molecule is provided, comprising (a) two heavy chains, each heavy chain comprising VH and CH1 domains and an Fc subunit capable of specifically binding to the Fab fragment of OX40, (b) two light chains, each light chain comprising VL and CL domains capable of specifically binding to the Fab fragment of OX40, and (c) a cross-fab fragment capable of specifically binding to FAP, comprising a VL-CH1 chain and a VH-CL chain, wherein the VL-CH1 chain is linked to the C-terminus of one of the two heavy chains in (a).

[0146] In one state, the VL-CH1 chain is attached to the C-terminus of the Fc pyrrhot heavy chain. In one state, the VL-CH1 chain is attached to the C-terminus of the Fc pyrrhot heavy chain containing amino acid-substituted S354C and T366W (numbered according to the Kabat EU index).

[0147] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:91; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:90; or (b) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; The sequence of NO:87 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:94; a first heavy chain comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:91; and a second heavy chain comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:90; or (c) two light chains, each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:87; a light chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:87; The sequence of NO:96 comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:91; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:90; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:90.In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two light chains, each comprising the amino acid sequence of SEQ ID NO:87; a light chain comprising the amino acid sequence of SEQ ID NO:88; a first heavy chain comprising the amino acid sequence of SEQ ID NO:91; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:90, or (b) two light chains, each comprising the amino acid sequence of SEQ ID NO:87; a light chain comprising the amino acid sequence of SEQ ID NO:94; a first heavy chain comprising the amino acid sequence of SEQ ID NO:91; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:90, or (c) two light chains, each comprising the amino acid sequence of SEQ ID NO:87; a light chain comprising the amino acid sequence of SEQ ID NO:96; a first heavy chain comprising the amino acid sequence of SEQ ID NO:91; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:90. The amino acid sequence of NO:90.

[0148] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:93; a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:97; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:98; or (b) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:98; The sequence of NO:93 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% an amino acid sequence identical to the sequence of SEQ ID NO:94; a first heavy chain comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% an amino acid sequence identical to the sequence of SEQ ID NO:97; and a second heavy chain comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% an amino acid sequence identical to the sequence of SEQ ID NO:98; or (c) two light chains, each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% an amino acid sequence identical to the sequence of SEQ ID NO:93; a light chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% an amino acid sequence identical to the sequence of SEQ ID NO:93; The sequence of NO:96 comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:97; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:98; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:98.In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two light chains, each comprising the amino acid sequence of SEQ ID NO:93; a light chain comprising the amino acid sequence of SEQ ID NO:88; a first heavy chain comprising the amino acid sequence of SEQ ID NO:97; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:98, or (b) two light chains, each comprising the amino acid sequence of SEQ ID NO:93; a light chain comprising the amino acid sequence of SEQ ID NO:94; a first heavy chain comprising the amino acid sequence of SEQ ID NO:97; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:98, or (c) two light chains, each comprising the amino acid sequence of SEQ ID NO:93; a light chain comprising the amino acid sequence of SEQ ID NO:96; a first heavy chain comprising the amino acid sequence of SEQ ID NO:97; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:98. The amino acid sequence of NO:98.

[0149] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 100; a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 88; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 99; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 102; or (b) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 102; The sequence of NO:100 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:94; a first heavy chain comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:99; and a second heavy chain comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:102; or (c) two light chains, each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:100; a light chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence identical to the sequence of SEQ ID NO:100; The sequence of NO:96 comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:99; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:102.In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two light chains, each comprising the amino acid sequence of SEQ ID NO:100; a light chain comprising the amino acid sequence of SEQ ID NO:88; a first heavy chain comprising the amino acid sequence of SEQ ID NO:99; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:102, or (b) two light chains, each comprising the amino acid sequence of SEQ ID NO:100; a light chain comprising the amino acid sequence of SEQ ID NO:94; a first heavy chain comprising the amino acid sequence of SEQ ID NO:99; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:102, or (c) two light chains, each comprising the amino acid sequence of SEQ ID NO:100; a light chain comprising the amino acid sequence of SEQ ID NO:96; a first heavy chain comprising the amino acid sequence of SEQ ID NO:99; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:102. The amino acid sequence of NO:102.

[0150] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 104; a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 88; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 103; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 106; or (b) two light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 106; The sequence of NO:104 contains at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:94; a first heavy chain contains at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:103; and a second heavy chain contains at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:106; or (c) two light chains, each containing at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:104; a light chain containing at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:104; The sequence of NO:96 comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:103; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:106; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:106.In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) two light chains, each comprising the amino acid sequence of SEQ ID NO:104; a light chain comprising the amino acid sequence of SEQ ID NO:88; a first heavy chain comprising the amino acid sequence of SEQ ID NO:103; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:106, or (b) two light chains, each comprising the amino acid sequence of SEQ ID NO:104; a light chain comprising the amino acid sequence of SEQ ID NO:94; a first heavy chain comprising the amino acid sequence of SEQ ID NO:103; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:106, or (c) two light chains, each comprising the amino acid sequence of SEQ ID NO:104; a light chain comprising the amino acid sequence of SEQ ID NO:96; a first heavy chain comprising the amino acid sequence of SEQ ID NO:103; and a second heavy chain comprising the amino acid sequence of SEQ ID NO:106. The amino acid sequence of ID NO:106. A bispecific antigen-binding molecule (3+1 format) that is trivalent when binding to OX40 and monovalent when binding to FAP.

[0151] In another embodiment, a bispecific antigen-binding molecule is provided, wherein the bispecific antigen-binding molecule comprises (aa) a first Fab fragment specifically binding to OX40, (ab) a second Fab fragment specifically binding to OX40, (ac) a third Fab fragment specifically binding to OX40, (b) a cross-Fab fragment specifically binding to FAP, which is fused to the C-terminus of a subunit of the Fc region, and (c) an Fc region composed of a first subunit and a second subunit capable of stable association, wherein the second Fab fragment (ab) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the first Fab fragment (aa), the VH-CH1 chain of the first Fab fragment (aa) is fused at its C-terminus to the N-terminus of the first subunit, and the third Fab fragment (ac) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit.

[0152] In one state, the bispecific antigen-binding molecule consists of: (aa) a first Fab fragment that specifically binds to OX40, (ab) a second Fab fragment that specifically binds to OX40, (ac) a third Fab fragment that specifically binds to OX40, (b) a cross-Fab fragment that specifically binds to FAP, which is fused to the C-terminus of a subunit of the Fc region, and (c) an Fc region consisting of a first subunit and a second subunit that can stably associate, wherein the second Fab fragment (ab) is fused to the N-terminus of the VH-CH1 chain of the first Fab fragment (aa) at the C-terminus, the VH-CH1 chain of the first Fab fragment (aa) is fused to the N-terminus of the first subunit at its C-terminus, and the third Fab fragment (ac) is fused to the N-terminus of the second subunit at the C-terminus of the Fab heavy chain.

[0153] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) a heavy chain comprising a VH-CH1 chain capable of specifically binding to a first Fab fragment of OX40 (the VH-CH1 chain is fused at its N-terminus to a VH-CH1 chain capable of specifically binding to a second Fab fragment of OX40 via a peptide linker) and an Fc subunit, (b) a heavy chain comprising a VH-CH1 domain capable of specifically binding to a Fab fragment of OX40, an Fc subunit, and a VH-CL chain capable of specifically binding to a Fab fragment of FAP (the VH-CL chain is fused at its C-terminus via a peptide linker), (c) three light chains, each light chain comprising a VL and CL domain capable of specifically binding to a Fab fragment of OX40, and (d) a light chain comprising a VL and CH1 domain capable of specifically binding to a Fab fragment of FAP.

[0154] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) a heavy chain comprising a VH-CH1 chain specifically binding to a first Fab fragment of OX40 (which is fused at its N-terminus to a VH-CH1 chain specifically binding to a second Fab fragment of OX40 via a peptide linker), an Fc subunit, and a VH-CL chain specifically binding to a Fab fragment of FAP (which is fused at its C-terminus via a peptide linker), (b) a heavy chain comprising a VH-CH1 domain specifically binding to a Fab fragment of OX40 and an Fc subunit, (c) three light chains, each light chain comprising a VL and CL domain specifically binding to a Fab fragment of OX40, and (d) a light chain comprising a VL and CH1 domain specifically binding to a Fab fragment of FAP.

[0155] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) a heavy chain comprising a VH-CH1 chain capable of specifically binding to a first Fab fragment of OX40 (the VH-CH1 chain being fused at its N-terminus to a VH-CH1 chain capable of specifically binding to a second Fab fragment of OX40 via a peptide linker) and an Fc subunit, (b) a heavy chain comprising a VH-CH1 domain capable of specifically binding to a Fab fragment of OX40, an Fc subunit, and a VL-CH1 chain capable of specifically binding to a Fab fragment of FAP (the VL-CH1 chain being fused at its C-terminus via a peptide linker), (c) three light chains, each light chain comprising VL and CL domains capable of specifically binding to a Fab fragment of OX40, and (d) a light chain comprising VH and CL domains capable of specifically binding to a Fab fragment of FAP.

[0156] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) a heavy chain comprising a VH-CH1 chain specifically binding to a first Fab fragment of OX40 (which is fused at its N-terminus to a VH-CH1 chain specifically binding to a second Fab fragment of OX40 via a peptide linker), an Fc subunit, and a VL-CH1 chain specifically binding to a Fab fragment of FAP (which is fused at its C-terminus via a peptide linker), (b) a heavy chain comprising a VH-CH1 domain specifically binding to a Fab fragment of OX40 and an Fc subunit, (c) three light chains, each light chain comprising a VL and CL domain specifically binding to a Fab fragment of OX40, and (d) a light chain comprising a VH and CL domain specifically binding to a Fab fragment of FAP.

[0157] In a specific state, the peptide linker is selected from GGGGS (SEQ ID NO:67), GGGGSGGGGS (SEQ ID NO:68), SGGGGSGGGG (SEQ ID NO:69), GGGGSGGGGGGG (SEQ ID NO:70), GSPGSSSSGS (SEQ ID NO:71), (G4S)3 (SEQ ID NO:72), (G4S)4 (SEQ ID NO:73), GSGSGSGS (SEQ ID NO:74), GSGSGNGS (SEQ ID NO:75), GGSGSGSG (SEQ ID NO:76), GGSGSG (SEQ ID NO:77), GGSG (SEQ ID NO:78), GGSGNGSG (SEQ ID NO:79), GGNGSGSG (SEQ ID NO:80), and GGNGSG (SEQ ID NO:81). Of particular interest are (G4S) (SEQ ID NO:67), (G4S)2 or GGGGSGGGGS (SEQ ID NO:68), (G4S)3 (SEQ ID NO:72) and (G4S)4 (SEQ ID NO:73).

[0158] In one state, a bispecific antigen-binding molecule is provided, comprising (a) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:86; a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:90; three light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; and a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; or (b) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; or (b) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; and a third light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; and a fourth light chain comprising an amino acid sequence that is at least about 90 The sequence of NO:86 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:90; a second heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:90; three light chains each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:87; and a light chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:94; or (c) a first heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:86; a second heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:86; and a light chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:86; The sequence of NO:90 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:87; three light chains, each comprising an amino acid sequence identical to the sequence of SEQ ID NO:96 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:96.In one state, a bispecific antigen-binding molecule is provided, comprising (a) a first heavy chain containing the amino acid sequence of SEQ ID NO:86; a second heavy chain containing the amino acid sequence of SEQ ID NO:90; three light chains, each containing the amino acid sequence of SEQ ID NO:87; and one light chain containing the amino acid sequence of SEQ ID NO:88; or (b) a first heavy chain containing the amino acid sequence of SEQ ID NO:86; a second heavy chain containing the amino acid sequence of SEQ ID NO:90; three light chains, each containing the amino acid sequence of SEQ ID NO:87; and one light chain containing the amino acid sequence of SEQ ID NO:94; or (c) a first heavy chain containing the amino acid sequence of SEQ ID NO:86; a second heavy chain containing the amino acid sequence of SEQ ID NO:90; three light chains, each containing the amino acid sequence of SEQ ID NO:87; and one light chain containing the amino acid sequence of SEQ ID NO:94. The amino acid sequence of NO:96.

[0159] In one state, a bispecific antigen-binding molecule is provided, comprising (a) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:97; a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:95; three light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:93; and a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88, or (b) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88. The sequence of NO:97 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:95; a second heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:95; three light chains each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:93; and a light chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:94; or (c) a first heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:97; a second ...5; a third light chain each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:97; a fourth light chain each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:97; a fifth light chain each comprising at least about 90%, 95 The sequence of NO:95 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:93; three light chains, each comprising an amino acid sequence identical to the sequence of SEQ ID NO:96 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences; and one light chain comprising an amino acid sequence identical to the sequence of SEQ ID NO:96 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences.In one state, a bispecific antigen-binding molecule is provided, comprising (a) a first heavy chain containing the amino acid sequence of SEQ ID NO:97; a second heavy chain containing the amino acid sequence of SEQ ID NO:95; three light chains, each containing the amino acid sequence of SEQ ID NO:93; and a light chain containing the amino acid sequence of SEQ ID NO:88, or (b) a first heavy chain containing the amino acid sequence of SEQ ID NO:97; a second heavy chain containing the amino acid sequence of SEQ ID NO:95; three light chains, each containing the amino acid sequence of SEQ ID NO:93; and a light chain containing the amino acid sequence of SEQ ID NO:94, or (c) a first heavy chain containing the amino acid sequence of SEQ ID NO:97; a second heavy chain containing the amino acid sequence of SEQ ID NO:95; three light chains, each containing the amino acid sequence of SEQ ID NO:93; and a light chain containing the amino acid sequence of SEQ ID NO:94. The amino acid sequence of NO:96.

[0160] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 99; a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 101; three light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 100; and a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 88, or (b) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 88, or (b) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 99. The sequence of NO:99 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:101; a second heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:101; three light chains each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:100; and a light chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:94; or (c) a first heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:99; a second ... third heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:99; a fourth heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:99; a fifth heavy chain comprising at least about 90%, 95 The sequence of NO:101 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:100; three light chains, each comprising an amino acid sequence identical to the sequence of SEQ ID NO:96 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:96.In one state, a bispecific antigen-binding molecule is provided, comprising (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO:99; a second heavy chain comprising the amino acid sequence of SEQ ID NO:101; three light chains, each comprising the amino acid sequence of SEQ ID NO:100; and a light chain comprising the amino acid sequence of SEQ ID NO:88; or (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO:99; a second heavy chain comprising the amino acid sequence of SEQ ID NO:101; three light chains, each comprising the amino acid sequence of SEQ ID NO:100; and a light chain comprising the amino acid sequence of SEQ ID NO:94; or (c) a first heavy chain comprising the amino acid sequence of SEQ ID NO:99; a second heavy chain comprising the amino acid sequence of SEQ ID NO:101; three light chains, each comprising the amino acid sequence of SEQ ID NO:100; and a light chain comprising the amino acid sequence of SEQ ID NO:94. The amino acid sequence of ID NO:96.

[0161] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:103; a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:105; three light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:104; and a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88, or (b) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:103; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; or (b) a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:103; and a third light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:104; and a fourth light chain comprising an amino acid sequence that is The sequence of NO:103 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:105; a second heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:105; three light chains each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:104; and a light chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:94; or (c) a first heavy chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:103; a second ... third light chain each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:103; a fourth light chain each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:103; a fifth light chain each comprising at The sequence of NO:105 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:104; three light chains, each comprising an amino acid sequence identical to the sequence of SEQ ID NO:96 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:96.In one state, a bispecific antigen-binding molecule is provided, comprising (a) a first heavy chain containing the amino acid sequence of SEQ ID NO:103; a second heavy chain containing the amino acid sequence of SEQ ID NO:105; three light chains, each containing the amino acid sequence of SEQ ID NO:104; and one light chain containing the amino acid sequence of SEQ ID NO:88; or (b) a first heavy chain containing the amino acid sequence of SEQ ID NO:103; a second heavy chain containing the amino acid sequence of SEQ ID NO:105; three light chains, each containing the amino acid sequence of SEQ ID NO:104; and one light chain containing the amino acid sequence of SEQ ID NO:94; or (c) a first heavy chain containing the amino acid sequence of SEQ ID NO:103; a second heavy chain containing the amino acid sequence of SEQ ID NO:105; and three light chains, each containing the amino acid sequence of SEQ ID NO:94. The amino acid sequence NO:104; and a light chain containing the amino acid sequence SEQ ID NO:96. A bispecific antigen-binding molecule (4+1 format) that is tetravalent for binding to OX40 and monovalent for binding to target cell antigens.

[0162] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising (a) four antigen-binding domains specifically binding to OX40, (b) one antigen-binding domain specifically binding to FAP, comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:5. (c) The amino acid sequence of NO:8; and (c) the Fc domain composed of a first subunit and a second subunit capable of stable association.

[0163] Therefore, a bispecific antigen-binding molecule is provided, wherein the bispecific antigen-binding molecule is tetravalently bound to OX40 and monovalently bound to FAP.

[0164] In one state, a bispecific antigen-binding molecule is provided, wherein four Fab fragments are specifically capable of binding to the antigen-binding domain of OX40, and each two of them are fused to each other at a heavy chain via a peptide linker, if applicable. In a specific state, the antigen-binding molecule comprises two heavy chains, each containing a VHCH1-peptide linker-VHCH1 fragment. In the specific state, the peptide linker has the amino acid sequence of SEQ ID NO:68.

[0165] In a single-state sample, the bispecific antigen-binding molecule consists of the following: (aa) specifically binding to the first Fab fragment of OX40, (ab) specifically binding to the second Fab fragment of OX40, (ac) specifically binding to the third Fab fragment of OX40, and (ad) specifically binding to the fourth Fab fragment of OX40. (b) a cross-Fab fragment capable of specifically binding to FAP, which fuses with the C-terminus of a subunit of the Fc region, and (c) an Fc region composed of a first subunit and a second subunit capable of stable association, wherein the second Fab fragment (ab) fuses with the N-terminus of the VH-CH1 chain of the first Fab fragment (aa) at the C-terminus of the VH-CH1 chain, the VH-CH1 chain of the first Fab fragment (aa) fuses with the N-terminus of the first subunit at its C-terminus, and the fourth Fab fragment (ad) fuses with the N-terminus of the VH-CH1 chain of the third Fab fragment (ac) at the C-terminus of the VH-CH1 chain, the VH-CH1 chain of the third Fab fragment (ac) fuses with the N-terminus of the second subunit at its C-terminus.

[0166] In one state, an antigen-binding molecule is provided, comprising: (a) four light chains, each light chain containing a VL and CL domain capable of specifically binding to a Fab fragment of OX40; (b) two heavy chains, each heavy chain containing a VH-CH1 domain (fused to the N-terminus of a VH-CH1 domain capable of specifically binding to a Fab fragment of OX40) and an Fc subunit capable of specifically binding to a Fab fragment of OX40; and (c) a cross-fab fragment capable of specifically binding to a FAP, wherein the VH-CL domain is linked to the C-terminus of one of the heavy chains via a peptide linker.

[0167] In one state, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:86; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:89; and (b) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:89; (c) Four light chains, each containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a first heavy chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; and a second heavy chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:89; and (d) four light chains, each containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; and a light chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87. The sequence of NO:96 comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:86; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:89; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:89.In one state, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:88; a first heavy chain containing the amino acid sequence of SEQ ID NO:86; and a second heavy chain containing the amino acid sequence of SEQ ID NO:89; (b) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:94; a first heavy chain containing the amino acid sequence of SEQ ID NO:86; and a second heavy chain containing the amino acid sequence of SEQ ID NO:89; or (c) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:96; a first heavy chain containing the amino acid sequence of SEQ ID NO:86; and a second heavy chain containing the amino acid sequence of SEQ ID NO:89. The amino acid sequence of NO:89.

[0168] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:93; a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:92; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:95; and (b) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:95; The sequence of NO:93 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:94; a first heavy chain comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:92; and a second heavy chain comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:95; or (c) four light chains, each comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:93; a light chain comprising at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequences identical to the sequence of SEQ ID NO:93; The sequence of NO:96 comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:92; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:95; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:95.In one state, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each containing the amino acid sequence of SEQ ID NO:93; one light chain containing the amino acid sequence of SEQ ID NO:88; a first heavy chain containing the amino acid sequence of SEQ ID NO:92; and a second heavy chain containing the amino acid sequence of SEQ ID NO:95; (b) four light chains, each containing the amino acid sequence of SEQ ID NO:93; one light chain containing the amino acid sequence of SEQ ID NO:94; a first heavy chain containing the amino acid sequence of SEQ ID NO:92; and a second heavy chain containing the amino acid sequence of SEQ ID NO:95; or (c) four light chains, each containing the amino acid sequence of SEQ ID NO:93; one light chain containing the amino acid sequence of SEQ ID NO:96; a first heavy chain containing the amino acid sequence of SEQ ID NO:92; and a second heavy chain containing the amino acid sequence of SEQ ID NO:95. The amino acid sequence of NO:95.

[0169] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:107; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:108; and (b) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; The sequence of NO:87 comprises at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% an amino acid sequence identical to the sequence of SEQ ID NO:87; a light chain comprising an amino acid sequence identical to the sequence of SEQ ID NO:94 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%; a first heavy chain comprising an amino acid sequence identical to the sequence of SEQ ID NO:107 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%; and a second heavy chain comprising an amino acid sequence identical to the sequence of SEQ ID NO:108 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%; or (c) four light chains, each comprising an amino acid sequence identical to the sequence of SEQ ID NO:87 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%; a light chain comprising an amino acid sequence identical to the sequence of SEQ ID NO:87 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%; and a light chain comprising an amino acid sequence identical to the sequence of SEQ ID NO:87 at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%; The sequence of NO:96 comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:107; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:108; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:108.In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:88; a first heavy chain containing the amino acid sequence of SEQ ID NO:107; and a second heavy chain containing the amino acid sequence of SEQ ID NO:108; or (b) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:94; a first heavy chain containing the amino acid sequence of SEQ ID NO:107; and a second heavy chain containing the amino acid sequence of SEQ ID NO:108; or (c) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:96; a first heavy chain containing the amino acid sequence of SEQ ID NO:107; and a second heavy chain containing the amino acid sequence of SEQ ID NO:108. The amino acid sequence of NO:108.

[0170] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:109; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:110; or (b) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:110; (c) Four light chains, each containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a light chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a first heavy chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; and a second heavy chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; and a light chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; and a light chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87. The sequence of NO:96 comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:109; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:110; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:110.In one state, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:88; a first heavy chain containing the amino acid sequence of SEQ ID NO:109; and a second heavy chain containing the amino acid sequence of SEQ ID NO:110; or (b) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:94; a first heavy chain containing the amino acid sequence of SEQ ID NO:109; and a second heavy chain containing the amino acid sequence of SEQ ID NO:110; or (c) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:96; a first heavy chain containing the amino acid sequence of SEQ ID NO:109; and a second heavy chain containing the amino acid sequence of SEQ ID NO:110. The amino acid sequence of NO:110.

[0171] In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a light chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:88; a first heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:111; and a second heavy chain comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:112; and (b) four light chains, each comprising an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; (c) Four light chains, each containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a light chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; a first heavy chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; and a second heavy chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; and a light chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87; and a light chain containing an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:87. The sequence of NO:96 comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:111; and the second heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:112.In another embodiment, a bispecific antigen-binding molecule is provided, comprising (a) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:88; a first heavy chain containing the amino acid sequence of SEQ ID NO:111; and a second heavy chain containing the amino acid sequence of SEQ ID NO:112; or (b) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:94; a first heavy chain containing the amino acid sequence of SEQ ID NO:111; and a second heavy chain containing the amino acid sequence of SEQ ID NO:112; or (c) four light chains, each containing the amino acid sequence of SEQ ID NO:87; one light chain containing the amino acid sequence of SEQ ID NO:96; a first heavy chain containing the amino acid sequence of SEQ ID NO:111; and a second heavy chain containing the amino acid sequence of SEQ ID NO:112. The amino acid sequence of NO:112. Modification of the Fc domain that reduces Fc receptor binding and / or effector function.

[0172] The bispecific antigen-binding molecule of the present invention further includes an Fc domain, which is composed of a first subunit and a second subunit capable of stable association.

[0173] In some forms, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0174] The Fc domain endows the bispecific antibodies of the present invention with favorable pharmacokinetic properties, including a long serum half-life (which facilitates good accumulation in target tissues) and a favorable tissue-to-blood distribution ratio. However, it can also cause the bispecific antibodies of the present invention to undesirably target cells expressing the Fc receptor, rather than targeting preferred antigen-carrying cells. Therefore, in certain embodiments, the Fc domain of the bispecific antibodies of the present invention exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the native IgG Fc domain, and specifically the IgG1 Fc domain or the IgG4 Fc domain. More specifically, the Fc domain is the IgG1 Fc domain.

[0175] In one of these states, the Fc domain (or a bispecific antigen-binding molecule containing the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% binding affinity to the Fc receptor compared to the native IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing the native IgG1 Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% effector function compared to the native IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing the native IgG1 Fc domain). In one state, the Fc domain (or the bispecific antigen-binding molecule of the present invention containing the Fc domain) does not substantially bind to the Fc receptor and / or induce effector function. In a particular state, the Fc receptor is an Fcγ receptor. In one state, the Fc receptor is a human Fc receptor. In one state, the Fc receptor is an activated Fc receptor. In specific states, Fc is regulated by the systemic activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one state, Fc is regulated by the systemic inhibitory Fc receptor. In specific states, Fc is regulated by the systemic inhibitory human Fcγ receptor, more specifically human FcγRIIB. In one state, the effector function is one or more of CDC, ADCC, ADCP, and intercytokine secretion. In a specific state, the effector function is ADCC. In one state, the Fc domain exhibits substantially similar binding affinity to the nascent Fc receptor (FcRn) compared to the native IgG1 Fc domain. When the Fc domain (or the bispecific antigen-binding molecule of the present invention containing the Fc domain) exhibits a binding affinity of more than about 70%, specifically more than about 80%, and more particularly more than about 90% of the natural IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention containing the natural IgG1 Fc domain) to FcRn, substantially similar binding to FcRn is achieved.

[0176] In a specific state, the Fc domain is engineered to have reduced binding affinity to the Fc receptor and / or reduced effector function compared to the unengineered Fc domain. In the specific state, the Fc domain of the bispecific antigen-binding molecule of the present invention contains one or more amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptor and / or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one state, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In another state, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In one state, compared to the bispecific antibody of the present invention containing an unengineered Fc domain, the bispecific antigen-binding molecule of the present invention containing an engineered Fc domain exhibits less than 20%, specifically less than 10%, and more specifically less than 5% binding affinity to the Fc receptor. In a specific state, the Fc receptor is a systemic Fcγ receptor. In other states, the Fc receptor is a systemic human Fc receptor. In one state, the Fc receptor is a systemic inhibitory Fc receptor. In a specific state, the Fc receptor is a systemic inhibitory human Fcγ receptor, more specifically human FcγRIIB. In some states, the Fc receptor is a systemic activating Fc receptor. In a specific state, the Fc receptor is a systemic activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some states, the binding affinity for complement components, specifically for C1q, is also reduced. In one state, the binding affinity for the nascent Fc receptor (FcRn) is not reduced. A substantially similar binding affinity to FcRn is achieved when the Fc domain (or the bispecific antigen-binding molecule of the present invention containing the Fc domain) exhibits a binding affinity greater than about 70% of the unengineered form of the Fc domain (or the bispecific antigen-binding molecule of the present invention containing the unengineered Fc domain) to the FcRn, i.e., the binding affinity of the Fc domain to the receptor is preserved. The Fc domain or the bispecific antigen-binding molecule of the present invention containing the Fc domain can exhibit this affinity greater than about 80% and even greater than about 90%. In some embodiments, the Fc domain of the bispecific antigen-binding molecule of the present invention is engineered to have reduced effector function compared to the unengineered Fc domain.Reduced effector functions may include (but are not limited to) one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immune complex-mediated antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling for apoptosis induction, reduced dendritic cell maturation, or reduced T cell initiation.

[0177] Antibodies with reduced effector function include those antibodies in which one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with alanine substituted at residues 265 and 297 (US Patent No. 7,332,581). Certain antibody variants that modify or weaken binding to FcR are described (e.g., US Patent No. 6,737,056; WO 2004 / 056312, and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0178] In one state, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331, and P329. In some states, the Fc domain comprises amino acid substitutions L234A and L235A ("LALA"). In one embodiment, the Fc domain is an IgG1 Fc domain, specifically a human IgG1 Fc domain. In one state, the Fc domain comprises an amino acid substitution at position P329. In a more specific state, the amino acid substitution is P329A or P329G, specifically P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and another amino acid substitution selected from the group consisting of: E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A, and P329G (“P329G LALA”). As described in PCT patent application WO 2012 / 130831 A1, the amino acid-substituted “P329G LALA” combination almost completely eliminates Fcγ receptor binding of the human IgG1 Fc domain. The document also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function). This antibody system contains IgG1 with mutations L234A and L235A or with mutations L234A, L235A, and P329G (numbered according to the EU index by Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0179] In one state, the Fc domain is the IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution (Kabat number) at position S228, specifically an amino acid substitution at S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising amino acid substitutions at L235E, S228P, and P329G. This amino acid substitution reduces in vivo Fab arm exchange of the IgG4 antibody (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).

[0180] Antibodies with extended half-lives and modified binding to neonatal Fc receptors (FcRn, which is responsible for transferring maternal IgG to the fetus, Guyer, RL et al., J. Immunol. 117 (1976) 587-593 and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434) are described in US 2005 / 0014934. These antibodies contain an Fc region having one or more substitutions that bind to the FcRn. These Fc variants include variants that have substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substituted Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan, AR, and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94 / 29351.

[0181] Binding to the Fc receptor can be readily determined, for example, using standard instruments (such as BIAcore instruments (GE Healthcare)) by ELISA or by surface plasma resonance (SPR), and Fc receptors such as [specific Fc receptors] can be obtained by recombinant expression. Suitable binding analyses are described herein. Alternatively, the binding affinity of the Fc domain or cell-activating bispecific antigen-binding molecules containing the Fc domain to the Fc receptor can be assessed using cell lines known to express a specific Fc receptor (such as human NK cells expressing the FcγIIIa receptor). The effector function of the Fc domain or the bispecific antigen-binding molecules of the present invention containing the Fc domain can be measured by methods known in this art. Suitable analyses for measuring ADCC are described herein. Other examples of in vitro analyses for evaluating the ADCC activity of molecules 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 analytical methods may be used (e.g., see ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells that can be used for these analyses include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in animal models, such as those described by Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0182] The following describes preferred embodiments of the bispecific antigen-binding molecule of the present invention, comprising an Fc domain modified to reduce Fc receptor binding and / or effector function. In one embodiment, the present invention relates to a bispecific antigen-binding molecule comprising (a) at least two antigen-binding domains specifically binding to OX40, (b) an antigen-binding domain specifically binding to FAP, and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antibody to the Fc receptor, and particularly to the Fcγ receptor. In another embodiment, the present invention relates to a bispecific antigen-binding molecule comprising (a) at least two antigen-binding domains specifically binding to OX40, (b) an antigen-binding domain specifically binding to FAP, and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, wherein the Fc domain comprises one or more amino acid substitutions that reduce effector function. In specific states, the Fc domains are human IgG1 subclass Fc domains with amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index). Fc domain modifications that promote heterodimerization.

[0183] The bispecific antigen-binding molecule of the present invention comprises different antigen-binding sites, which are fused to one or the other of the two subunits of the Fc domain, thereby allowing the two subunits of the Fc domain to be contained in two inconsistent polypeptide chains. Recombination of these polypeptides co-exists and subsequent dimerization produces several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antigen-binding molecule of the present invention in recombination production, it is advantageous to introduce modifications that promote the association of the desired polypeptide into the Fc domain of the bispecific antigen-binding molecule of the present invention.

[0184] Therefore, in a specific state, the present invention relates to a bispecific antigen-binding molecule comprising (a) at least two antigen-binding domains specifically binding to OX40, (b) an antigen-binding domain specifically binding to FAP, and (c) an Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein the Fc domain includes modifications that promote the association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one state, the modification is in the CH3 domain of the Fc domain.

[0185] In a specific embodiment, the modification is a so-called "bulge-pore" modification, wherein one of the two subunits of the Fc domain contains a "bulge" modification, and the other of the two subunits of the Fc domain contains a "pore" modification. Therefore, the present invention relates to a bispecific antigen-binding molecule comprising (a) at least two antigen-binding domains specifically capable of binding to OX40, (b) an antigen-binding domain specifically capable of binding to FAP, and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, wherein, according to the bulge-pore method, the first subunit of the Fc domain contains a bulge and the second subunit of the Fc domain contains a pore. In a specific state, the first subunit of the Fc domain contains amino acid substitutions of S354C and T366W (EU number), and the second subunit of the Fc domain contains amino acid substitutions of Y349C, T366S, and Y407V (numbered according to the Kabat EU index).

[0186] The bulge-pore technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a protrusion (“bulge”) at the interface of a first polypeptide and a corresponding cavity (“pore”) at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing the small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0187] Therefore, in one state, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen-binding molecule of the present invention, amino acid residues are replaced by amino acid residues with larger side chain volumes, thereby generating a protrusion within the CH3 domain of the first subunit. This protrusion can be located in a cavity within the CH3 domain of the second subunit. Furthermore, in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby generating a cavity within the CH3 domain of the second subunit. The protrusion within the CH3 domain of the first subunit can be located within this cavity. The protrusion and cavity can be generated, for example, by site-specific mutagenesis altering the nucleic acid encoding the polypeptide or by peptide synthesis. In one specific state, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced by 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 by a valine residue (Y407V). In another state, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A).

[0188] In another state, the serine residue at position 354 in the first subunit of the Fc domain is further substituted with a cysteine ​​residue (S354C), and the tyrosine residue at position 349 in the second subunit of the Fc domain is further substituted with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, which further stabilizes the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a specific state, the first subunit of the Fc domain contains amino acid substitutions of S354C and T366W (EU number), and the second subunit of the Fc domain contains amino acid substitutions of Y349C, T366S, and Y407V (numbered according to the Kabat EU index).

[0189] In the substitutional state, as described in, for example, PCT Publication WO 2009 / 089004, modifications that promote the association of the first and second subunits of the Fc domain include modifications that mediate electrostatic traction effects. Typically, this method involves replacing one or more amino acid residues at the interface between the two Fc domain subunits with charged amino acid residues, making homodimer formation electrostatically unfavorable but heterodimerization electrostatically favorable.

[0190] The C-terminus of the heavy chain of the bispecific antibody reported herein may be a complete C-terminus terminated with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two C-terminal amino acid residues have been removed. In a preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus terminated with PG. In one embodiment of all the embodiments reported herein, the bispecific antibody comprising a heavy chain including the C-terminal CH3 domain as specified herein contains a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of all the embodiments reported herein, the bispecific antibody comprising a heavy chain including the C-terminal CH3 domain as specified herein contains a C-terminal glycine residue (G446, numbered according to the Kabat EU index). Modifications in the Fab domain

[0191] In one embodiment, the present invention relates to a bispecific antigen-binding molecule comprising (a) at least two Fab fragments specifically binding to OX40, (b) a Fab fragment specifically binding to FAP, and (c) an Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein in one Fab fragment, either the variable domains VH and VL or the constant domains CH1 and CL are exchanged. The bispecific antibody is prepared according to the Crossmab technique.

[0192] Multispecific antibodies (CrossMab VH-VL or CrossMab CH-CL) with domain substitution / exchange in one binding arm are described in detail in WO2009 / 080252 and Schaefer, W. et al., PNAS, 108 (2011) 11187-1191. They significantly reduce byproducts resulting from mismatches between the light chain targeting the first antigen and the incorrect heavy chain targeting the second antigen (compared to methods that do not utilize this domain exchange).

[0193] In one embodiment, the present invention relates to a bispecific antigen-binding molecule comprising (a) at least two Fab fragments capable of specifically binding to OX40, (b) a Fab fragment capable of specifically binding to FAP, and (c) an Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein in one Fab fragment, constant domains CL and CH1 substitute for each other, such that the CH1 domain is part of a light chain and the CL domain is part of a heavy chain. More specifically, in the second Fab fragment capable of specifically binding to a target cell antigen, constant domains CL and CH1 substitute for each other, such that the CH1 domain is part of a light chain and the CL domain is part of a heavy chain.

[0194] In a specific state, the present invention relates to a bispecific antigen-binding molecule comprising (a) at least two Fab fragments capable of specifically binding to OX40, and (b) a Fab fragment capable of specifically binding to FAP, wherein in the Fab fragment capable of specifically binding to FAP, constant domains CL and CH1 substitute for each other, such that the CH1 domain is part of the light chain and the CL (Cκ) domain is part of the heavy chain.

[0195] Therefore, in one state, the present invention comprises a bispecific antigen-binding molecule comprising (a) two light chains and two heavy chains of an antibody, comprising two Fab fragments capable of specifically binding to OX40 and an Fc region, and (b) a cross-Fab fragment capable of specifically binding to FAP, which is fused to the C-terminus of a subunit of the Fc region.

[0196] In another state, and to further improve correct pairing, a bispecific antigen-binding molecule comprising (a) at least two Fab fragments specifically binding to OX40, (b) a cross-Fab fragment specifically binding to FAP, and (c) an Fc domain composed of a first and a second subunit capable of stable association may contain different charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the cross- or non-cross-connected CH1 and CL domains. In a particular state, the invention relates to a bispecific antigen-binding molecule wherein, in one CL domain, the amino acid at position 123 (EU number) has been replaced by arginine (R) and / or the amino acid at position 124 (EU number) has been replaced by lysine (K), and in one CH1 domain, the amino acids at positions 147 (EU number) and / or 213 (EU number) have been replaced by glutamic acid (E). Exemplary antibodies of the invention

[0197] In one embodiment, the present invention provides novel antibodies and antibody fragments that specifically bind to FAP. These antibodies bind to different antigenic determinants compared to known FAP antibodies 4B9 or 28H1, making them particularly suitable for incorporation into bispecific antigen-binding molecules that can be used in combination with other FAP-targeting molecules. These novel antibodies are further characterized by their ability to be produced in higher quantities and with higher titers, exhibiting high thermal stability (as measured by aggregation temperature Tagg), or presumably possessing excellent PK properties and binding to human FAP with high affinity as measured by Biacore analysis.

[0198] In one state, an antibody (pure line 212) specifically binding to FAP is provided, wherein the antibody comprises a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing an amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing an amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, (v) CDR-L2 containing an amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3 containing an amino acid sequence of SEQ ID NO:8.

[0199] In one state, a humanized antibody specifically binding to FAP is provided, wherein the antibody comprises a heavy chain variable region (VHFAP), the VHFAP comprising (i) CDR-H1, which comprises an amino acid sequence of SEQ ID NO:3, (ii) CDR-H2, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3, which comprises an amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP), the VLFAP comprising (iv) CDR-L1, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, (v) CDR-L2, which comprises an amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3, which comprises an amino acid sequence of SEQ ID NO:8.

[0200] In another state, an antibody is provided that competes with an antibody that specifically binds to the FAP, wherein the antibody comprises any of the heavy chain variable regions (VHFAPs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20; and any of the light chain variable regions (VLFAPs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.

[0201] In one state, an antibody is provided that competes with an antibody that specifically binds to FAP, wherein the antibody comprises a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:15 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO:21.

[0202] In another state, an antibody specifically binding to FAP is provided, wherein the antibody comprises (a) a heavy chain variable region (VHFAP) comprising the amino acid sequence of SEQ ID NO:15 and a light chain variable region (VLFAP) comprising the amino acid sequence of SEQ ID NO:21, (b) a heavy chain variable region (VHFAP) comprising the amino acid sequence of SEQ ID NO:16 and a light chain variable region (VLFAP) comprising the amino acid sequence of SEQ ID NO:21, (c) a heavy chain variable region (VHFAP) comprising the amino acid sequence of SEQ ID NO:16 and a light chain variable region (VLFAP) comprising the amino acid sequence of SEQ ID NO:22, or (d) a heavy chain variable region (VHFAP) comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region (VLFAP) comprising the amino acid sequence of SEQ ID NO:25.

[0203] In another state, an antibody that specifically binds to FAP is provided, comprising a heavy chain variable region (VHFAP) comprising the amino acid sequence of SEQ ID NO:15 and a light chain variable region (VLFAP) comprising the amino acid sequence of SEQ ID NO:21.

[0204] In another embodiment, the present invention provides novel antibodies and antibody fragments that specifically bind to OX40. These antibodies are variants of the OX40 antibody 49B4 and have fewer positive charge patches compared to 49B4. These novel antibodies should have improved PK properties compared to 49B4 and, as measured by Biacore analysis, bind to human OX40 with high affinity.

[0205] Therefore, a humanized antibody specifically binding to OX40 is provided, wherein the antibody comprises (a) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:59 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34, (b) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:60 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34, and (c) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO:61 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:34. Polynucleotide

[0206] The present invention further provides isolated nucleic acids encoding bispecific antigen-binding molecules or fragments thereof as described herein, or isolated nucleic acids encoding antibodies as described herein.

[0207] The isolated polynucleotide encoding the bispecific antigen-binding molecule of the present invention can be expressed as a single polynucleotide encoding the entire antigen-binding molecule or as multiple (e.g., two or more) co-expressed polynucleotides. The polypeptides encoded by the co-expressed polynucleotides can associate via, for example, disulfide bonds or other means to form a functional antigen-binding molecule. For example, the light chain portion of an immunoglobulin can be encoded by a single polynucleotide from the heavy chain portion of that immunoglobulin. In co-expression, the heavy chain polypeptide will associate with the light chain polypeptide to form an immunoglobulin.

[0208] In some samples, the isolated polynucleotide encodes a polypeptide contained in the bispecific molecule of the present invention as described herein.

[0209] In one state, the present invention relates to an isolated polynucleotide encoding a bispecific antigen-binding molecule comprising: (a) at least two antigen-binding domains specifically binding to OX40; (b) an antigen-binding domain specifically binding to FAP, comprising: a heavy chain variable region (VHFAP) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO:3, (ii) CDR-H2 containing the amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1 containing the amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, and (v) CDR-L2 containing the amino acid sequence of SEQ ID NO:5. (vi) the amino acid sequence of NO:7, and (vi) CDR-L3, which contains the amino acid sequence of SEQ ID NO:8; and (c) the Fc domain composed of a first subunit and a second subunit capable of stable association.

[0210] In some embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded. Recombination Method

[0211] The bispecific antigen-binding molecule of the present invention can be obtained, for example, by recombination. For recombination, one or more polynucleotides encoding a bispecific antigen-binding molecule or a polypeptide fragment thereof are provided. The one or more polynucleotides encoding the bispecific antigen-binding molecule are isolated and inserted into one or more vectors for further selection and / or expression in host cells. This polynucleotide can be easily isolated and sequenced using conventional procedures. In one embodiment of the present invention, a vector comprising one or more of the polynucleotides of the present invention, preferably an expression vector, is provided. Expression vectors containing the coding sequence of the bispecific antigen-binding molecule (fragment) and appropriate transcription / translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA technology, synthetic techniques, and in vivo recombination / genetic recombination. For example, see the techniques described in the following literature: Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989). Expression vectors can be plasmids, part of a virus, or a nucleic acid fragment. Expression vectors include an expression cassette in which a polynucleotide (i.e., the coding region) encoding a bispecific antigen-binding molecule or a polypeptide fragment thereof is selectively and operatively associated with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of nucleic acid consisting of codons that translate into amino acids. Although "stop codons" (TAG, TGA, or TAA) are not translated into amino acids, if present, they can be considered part of a coding region, while any side sequences (e.g., promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, and the like) are not part of a coding region. Two or more coding regions may exist in a single polynucleotide construct (e.g., on a single vector) or in separate polynucleotide constructs (e.g., on separate (different) vectors). Furthermore, any vector may contain a single coding region or may contain two or more coding regions; for example, the vectors of the present invention may encode one or more polypeptides, which are subsequently translated or co-translated and separated into the final protein via proteolytic cleavage. Additionally, the vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions fused or not fused to polynucleotides encoding the bispecific antigen-binding molecule of the present invention, its polypeptide fragments, or their variants or derivatives thereof.Heterologous coding regions include (but are not limited to) specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association occurs when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in such a way that the expression of the gene product is under the influence or control of those regulatory sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably associated" when the promoter function induces transcription of mRNA encoding the desired gene product, and the bonding between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to guide the expression of the gene product or to interfere with the transcription of the DNA template. Therefore, if a promoter can enable the transcription of a nucleic acid encoding a polypeptide, the promoter region will operably associate with that nucleic acid. A promoter can be a cell-specific promoter that guides substantial transcription of DNA only in a predetermined cell. In addition to promoters, other transcriptional control elements (e.g., enhancers, operants, repressors, and transcription termination signals) can also operably associate with polynucleotides to guide cell-specific transcription.

[0212] This article discloses suitable promoters and other transcriptional control regions. Those skilled in the art are familiar with various transcriptional control regions. These include (but are not limited to) transcriptional control regions that function in vertebrate cells, such as (but not limited to) promoter and enhancer segments derived from: cytomegaloviruses (e.g., the immediate early promoter linked to intron-A), simian virus 40 (e.g., the early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (e.g., actin, heat shock proteins, bovine growth hormone, and rabbit γ-globin) and other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., the tetracycline inducible promoter). Similarly, those skilled in the art are familiar with various translational control elements. These translation control elements include (but are not limited to) ribosome binding sites, translation start and stop codons, and elements derived from the viral system (specifically, internal ribosome entry sites or IRES, also known as CITE sequences). The cassette may also include other features such as origins of replication and / or chromosomal integration elements (such as retroviral long terminal repeats (LTRs) or adeno-associated virus (AAV) terminal inverted repeats (ITRs)).

[0213] The polynucleotide and nucleic acid coding regions of the present invention can associate with other coding regions that encode secretory peptides or signal peptides, which guide the secretion of polypeptides encoded by the polynucleotides of the present invention. For example, if the secretion of a bispecific antigen-binding molecule or its polypeptide fragment is desired, the DNA encoding the signal sequence can be placed upstream of the nucleic acid encoding the bispecific antigen-binding molecule or its polypeptide fragment of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have signal peptides or secretory leader sequences that cleave from the mature protein once the growing protein chain begins to exit through the rough endoplasmic reticulum. Those skilled in the art will understand that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which cleaves from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. In some embodiments, a natural signal peptide (e.g., an immunoglobulin heavy chain or light chain signal peptide) is used, or a functional derivative of the sequence is used, which retains the ability to guide the secretion of the polypeptide with which it is operatively associated. Alternatively, a heterologous mammalian signal peptide or its functional derivative can be used. For example, the wild-type leader sequence can be replaced by a leader sequence from human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0214] DNA encoding a short protein sequence may be incorporated within or at the end of a polynucleotide encoding the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof. This short protein sequence may be used to facilitate subsequent purification (e.g., histidine tagging) or to help label fusion proteins.

[0215] In another embodiment of the invention, a host cell comprising one or more of the polynucleotides of the invention is provided. In some embodiments, a host cell comprising one or more vectors of the invention is provided. These polynucleotides and vectors may be incorporated individually or in combination into any of the features described herein, respectively, associated with the polynucleotides and vectors. In one embodiment, the host cell comprises a vector comprising a polynucleotide (e.g., transformed or transfected therein) encoding a portion of the bispecific antigen-binding molecule of the invention. As used herein, the term "host cell" refers to any type of cellular system that can be engineered to generate the fusion protein or fragments thereof of the invention. Host cells suitable for replicating antigen-binding molecules and supporting their expression are well known in the art. Such cells can be transfected or transduced with specific expression vectors as needed, and large quantities of vector-containing cells can be grown for inoculation into large-scale fermenters to obtain sufficient quantities of antigen-binding molecules for clinical applications. Suitable host cells include prokaryotic microorganisms (such as Escherichia coli) or various eukaryotic cells (such as Chinese hamster ovary cells (CHO), insect cells, or the like). For example, peptides can be produced in bacteria, specifically when glycosylation is not required. After expression, the peptides can be isolated from the soluble fraction of the bacterial cell sap and can be further purified. In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeasts) are also suitable colonization or expression hosts for peptide-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in peptides with partial or complete human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006).

[0216] Suitable host cells for expressing (glycosylated) peptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Several baculovirus strains have been identified that can be used in conjunction with insect cells, specifically for transfecting fall armyworm (Spodoptera frugiperda) cells. Plant cell cultures can also be used as hosts. For example, see U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (depicting PLATNIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7), human embryonic kidney cell lines (293 or 293T cells, as described in Graham et al., J Gen Virol 36, 59 (1977)), young hamster kidney cells (BHK), mouse Support cells (TM4 cells, as described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), and TRI cells (as described in Mather et al., Annals NYAcad Sci 383, 44-68). (described in 1982), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / O. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).Host cells include cultured cells (to name just a few, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells), and also include cells contained within genetically modified animals, genetically modified plants, or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphoid cells (e.g., Y0, NSO, Sp20 cells). Standard techniques for expressing exogenous genes in such systems are known in this art. Cells expressing polypeptides containing either the heavy or light chain of immunoglobulins can be engineered to also express the other of the immunoglobulin chains, such that the expressed product is an immunoglobulin having both heavy and light chains.

[0217] In one embodiment, a method for generating the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof is provided, wherein the method comprises culturing a host cell as provided herein under conditions suitable for expressing the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof, the host cell containing a polynucleotide encoding the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof, and recovering the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof from the host cell (or host cell culture medium).

[0218] The bispecific molecules of the present invention prepared as described herein can be purified by techniques known in this art, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, particle size separation chromatography, and the like. The actual conditions for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens bound to the bispecific antigen-binding molecules can be used. For example, for affinity chromatography purification of the fusion protein of the present invention, a matrix containing protein A or protein G can be used. The antigen-binding molecules can be separated by sequential protein A or G affinity chromatography and particle size separation chromatography, essentially as described in the examples. The purity of the bispecific antigen-binding molecules or fragments thereof can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-performance liquid chromatography, and the like. For example, as demonstrated by reducing and non-reducing SDS-PAGE, the bispecific antigen-binding molecules exhibited in the examples are shown to be complete and properly assembled. Analysis

[0219] The binding properties and / or biological activity of the antigen-binding molecules provided herein can be characterized by various analyses known in this art. Specifically, characterization is performed by analyses described in more detail in the examples. 1. Binding Analysis

[0220] For example, the binding of the bispecific antigen-binding molecule provided herein to the corresponding target-expressing cells can be assessed using a mouse fibroblast cell line expressing human fibroblast activation protein (FAP) and flow cytometry (FACS) analysis. The binding of the bispecific antigen-binding molecule provided herein to OX40 can be determined using activated human PBMCs as described in Example 3.1. 2. Activity Assay

[0221] The biological activity of the bispecific antigen-binding molecule of the present invention was tested. Biological activity may include the efficacy and specificity of the bispecific antigen-binding molecule. Efficacy and specificity were demonstrated by an analysis showing that activating signaling was achieved via the OX40 receptor upon binding to the target antigen. Furthermore, as illustrated in Example 4.1, the stimulation of OX40 signaling was measured by induced NFκB activation in human OX40-positive NFκB reporter cells. Pharmaceutical compositions, formulations, and routes of administration.

[0222] In another embodiment, the present invention provides a pharmaceutical composition comprising any of the bispecific antigen-binding molecules provided herein, for example, for use in any of the treatment methods described below. In one embodiment, the pharmaceutical composition comprises any of the bispecific antigen-binding molecules provided herein and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises any of the bispecific antigen-binding molecules provided herein and at least one additional therapeutic agent, for example, as described below.

[0223] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more bispecific antigen-binding molecules dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" means a molecular entity or composition that, when administered to animals such as humans as appropriate, is generally non-toxic to the recipient at the doses and concentrations used, i.e., will not produce adverse, allergic, or other adverse reactions. Those skilled in the art will recognize from this disclosure the preparation of pharmaceutical compositions containing at least one of the bispecific antigen-binding molecules of the present invention and, where applicable, additional active ingredients, as illustrated in Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. Specifically, the composition is a lyophilized formulation or an aqueous solution. As used herein, "medically acceptable excipients" include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers, and combinations thereof, as known to those skilled in the art.

[0224] Non-enteral compositions include those designed for administration by injection, such as subcutaneous, intradermal, intralesional, intravenous, intra-arterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the bispecific antigen-binding molecule of the present invention can be formulated in an aqueous solution, preferably a physiologically compatible buffer (such as Hank's solution, Ringer's solution, or physiological saline buffer). The solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the bispecific antigen-binding molecule may be in powder form and prepared with a suitable medium (e.g., sterile pyrogen-free water) prior to use. A sterile injectable solution is prepared by incorporating the antigen-binding molecule of the present invention, along with (as needed) various other components listed below, into a suitable solvent in the desired amount. Sterility can be readily achieved, for example, by filtration through a sterile filter membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile medium containing a basic dispersion medium and / or other components. In the case of preparing sterile powders for sterile injectable solutions, suspensions, or emulsions, a preferred preparation method is vacuum drying or freeze-drying, which produces a powder of the active ingredient plus any other desired components from a previously sterile filtered liquid medium. The liquid medium should be appropriately buffered as needed, and the liquid diluent should be made isotonic with sufficient saline or glucose before injection. The composition must be stable under manufacturing and storage conditions and protected against microbial contamination such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a minimum safe level, for example, less than 0.5 ng / mg protein. Suitable excipients that are pharmaceutically acceptable include (but are not limited to): buffers, such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; benzalkonium chloride; benzyl chloride). Chloride); phenol; butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; 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, glutamic acid, aspartic acid, histamine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming ions, such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, polydextrose, or similar substances. Depending on the application, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions. Alternatively, suspensions of active compounds can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or mediators include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes.

[0225] The active ingredient may be encapsulated, for example, in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules) prepared by cohesion techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in coarse drop emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained-release formulations may be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix containing a solid hydrophobic polymer of a polypeptide, wherein the matrix is ​​in the form of a molded article, such as a membrane or microcapsule. In certain embodiments, the absorption of the injectable composition may be prolonged by using an absorption-delaying agent (such as aluminum monostearate, gelatin, or a combination thereof) in the composition.

[0226] The exemplary pharmaceutically acceptable excipients described herein further include interstitial drug dispersants, 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 Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycans (such as chondroitinase).

[0227] 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 WO2006 / 044908, the latter of which include histidine-acetate buffer.

[0228] In addition to the compositions described above, antigen-binding molecules can also be formulated into storage formulations. These long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, fusion proteins can be formulated using suitable polymeric or hydrophobic materials (e.g., formulated as emulsions acceptable in oils) or ion exchange resins, or formulated as poorly soluble derivatives, such as poorly soluble salts.

[0229] Pharmaceutical compositions comprising the bispecific antigen-binding molecule of the present invention can be manufactured using conventional mixing, dissolving, emulsifying, encapsulating, embedding, or lyophilizing processes. The pharmaceutical compositions can be formulated conventionally using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate the processing of proteins into pharmaceutically usable formulations. The appropriate formulation depends on the chosen route of administration.

[0230] Bispecific antigen-binding molecules can be formulated into compositions in the form of free acids or bases, neutral or salts. Pharmaceutically acceptable salts are those that substantially retain the biological activity of the free acid or base. These salts include acid addition salts, such as those forming with the free amino group of a protein composition, or those forming with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, or mandelic acid). Salts forming with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts are often more soluble in aqueous and other protic solvents than their corresponding free base forms.

[0231] The compositions herein may also contain one or more active ingredients for the specific indications being treated, preferably complementary active ingredients that do not adversely affect each other. These active ingredients are suitable to be present in combination in an amount effective for the intended purpose.

[0232] The formulation intended for in vivo administration is typically sterile. Sterility can be easily achieved, for example, by filtration through a sterile filter membrane. Treatment methods and compositions

[0233] Any of the bispecific antigen-binding molecules provided herein can be used in therapeutic applications. For use in therapeutic applications, the bispecific antigen-binding molecules of this invention can be formulated, administered, and given in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, etiology, site of delivery, method of administration, timing of administration, and other factors known to the practicing physician.

[0234] In one state, the present invention provides a bispecific antigen-binding molecule for use as a pharmaceutical agent.

[0235] In other embodiments, the bispecific antigen-binding molecule of the present invention is provided for the following purposes: (i) inducing immune stimulation, (ii) stimulating tumor-specific T cell responses, (iii) inducing apoptosis of tumor cells, (iv) treating cancer, (v) delaying cancer progression, (vi) prolonging the survival of patients with cancer, and (vii) treating infections. In certain embodiments, the bispecific antigen-binding molecule of the present invention is provided for treating diseases, and specifically for treating cancer.

[0236] In some embodiments, the present invention provides a bispecific antigen-binding molecule for use in a treatment method. In one embodiment, the present invention provides a bispecific antigen-binding molecule as described herein for treating a disease in an individual in need. In some embodiments, the present invention provides a bispecific antigen-binding molecule for treating an individual suffering from a disease, the method comprising administering a therapeutically effective amount of the bispecific antigen-binding molecule to the individual. In some embodiments, the disease to be treated is cancer. The individual in need of treatment, the patient, or the "individual" is generally a mammal, more specifically a human.

[0237] In one embodiment, a method is provided for the following purposes: i) inducing immune stimulation, (ii) stimulating a tumor-specific T cell response, (iii) inducing apoptosis of tumor cells, (iv) treating cancer, (v) delaying cancer progression, (vi) prolonging the survival of a patient with cancer, or (vii) treating an infection, wherein the method comprises administering a therapeutically effective amount of the bispecific antigen-binding molecule of the present invention to an individual in need.

[0238] In another embodiment, the present invention provides the use of the bispecific antigen-binding molecule of the present invention for manufacturing or preparing a medicament for treating a disease in an individual in need. In one embodiment, the method of using the medicament to treat a disease includes administering a therapeutically effective amount of the medicament to an individual suffering from the disease. In some embodiments, the disease to be treated is a proliferative disorder, specifically cancer. Examples of cancer include (but are not limited to) bladder cancer, 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, stomach cancer, prostate cancer, leukemia, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other examples of cancer include carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Other cell proliferation disorders that can be treated with the bispecific antigen-binding molecules or antibodies of this invention include (but are not limited to) tumors located in the following sites: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest region, and genitourinary system. This also includes precancerous lesions or cancer metastases. In some embodiments, the cancer is selected from the group consisting of: renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer. Those skilled in the art will readily recognize that in many cases, the bispecific antigen-binding molecules or antibodies of this invention may not provide a cure, but may provide certain benefits. In some cases, beneficial physiological changes are also considered therapeutically beneficial. Therefore, in some cases, the amount of the bispecific antigen-binding molecule or antibody of the present invention that provides physiological changes is considered an "effective amount" or a "therapeutic effective amount".

[0239] For the prevention or treatment of disease, the appropriate dosage of the bispecific antigen-binding molecule of the present 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 patient's weight, the specific molecule, the severity and course of the disease, whether the bispecific antigen-binding molecule of the present invention is administered for preventive or therapeutic purposes, prior or concurrent therapeutic interventions, the patient's clinical history and response to the bispecific antigen-binding molecule, and the attending physician's decision. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual. Various administration schedules are considered herein, including (but not limited to) single or multiple administrations at various time points, concentrated administration, and pulsed infusion.

[0240] The bispecific antigen-binding molecule of the present invention is suitable for single or series of treatments administered to a patient. Depending on the type and severity of the disease, whether administered, for example, by one or more separate administrations or by continuous infusion, a bispecific antigen-binding molecule of about 1 µg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg) may be an initial candidate dose for administration to the patient. Depending on the factors mentioned above, a typical daily dose range may be about 1 µg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the disease, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the bispecific antigen-binding molecule of the present invention will be in the range of about 0.005 mg / kg to about 10 mg / kg. In other instances, the dosage administered may also include approximately 1 μg / kg body weight, approximately 5 μg / kg body weight, approximately 10 μg / kg body weight, approximately 50 μg / kg body weight, approximately 100 μg / kg body weight, approximately 200 μg / kg body weight, approximately 350 μg / kg body weight, approximately 500 μg / kg body weight, approximately 1 mg / kg body weight, approximately 5 mg / kg body weight, approximately 10 mg / kg body weight, approximately 50 mg / kg body weight, approximately 100 mg / kg body weight, approximately 200 mg / kg body weight, approximately 350 mg / kg body weight, approximately 500 mg / kg body weight, or approximately 1000 mg / kg body weight or more, and any range derived therefrom. In examples of derivable ranges derived from the values ​​listed herein, based on the aforementioned values, a range may be administered from approximately 0.1 mg / kg body weight to approximately 20 mg / kg body weight, approximately 5 μg / kg body weight to approximately 1 mg / kg body weight, etc. Therefore, patients can be administered one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof). These doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about 2 to about 20, or for example about 6, doses of the fusion protein). In a specific formulation, the bispecific antigen-binding molecule will be administered every three weeks. A higher loading dose can be administered at the start, followed by one or more lower doses. However, other dosing regimens may be used. Progression of this therapy can be easily monitored using conventional techniques and analyses.

[0241] The bispecific antigen-binding molecules of the present invention are generally used in an amount that effectively achieves the intended purpose. For the treatment or prevention of disease conditions, the bispecific antigen-binding molecules of the present invention or their pharmaceutical compositions are administered or applied in a therapeutically effective amount. The determination of the therapeutically effective amount is well known to those skilled in the art, and can be determined in particular based on the detailed disclosure provided herein. For systemic administration, the therapeutically effective dose can first be estimated from in vitro analyses (such as cell culture analyses). The dose can then be adjusted in animal models to achieve a range of circulating concentrations, including the IC50 as determined in cell culture. This information can be used to more accurately determine the dose usable in humans. Initial doses can also be estimated from in vivo data (e.g., animal models) using techniques well known in the art. Those skilled in the art can easily optimize human administration based on animal data.

[0242] The dosage and interval can be individually adjusted to provide plasma levels of the bispecific antigen-binding molecule of the present invention sufficient to maintain a therapeutic effect. Commonly used patient doses for administration by injection range from about 0.1 mg / kg / day to 50 mg / kg / day, typically from about 0.1 mg / kg / day to 1 mg / kg / day. Therapeuticly effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by HPLC. In cases of local administration or selective uptake, the effective local concentration of the bispecific antigen-binding molecule or antibody of the present invention may be independent of plasma concentration. Those skilled in the art can optimize the therapeutically effective local dose without extensive experimentation.

[0243] The therapeutically effective dose of the bispecific antigen-binding molecule of the present invention described herein will generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the fusion protein can be determined in cell cultures or laboratory animals using standard pharmaceutical procedures. The LD50 (lethal dose to 50% of the population) and ED50 (the therapeutically effective dose to 50% of the population) can be determined using cell culture analysis and animal studies. The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Bispecific antigen-binding molecules exhibiting a larger therapeutic index are preferred. In one state, the bispecific antigen-binding molecule or antibody of the present invention exhibits a high therapeutic index. Data obtained from cell culture analysis and animal studies can be used to determine the dosage range suitable for humans. The dose is preferably within the circulating concentration range including the ED50, with very low or no toxicity. The dose can vary within this range depending on various factors such as the dosage form used, the route of administration, the individual's disease, and the like. The actual preparation, route of administration, and dosage can be selected by the individual physician based on the patient's condition (see, for example, Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Chapter 1, page 1, which is incorporated herein by reference in its entirety).

[0244] The attending physician of a patient treated with the fusion protein of this invention will be aware of the need to terminate, interrupt, or adjust the administration method and timing due to toxicity, organ dysfunction, and the like. Conversely, if the clinical response is insufficient (excluding toxicity), the attending physician will also be aware of the need to adjust the treatment to a higher level. In the management of the disease of concern, the order of magnitude of the administered dose will vary depending on the severity of the disease to be treated, the route of administration, and the like. For example, the severity of the disease can be assessed in part by standard prognostic assessment methods. In addition, the dose and (possibly) the frequency of dose will also vary according to the individual patient's age, weight, and response. Other agents and treatments

[0245] In the therapy, the bispecific antigen-binding molecule of the present invention may be administered in combination with one or more other agents. For example, the bispecific antigen-binding molecule of the present invention may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent that can be administered to treat symptoms or diseases in an individual who requires such treatment. This additional therapeutic agent may contain any active ingredient suitable for the specific indication being treated, preferably complementary active ingredients that do not adversely affect each other. In some embodiments, the additional therapeutic agent is another anticancer agent, such as a microtubule disruptor, antimetabolite, topoisomerase inhibitor, DNA intercalator, alkylating agent, hormone therapy, kinase inhibitor, receptor antagonist, tumor cell apoptosis activator, or antiangiogenic agent. In some embodiments, the additional therapeutic agent is an immunomodulator, cell growth inhibitor, cell adhesion inhibitor, cytotoxic agent, or cell growth inhibitor, apoptosis activator, or agent that increases the sensitivity of cells to apoptosis inducers.

[0246] Therefore, the present invention provides a bispecific antigen-binding molecule for treating cancer or a pharmaceutical composition comprising the present invention, wherein the bispecific antigen-binding molecule is administered in combination with a chemotherapeutic agent, radiation and / or other agents for cancer immunotherapy.

[0247] These other agents are suitable to be present in combination in an amount effective for the intended purpose. The effective amount of these other agents depends on the amount of fusion protein used, the type of disease or treatment, and other factors discussed above. The bispecific antigen-binding molecules or antibodies of the present invention are generally used at the same dose and via the route of administration as described herein, or at about 1% to 99% of the dose described herein, or at any dose and via any route determined empirically / clinically as appropriate. Other agents for cancer immunotherapy may also include vaccines, TLR receptor (TLR) agents, and oncolytic viruses.

[0248] The above-described combination therapies cover combination administration (in which two or more therapeutic agents are included in the same or separate compositions) and separate administration, in which the bispecific antigen-binding molecule or antibody of the present invention may be administered before, simultaneously with and / or after administration of additional therapeutic agents and / or adjuvants.

[0249] In another embodiment, a bispecific antigen-binding molecule as described above is provided for the treatment of cancer, wherein the bispecific antigen-binding molecule is administered in combination with another immunomodulator.

[0250] The term "immunomodulator" refers to any substance that affects the immune system, including monoclonal antibodies. The molecules of the present invention can be considered immunomodulators. Immunomodulators can be used as anti-tumor agents for the treatment of cancer. In one embodiment, the immunomodulator includes (but is not limited to) anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD1 antibodies (e.g., nivorumab or pembrolizumab), PD-L1 antibodies (e.g., atezolizumab, avelumab, or durvalumab), ICOS antibodies, 4-1BB antibodies, and GITR antibodies. In another embodiment, a bispecific antigen-binding molecule as described above is provided for the treatment of cancer, wherein the bispecific antigen-binding molecule is administered in combination with an agent that blocks PD-L1 / PD-1 interaction. In one embodiment, the agent that blocks PD-L1 / PD-1 interaction is an anti-PD-L1 antibody or an anti-PD1 antibody. More specifically, the agent blocking PD-L1 / PD-1 interaction is an anti-PD-L1 antibody, specifically an anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, pembrolizumab, and nivorumab. In one specific sample, the agent blocking PD-L1 / PD-1 interaction is atezolizumab (MPDL3280A, RG7446). In another sample, the agent blocking PD-L1 / PD-1 interaction is an anti-PD-L1 antibody comprising the heavy chain variable domain VH (PDL-1) of SEQ ID NO:149 and the light chain variable domain VL (PDL-1) of SEQ ID NO:150. In another embodiment, the agent blocking PD-L1 / PD-1 interaction is an anti-PD-L1 antibody comprising the heavy chain variable domain VH (PDL-1) of SEQ ID NO:151 and the light chain variable domain VL (PDL-1) of SEQ ID NO:152. In yet another embodiment, the agent blocking PD-L1 / PD-1 interaction is an anti-PD1 antibody, specifically an anti-PD1 antibody selected from pembrolizumab or nivorumab. These other agents are suitable to be present in combination at an amount effective for the intended purpose. The effective amount of these other agents depends on the amount of bispecific antigen-binding molecule used, the type of disease or treatment, and other factors discussed above. Bispecific antigen-binding molecules as described above are generally used at the same dose and via the route of administration as described herein, or at about 1% to 99% of the dose described herein, or at any dose and via any route determined empirically / clinically as appropriate.

[0251] In one embodiment, a bispecific activating OX40 antigen-binding molecule or pharmaceutical composition for treating cancer is provided, wherein the bispecific activating OX40 antigen-binding molecule is used for combined administration with a T-cell activating anti-CD3 bispecific antibody. In another embodiment, a T-cell activating anti-CD3 bispecific antibody system specific to tumor-associated antigens is provided, comprising an anti-CEA / anti-CD3 bispecific antibody.

[0252] In a specific state, the anti-CD3 bispecific antibody used in combination comprises a first antigen-binding domain comprising: a heavy chain variable region (VHCD3) comprising the CDR-H1 sequence of SEQ ID NO:117, the CDR-H2 sequence of SEQ ID NO:118, and the CDR-H3 sequence of SEQ ID NO:119; and / or a light chain variable region (VLCD3) comprising the CDR-L1 sequence of SEQ ID NO:120, the CDR-L2 sequence of SEQ ID NO:121, and the CDR-L3 sequence of SEQ ID NO:122. More specifically, the anti-CD3 bispecific antibody comprises a first antigen-binding domain comprising a heavy chain variable region (VHCD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:123 and / or a light chain variable region (VLCD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:124. In another embodiment, the anti-CD3 bispecific antibody comprises a heavy chain variable region (VHCD3) comprising the amino acid sequence of SEQ ID NO:123 and / or a light chain variable region (VLCD3) comprising the amino acid sequence of SEQ ID NO:124.

[0253] In another embodiment, the anti-CD3 bispecific antibody used in combination comprises a first antigen-binding domain comprising: a heavy chain variable region (VHCD3) comprising the CDR-H1 sequence of SEQ ID NO:125, the CDR-H2 sequence of SEQ ID NO:126, and the CDR-H3 sequence of SEQ ID NO:127; and / or a light chain variable region (VLCD3) comprising the CDR-L1 sequence of SEQ ID NO:128, the CDR-L2 sequence of SEQ ID NO:129, and the CDR-L3 sequence of SEQ ID NO:130. More specifically, the anti-CD3 bispecific antibody includes a first antigen-binding domain comprising a heavy chain variable region (VHCD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:131 and / or a light chain variable region (VLCD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:132. In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VHCD3) comprising the amino acid sequence of SEQ ID NO:131 and / or a light chain variable region (VLCD3) comprising the amino acid sequence of SEQ ID NO:132.

[0254] In another embodiment, the anti-CD3 bispecific antibody used in combination comprises a first antigen-binding domain comprising: a heavy chain variable region (VHCD3) comprising the CDR-H1 sequence of SEQ ID NO:133, the CDR-H2 sequence of SEQ ID NO:134, and the CDR-H3 sequence of SEQ ID NO:135; and / or a light chain variable region (VLCD3) comprising the CDR-L1 sequence of SEQ ID NO:136, the CDR-L2 sequence of SEQ ID NO:137, and the CDR-L3 sequence of SEQ ID NO:138. More specifically, the anti-CD3 bispecific antibody includes a first antigen-binding domain comprising a heavy chain variable region (VHCD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:139 and / or a light chain variable region (VLCD3) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:140. In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises a heavy chain variable region (VHCD3) comprising the amino acid sequence of SEQ ID NO:139 and / or a light chain variable region (VLCD3) comprising the amino acid sequence of SEQ ID NO:140.

[0255] In one specific embodiment, the anti-CEA / anti-CD3 bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 141, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 142, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 143, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 144. In another specific embodiment, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 141, the polypeptide sequence of SEQ ID NO: 142, the polypeptide sequence of SEQ ID NO: 143, and the polypeptide sequence of SEQ ID NO: 144 (CEA TCB).

[0256] In another specific embodiment, the anti-CEA / anti-CD3 bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:145, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:146, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:147, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:148. In another specific embodiment, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO:145, the polypeptide sequence of SEQ ID NO:146, the polypeptide sequence of SEQ ID NO:147, and the polypeptide sequence of SEQ ID NO:148 (CEACAM5 TCB).

[0257] Certain bispecific antibodies are further described in PCT Publication No. WO 2014 / 131712 A1. In another embodiment, the anti-CEA / anti-CD3 bispecific antibody may also include a bispecific T-cell adaptor (BiTE®). In another embodiment, the anti-CEA / anti-CD3 bispecific antibody system is the bispecific antibody described in WO 2007 / 071426 or WO 2014 / 131712.

[0258] The above-mentioned combination therapies cover combined administration (where two or more therapeutic agents are included in the same or separate compositions) and separate administration, in which case the bispecific antigen-binding molecule may be administered before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. Products

[0259] In another embodiment of the invention, an article is provided containing material that can be used to treat, prevent, and / or diagnose the conditions described above. The article comprises a container and a label or packaging insert located on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. These containers can be formed from a variety of materials such as glass or plastic. The container contains a composition that, on its own or in combination with another composition, is effective for treating, preventing, and / or diagnosing a disease, and may have a sterile infusion port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one active agent in the composition is a bispecific antigen-binding molecule of the present invention.

[0260] A label or packaging insert indicates that the composition is used to treat a selected disease. Furthermore, the article may comprise (a) a first container containing the composition, wherein the composition contains the bispecific antigen-binding molecule of the present invention; and (b) a second container containing the composition, wherein the composition contains another cytotoxic agent or other therapeutic agent. The article in this embodiment of the invention may further comprise a packaging insert indicating that the composition can be used to treat a specific disease.

[0261] Alternatively, the article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. Table B (Sequence): SEQ ID NO: name sequence 1 hu OX40 Uniprot number P43489, aa 29-214 LH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ NTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICED RDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 2 hu FAP Uniprot ID Q12884, Version 168 MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTM KSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGREN KIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAM IASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYI KDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPIST LHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIA LVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSD 3 FAP (212) CDR-H1 DYNMD 4 FAP (212) CDR-H2 DIYPNTGGTIYNQKFKG 5 FAP (212) CDR-H3 FRGIHYAMDY 6 FAP (212) CDR-L1 RASESVDNYGLSFIN 7 FAP (212) CDR-L2 GTSNRGS 8 FAP (212) CDR-L3 QQSNEVPYT 9 FAP (212) VH EVLLQQSGPELVKPGASVKIACKASGYTLTDYNMDWVRQSHGKSLEWIGDIYPNTGGTIYNQKFKGKATLTIDKSSSTAYMDLRSLTSEDTAVYYCTRFRGIHYAMDYWGQGTSVTVSS 10 FAP (212) VL DIVLTQSPVSLAVSLGQRATISCRASESVDNYGLSFINWFQQKPGQPPKLLIYGTSNRGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSNEVPYTFGGGTNLEIK 11 FAP (VH1G3a) CDR-H2 DIYPNTGGTIYAQKFQG 12 FAP (VH2G3a) CDR-H2 DIYPNTGGTIYADSVKG 13 FAP (VL1G3a) CDR-L1 RASESVDNYGLSFLA 14 FAP (VL2G3a) CDR-L1 RASESIDNYGLSFLN 15 FAP (VH1G1a) See Table 10 16 FAP (VH1G2a) See Table 10 17 FAP (VH1G3a) See Table 10 18 FAP (VH2G1a) See Table 10 19 FAP (VH2G2a) See Table 10 20 FAP (VH2G3a) See Table 10 twenty one FAP (VL1G1a) See Table 10 twenty two FAP (VL1G2a) See Table 10 twenty three FAP (VL1G3a) See Table 10 twenty four FAP (VL2G1a) See Table 10 25 FAP (VL2G2a) See Table 10 26 FAP (VL2G3a) See Table 10 27 OX40 (49B4) CDR-H1 SYAIS 28 OX40 (49B4) CDR-H2 GIIPIFGTANY​​AQKFQG 29 OX40 (49B4) CDR-H3 EYYRGPYDY 30 OX40 (49B4) CDR-L1 RASQSISSWLA 31 OX40 (49B4) CDR-L2 DASSLES 32 OX40 (49B4) CDR-L3 QQYSSQPYT 33 OX40 (49B4) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSS 34 OX40 (49B4) VL DIQMTQSPSTLSSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIK 35 OX40 (CLC563) CDR-H1 SYAMS 36 OX40 (CLC563) CDR-H2 AISGSGGSTYYADSVKG 37 OX40 (CLC563) CDR-H3 DVGAFDY 38 OX40 (CLC563) CDR-L1 RASQSVSSSYLA 39 OX40 (CLC563) CDR-L2 GASSRAT 40 OX40 (CLC563) CDR-L3 QQYGSSPLT 41 OX40 (CLC563) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSS 42 OX40 (CLC563) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK 43 OX40 (MOXR0916) CDR-H1 DSYMS 44 OX40 (MOXR0916) CDR-H2 DMYPDNGDSSYNQKFRE 45 OX40 (MOXR0916) CDR-H3 APRWYFSV 46 OX40 (MOXR0916) CDR-L1 RASQDISNYLN 47 OX40 (MOXR0916) CDR-L2 YTSRLRS 48 OX40 (MOXR0916) CDR-L3 QQGHTLPPT 49 OX40 (MOXR0916) VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS 50 OX40 (MOXR0916) VL DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIK 51 OX40 (8H9) CDR-H1 SYAIS 52 OX40 (8H9) CDR-H2 GIIPIFGTANYAQKFQG 53 OX40 (8H9) CDR-H3 EYGWMDY 54 OX40 (8H9) CDR-L1 RASQSISSWLA 55 OX40 (8H9) CDR-L2 DASSLES 56 OX40 (8H9) CDR-L3 QQYLTYSRFT 57 OX40 (8H9) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSS 58 OX40 (8H9) VL DIQMTQSPSTLSSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYLTYSRFTFGQGTKVEIK 59 OX40 (49B4_K73E) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSS 60 OX40 (49B4_K23T_K73E) VH QVQLVQSGAEVKKPGSSVKVSCTASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSS 61 OX40 (49B4_K23E_K73E) VH QVQLVQSGAEVKKPGSSVKVSCEASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSS 62 hu FAP extracellular domain + poly-lys tag + his6 tag RPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH 63 Mouse FAP Uniprot number P97321 64 Mouse FAP extracellular domain + poly-lys tag + his6 tag RPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISEQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVLSICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGILSGRSQNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH 65 Cynomolgus monkey FAP extracellular domain + poly-lys tag + his6 tag RPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH 66 Mouse OX40 Uniprot accession number P47741, version 143 MYVWVQQPTALLLLALLTLGVTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDTLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGN NQACKPWTNCTLSGKQTRHPASDSLDAVCEDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPPSPPTLVTPEGPAFAVLLGLGLGLLAPLTVLLALYLLRKAWRLPNTPKPCWGNSFRTPIQEEHTDAHFTLAKI 67 Peptide linker (G4S) GGGGS 68 Peptide linker (G4S)2 GGGGSGGGGS 69 Peptide linker (SG4)2 SGGGGSGGGG 70 Peptide linker G4(SG4)2 GGGGSGGGGSGGGG 71 peptide linkers GSPGSSSSGS 72 (G4S)3 peptide linker GGGGSGGGGSGGGGS 73 (G4S)4 peptide linker GGGGSGGGGSGGGGSGGGGS 74 peptide linkers GSGSGSGS 75 peptide linkers GSGSGNGS 76 peptide linkers GGSGSGSG 77 peptide linkers GGSGSG 78 peptide linkers GGSG 79 peptide linkers GGSGNGSG 80 peptide linkers GGNGSGSG 81 peptide linkers GGNGSG 82 Fc convex chain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 83 Fc Hole Chain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 84 Receptor framework IGHJ6*01 / 02 YYYYYGMDVWGQGTTVTVSS 85 Receptor framework IGKJ4*01 / 02 LTFGGGTKVEIK 86 OX40(49B4) VHCH1- OX40(49B4) VHCH1- Fc bulge_PGLALA- FAP(1G1a) VHCL See Table 13 87 OX40 (49B4) Light Chain See Table 13 88 FAP(1G1a) VLCH1-Light Chain See Table 13 89 OX40(49B4) VHCH1-OX40(49B4) VHCH1-Fc Hole_PGLALA See Table 13 90 OX40(49B4) VHCH1- Fc Hole_PGLALA See Table 13 91 OX40(49B4) VHCH1- Fc_PGLALA- FAP(1G1a) VHCL See Table 13 92 OX40(CLC563) VHCH1- OX40(CLC563) VHCH1- Fc Bump_PGLALA- FAP(1G1a) VHCL See Table 13 93 OX40 (CLC563) Light Chain See Table 13 94 FAP(1G1a) VLCH1-Light Chain (EPKSCD) See Table 13 95 OX40(CLC563) VHCH1-OX40(CLC563) VHCH1-Fc Hole_PGLALA See Table 13 96 FAP(1G1a) VLCH1-Light Chain (EPKSCS) See Table 13 97 OX40(CLC563) VHCH1- Fc PTFE_PGLALA- FAP(1G1a) VHCL See Table 13 98 OX40(CLC563) VHCH1 Fc Hole_PGLALA See Table 13 99 OX40(MOXR0916) VHCH1- Fc PTFE_PGLALA- FAP(1G1a) VHCL See Table 13 100 OX40 (MOXR0916) Light Chain See Table 13 101 OX40(MOXR0916) VHCH1- OX40(MOXR0916) VHCH1- Fc Hole_PGLALA- See Table 13 102 OX40(MOXR0916) VHCH1- Fc Hole_PGLALA See Table 13 103 OX40(8H9) VHCH1- Fc convex_PGLALA-FAP(1G1a) VHCL See Table 13 104 OX40(8H9) Light Chain See Table 13 105 OX40(8H9) VHCH1- OX40(8H9) VHCH1- Fc hole_PGLALA See Table 13 106 OX40(8H9) VHCH1- Fc Hole_PGLALA See Table 13 107 OX40(49B4_K73E) VHCH1- OX40(49B4_K73E) VHCH1- Fc bulge_PGLALA- FAP(1G1a) VHCL See Table 15 108 OX40(49B4_K73E) VHCH1-OX40(49B4_K73E) VHCH1-Fc Hole_PGLALA See Table 15 109 OX40(49B4_K23T_K73E) VHCH1- OX40(49B4_K23T_K73E) VHCH1- Fc bulge_PGLALA- FAP(1G1a) VHCL See Table 15 110 OX40(49B4_K23T_K73E) VHCH1-OX40(49B4_K23T_K73E) VHCH1-Fc Hole_PGLALA See Table 15 111 OX40(49B4_K23E_K73E) VHCH1- OX40(49B4_K23E_K73E) VHCH1- Fcprotuberance_PGLALA- FAP(1G1a) VHCL See Table 15 112 OX40(49B4_K23E_K73E) VHCH1-OX40(49B4_K23E_K73E) VHCH1-Fc Hole_PGLALA See Table 15 113 OX40(49B4) VHCH1- OX40(49B4) VHCH1- Fc Hole_PGLALA - FAP(4B9) VL QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEP EDFAVYYCQQGIMLPPTFGQGTKVEIK 114 OX40(49B4) VHCH1 - OX40(49B4) VHCH1 - Fc Ridge_PGLALA - FAP(4B9) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLASSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSGGGSGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 115 OX40 (49B4) VLCκ DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 116 OX40(49B4) VHCH1- OX40(49B4) VHCH1- IgG1 Fc _PGLALA QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 117 CD3 CDR-H1 TYAMN 118 CD3 CDR-H2 RIRSKYNNYATYYADSVKG 119 CD3 CDR-H2 HGNFGNSYVSWFAY 120 CD3 CDR-L1 GSSTGAVTTSNYAN 121 CD3 CDR-L2 GTNKRAP 122 CD3 CDR-L3 ALWYSNLWV 123 CD3 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 124 CD3 VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 125 CD3 (Cl22) CDR-H1 SYAMN 126 CD3 (Cl22) CDR-H2 RIRSKYNNYATYYADSVKG 127 CD3 (Cl22) CDR-H3 HTTFPSSYVSYYGY 128 CD3 (Cl22) CDR-L1 GSSTGAVTTSNYAN 129 CD3 (Cl22) CDR-L2 GTNKRAP 130 CD3 (Cl22) CDR-L3 ALWYSNLWV 131 CD3 (Cl22) VH EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSS 132 CD3 (Cl22) VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 133 CD3 (V9) CDR-H1 GYSFTGYTMN 134 CD3 (V9) CDR-H2 LINPYKGVSTYNQKFKD 135 CD3 (V9) CDR-H3 SGYYGDSDWYFDV 136 CD3 (V9) CDR-L1 RASQDIRNYLN 137 CD3 (V9) CDR-L2 YTSRLES 138 CD3 (V9) CDR-L3 QQGNTLPWT 139 CD3 (V9) VH EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS 140 CD3 (V9) VL DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK 141 Light chain "CEA , , CH1A1A 98 / 99 , CH2527 」 (CEA TCB) DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 142 Humanized light chain CD3 CH2527 (Crossed fab, VL-CH1) (CEA TCB) QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 143 CEA CH1A1A 98 / 99 — Humanized CD3 CH2527 (Crossed fab VH-Ck) — Fc (Convex) P329GLALA (CEA TCB) QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 144 CEA CH1A1A 98 / 99 (VH-CH1)—Fc (Hole) P329GLALA (CEA TCB) QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 145 CD3 VH-CL (CEACAM5 TCB) EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 146 Humanized CEA VH-CH1(EE)-Fc (Hole, P329G LALA) (CEACAM5 TCB) QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVRQAPGQGLEWMGRIDPANGNSKYVPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 147 Humanized CEA VH-CH1(EE)-CD3 VL-CH1-Fc (Carina, P329G LALA) (CEACAM5 TCB) QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVRQAPGQGLEWMGRIDPANGNSKYVPKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 148 Humanized CEA VL-CL(RK) (CEACAM5 TCB) EIVLTQSPATLSLSPGERATLSCRAGESVDIFGVGFLHWYQQKPGQAPRLLIYRASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQTNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 149 VH (PD-L1) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 156 Fc IgG1, African American allotype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ​​​​ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 158 Fc IgG3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK 159 Fc IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 160 Full-length hinge DKTHTCPXCP, where X is S or P 161 medium hinge HTCPXCP, where X is S or P 162 short hinge CPXCP, where X is S or P 163 CH1 connector C-terminus EPKSC 164 CH1 linker C-terminal D variant EPKSCD 165 CH1 linker C-terminal S variant EPKSCS 166 CH1 domain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

[0262] The following numbered paragraphs (paragraph, para) describe the features of the present invention: 1. A bispecific antigen-binding molecule comprising (a) at least two antigen-binding domains specifically binding to OX40, and (b) an antigen-binding domain specifically binding to fibroblast activation protein (FAP), comprising: a heavy chain variable region (VHFAP) comprising (i) CDR-H1, comprising an amino acid sequence of SEQ ID NO:3, (ii) CDR-H2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:11 and SEQ ID NO:12, and (iii) CDR-H3, comprising an amino acid sequence of SEQ ID NO:5; and a light chain variable region (VLFAP) comprising (iv) CDR-L1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13 and SEQ ID NO:14, and (v) CDR-L2, comprising the amino acid sequence of SEQ ID NO:7, and (vi) CDR-L3, comprising the amino acid sequence of SEQ ID NO:8; and (c) an Fc region consisting of a first subunit and a second subunit capable of stable association. 2. The bispecific antigen-binding molecule of paragraph 1, wherein the Fc region comprises one or more amino acid substitutions that reduce the binding affinity of the antibody to the Fc receptor and / or effector function. 3. The bispecific antigen-binding molecule of paragraph 1 or 2, wherein the antigen-binding domain capable of specifically binding to the FAP comprises: a heavy chain variable region (VHFAP) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9; and a light chain variable region (VLFAP) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:10. 4. A bispecific antigen-binding molecule as described in any of paragraphs 1 to 3, wherein the antigen-binding domain capable of specifically binding to the FAP includes a heavy chain variable region (VHFAP) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, a...

Claims

1. A bispecific antigen-binding molecule comprising (a) at least two antigen-binding domains specifically binding to OX40, wherein the antigen-binding domains specifically binding to OX40 comprise (i) a heavy chain variable region (VHOX40) comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 27, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (VLOX40) comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO:

29. The amino acid sequence of SEQ ID NO: 32, or (ii) heavy chain variable region (VHOX40), which comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 37; and light chain variable region (VLOX40), which comprises (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO: 40, or (iii) heavy chain variable region (VHOX40), which comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and (iii) CDR-H ... and (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO: 40, or (iii) heavy chain variable region (VHOX40), which comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID (ii) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 44, and (iii) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (VLOX40), which contains (iv) CDR-L1, which contains the amino acid sequence of SEQ ID NO: 46, (v) CDR-L2, which contains the amino acid sequence of SEQ ID NO: 47, and (vi) CDR-L3, which contains the amino acid sequence of SEQ ID NO: 48, or (iv) a heavy chain variable region (VHOX40), which contains (i) CDR-H1, which contains the amino acid sequence of SEQ ID NO: 51, (ii) CDR-H2, which contains the amino acid sequence of SEQ ID NO: 52, and (iii) CDR-H3, which contains the amino acid sequence of SEQ ID NO: 53;And a light chain variable region (VLOX40), comprising (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO: 56; (b) an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), comprising: a heavy chain variable region (VHFAP) containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (VLFAP) containing the amino acid sequence of SEQ ID NO: 21; and (c) an Fc region composed of a first subunit and a second subunit capable of stable association, wherein the Fc region contains one or more amino acid substitutions that reduce the binding affinity of the antibody to the Fc receptor and / or effector function.

2. The bispecific antigen-binding molecule of claim 1, wherein the antigen-binding domain capable of specifically binding to OX40 comprises (i) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO: 34, or (ii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO: 42, or (iii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO: 49 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO: 50, or (iv) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO: 57 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:

58.

3. The bispecific antigen-binding molecule of claim 1, wherein the antigen-binding domain capable of specifically binding to OX40 comprises (i) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO: 59 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO: 34, or (ii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO: 60 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO: 34, or (iii) a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO: 61 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:

34.

4. The bispecific antigen-binding molecule of claim 1 or 2, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VHOX40) comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable region (VLOX40) comprising the amino acid sequence of SEQ ID NO:

42.

5. A bispecific antigen-binding molecule as requested in item 1 or 2, wherein the Fc region is an IgG Fc region.

6. A bispecific antigen-binding molecule as requested in item 1 or 2, wherein the Fc region is an IgG1 Fc region or an IgG4 Fc region.

7. A bispecific antigen-binding molecule as requested in claim 1 or 2, wherein the Fc region is the human IgG1 subclass Fc region with amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

8. The bispecific antigen-binding molecule of claim 1 or 2, wherein the bispecific antigen-binding molecule comprises (a) at least two Fab fragments specifically binding to OX40, each linked to the N-terminus of a subunit of the Fc region, and (b) a cross-Fab fragment specifically binding to FAP, fused to the C-terminus of a subunit of the Fc region, and (c) the Fc region consisting of a first subunit and a second subunit capable of stable association.

9. The bispecific antigen-binding molecule of claim 8, wherein the VH-Cκ chain of the cross-fab fragment of the FAP that specifically binds to the FAP is fused to the C-terminus of a subunit of the Fc region.

10. The bispecific antigen-binding molecule of claim 1 or 2, comprising: (aa) a first Fab fragment capable of specifically binding to OX40, (ab) a second Fab fragment capable of specifically binding to OX40, (b) a cross-Fab fragment capable of specifically binding to FAP, which is fused to the C-terminus of a subunit of the Fc region, and (c) the Fc region comprising the first and second subunits capable of stable association, wherein the first Fab fragment (aa) is fused to the N-terminus of the first subunit at the C-terminus of the VH-CH1 chain and the second Fab fragment (ab) is fused to the N-terminus of the second subunit at the C-terminus of the VH-CH1 chain.

11. The bispecific antigen-binding molecule of claim 1 or 2, comprising: (aa) a first Fab fragment specifically binding to OX40, (ab) a second Fab fragment specifically binding to OX40, (ac) a third Fab fragment specifically binding to OX40, (b) a cross-Fab fragment specifically binding to FAP, fused to the C-terminus of a subunit of the Fc region, and (c) the Fc region comprising the first and second subunits capable of stable association, wherein the second Fab fragment (ab) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the first Fab fragment (aa), the VH-CH1 chain of the first Fab fragment (aa) is fused at its C-terminus to the N-terminus of the first subunit, and the third Fab fragment (ac) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit.

12. The bispecific antigen-binding molecule of claim 1 or 2, comprising: (aa) a first Fab fragment specifically binding to OX40, (ab) a second Fab fragment specifically binding to OX40, (ac) a third Fab fragment specifically binding to OX40, (ad) a fourth Fab fragment specifically binding to OX40, (b) a cross-Fab fragment specifically binding to FAP, fused to the C-terminus of a subunit of the Fc region, and (c) the first and second subunits capable of stable association. The Fc region is composed of the second Fab segment (ab), which fuses with the N-terminus of the VH-CH1 chain of the first Fab segment (aa) at the C-terminus of the VH-CH1 chain. The VH-CH1 chain of the first Fab segment (aa) then fuses with the N-terminus of the first subunit at its C-terminus. The fourth Fab segment (ad) fuses with the N-terminus of the VH-CH1 chain of the third Fab segment (ac) at the C-terminus of the VH-CH1 chain. The VH-CH1 chain of the third Fab segment (ac) then fuses with the N-terminus of the second subunit at its C-terminus.

13. A bispecific antigen-binding molecule as claimed in claim 1 or 2, comprising (a) a heavy chain comprising a VH-CH1 chain specifically binding to a first Fab fragment of OX40, wherein the VH-CH1 chain of the first Fab fragment is fused at its N-terminus via a peptide linker to a VH-CH1 chain specifically binding to OX40, and an Fc subunit; (b) a heavy chain comprising a VH-CH1 domain specifically binding to a Fab fragment of OX40, an Fc subunit, and a VH-CL chain specifically binding to a Fab fragment of FAP, wherein the VH-CL chain of the Fab fragment is fused at its C-terminus via a peptide linker; (c) three light chains, each light chain comprising a VL and CL domain specifically binding to a Fab fragment of OX40; and (d) a light chain comprising a VL and CH1 domain specifically binding to a Fab fragment of FAP.

14. A bispecific antigen-binding molecule as claimed in claim 1 or 2, comprising (a) a first heavy chain containing the amino acid sequence of SEQ ID NO: 97; a second heavy chain containing the amino acid sequence of SEQ ID NO: 95; three light chains, each light chain containing the amino acid sequence of SEQ ID NO: 93; and a light chain containing the amino acid sequence of SEQ ID NO: 88, or (b) a first heavy chain containing the amino acid sequence of SEQ ID NO: 97; a second heavy chain containing the amino acid sequence of SEQ ID NO: 95; three light chains, each light chain containing the amino acid sequence of SEQ ID NO: 93; and a light chain containing the amino acid sequence of SEQ ID NO: 94, or (c) a first heavy chain containing the amino acid sequence of SEQ ID NO: 97; a second heavy chain containing the amino acid sequence of SEQ ID NO: 95; three light chains, each light chain containing the amino acid sequence of SEQ ID NO: 93; and a light chain containing the amino acid sequence of SEQ ID NO:

94. The amino acid sequence of NO:

96.

15. An isolated nucleic acid encoding a bispecific antigen-binding molecule as claimed in any one of claims 1 to 14.

16. A representation vector comprising the isolated nucleic acid as claimed in claim 15.

17. A host cell comprising the isolated nucleic acid as claimed in claim 15 or the expression vector as claimed in claim 16.

18. A method for generating a bispecific antigen-binding molecule, comprising culturing a host cell as claimed in claim 17 under conditions suitable for expressing the bispecific antigen-binding molecule and isolating the bispecific antigen-binding molecule.

19. A pharmaceutical composition comprising a bispecific antigen-binding molecule as claimed in any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, further comprising an additional therapeutic agent.

21. Use of a bispecific antigen-binding molecule as claimed in any one of claims 1 to 14 or a pharmaceutical composition as claimed in claim 19, for the manufacture of an agent for: (i) inducing immune stimulation, (ii) stimulating tumor-specific T cell responses, (iii) inducing apoptosis of tumor cells, (iv) treating cancer, (v) delaying cancer progression, (vi) prolonging the survival of patients with cancer, and (vii) treating infections.

22. As claimed in claim 21, wherein the agent is used to treat cancer.

23. The use as claimed in claim 22, wherein the agent is administered in combination with a chemotherapy agent, radiation and / or other agents used for cancer immunotherapy.

24. As claimed in claim 22, wherein the bispecific activating OX40 antigen-binding molecule is used in combination with a T-cell activating anti-CD3 bispecific antibody for administration.

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