Bispecific antigen-binding molecules targeting OX40 and FAP

Abstract

The present invention relates to novel bispecific antigen-binding molecules comprising at least two antigen-binding domains capable of specifically binding to OX40 and a particular antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), as well as methods for producing and using these molecules.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to novel bispecific antigen-binding molecules comprising at least two antigen-binding domains capable of specifically binding to OX40 and an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), and an Fc domain, particularly an Fc domain comprising one or more amino acid substitutions that reduce Fc receptor binding and / or effector function. Further aspects of the invention are methods of producing and using these molecules. [Background technology]

[0002] background Cancer remains one of the leading causes of death worldwide, despite several novel agents providing survival benefits to patients. Despite advances in treatment options, the prognosis for patients with advanced cancer remains poor. Management of most advanced solid tumors remains challenging due to poor prognosis for many cancer indications and high rates of tumor recurrence or distant metastasis. Patients with advanced solid tumors have clearly benefited from checkpoint inhibitor (CPI) therapy. Even after treatment cessation, CPI therapy can extend overall survival in a subset of patients, likely through the generation of memory immune responses. While this has transformed the treatment landscape in many types of cancer, unfortunately, 60–80% of patients with metastatic disease do not derive long-term benefit from this type of cancer immunotherapy. Consequently, there is a persistent and urgent medical need to develop novel and optimal therapies that can be added to existing treatments to enhance cancer patient survival without causing unacceptable toxicity.

[0003] The immunosuppressive microenvironment in certain tumors is characterized by high levels of co-inhibitory signals, such as PD-L1, but low expression of OX40 ligand. OX40 (CD134; TNFRSF4) is a member of the tumor necrosis factor (TNF) receptor superfamily that is transiently expressed by T cells upon T cell receptor (TCR) engagement. OX40 engagement is a key pathway for T cells to bind OX40L.+ It bidirectionally regulates interactions with antigen-presenting cells (e.g., B cells, dendritic cells (DCs), and monocytes). In the context of TCR engagement, OX40 primarily regulates CD4 + Not just CD8 + OX40 also provides costimulatory signals to effector T cells, leading to enhanced proliferation, survival, and effector function (e.g., cytokine secretion). Conversely, OX40 signaling leads to functional inhibition and loss of regulatory T cells. OX40 agonism counteracts TGF-β effects (e.g., by preventing FoxP3 induction) and reduces IL-10 secretion. In mouse tumor models, OX40 binding by agonistic anti-OX40 antibodies can promote antitumor T-cell responses, tumor shrinkage, and a reproducible abscopal effect. While the efficacy of OX40 agonists as monotherapy has generally been low, robust antitumor efficacy has been achieved in combination with immunogenic treatments (chemotherapy, radiation, and vaccination), checkpoint inhibitors (PD-1, CTLA-4), and other costimulatory agonists such as 4-1BB, ICOS, or GITR.

[0004] Fibroblast activation protein alpha (FAP) is a serine protease highly expressed on the cell surface of cancer-associated stromal cells in >90% of human epithelial malignancies, on reticular fibroblasts in T cell-priming regions of lymph nodes, and can be found on activated fibroblasts in normal tissues. Its high prevalence in various cancer indications allows its use as a targeting moiety for drugs that should accumulate within the tumor environment.

[0005] One approach to specifically restore OX40 costimulation in the tumor microenvironment is a bispecific antibody composed of at least one antigen-binding domain for fibroblast activation protein (FAP) in the tumor stroma and at least one antigen-binding domain for OX40. For example, such bispecific antibodies are described in International Publication Nos. 2017 / 055398 and 2017 / 060144. Crosslinking and surface immobilization of such bispecific molecules with cell surface FAP creates a highly agonistic matrix for OX40-positive T cells, which supports NFκB-mediated effector function and can replace OX40 ligand ligation. High FAP expression has been reported in either tumor cells themselves or immunosuppressive cancer-associated fibroblasts (CAFs) for a number of human tumor indications. Thus, there is a need for improved FAP-targeting OX40 bispecific antibodies that have superior pharmacological properties (e.g., better shelf life), are less immunogenic, and have fewer non-specific interactions (e.g., hypersensitivity reactions or uncontrolled cytokine release). Summary of the Invention

[0006] Summary of the Invention The present invention relates to novel bispecific antigen-binding molecules capable of specifically binding to OX40 and fibroblast activation protein (FAP) with improved properties, whereby OX40 costimulation is achieved by crosslinking FAP expressed on tumor stromal cells and potentially also via FAP expressed intermediately in secondary lymphoid tissues. Thus, the antigen-binding molecules of the present invention not only effectively induce OX40 but also induce OX40 highly selectively at the desired site, overcoming the need for FcγR crosslinking and thereby reducing side effects. The novel bispecific antigen-binding molecules contain a novel FAP antigen-binding domain fused to the C-terminus of the Fc domain and are characterized by improved pharmacokinetic properties.

[0007] The bispecific antigen-binding molecules of the present invention combine at least two antigen-binding domains capable of specifically binding to the costimulatory TNF receptor family member OX40 with an antigen-binding domain targeting fibroblast activation protein (FAP), including the new mouse anti-human FAP clone 212 and its humanized variants. These bispecific antigen-binding molecules are OX40 agonists, and are advantageous because they can bind to FAP with high affinity, thereby preferably activating costimulatory OX40 receptors near tumor sites where FAP is expressed. The molecules are further designed to have a favorable pharmacokinetic profile to optimize treatment regimens, improving the balance of safety and efficacy.

[0008] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) at least two antigen-binding domains capable of specifically binding to OX40; (b) an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), (i) a heavy chain variable region (V) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (ii) a 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) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5. H a light chain variable region (V FAP) comprising (iv) a 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, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; L an antigen-binding domain comprising FAP; (c) an Fc region composed of a first subunit and a second subunit capable of stably associating; The present invention provides a bispecific antigen-binding molecule comprising:

[0009] In one embodiment, the Fc region comprises one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antibody to an Fc receptor.

[0010] In a further embodiment, the antigen-binding domain capable of specifically binding to a FAP comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9. H A light chain variable region (V) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10. L In one embodiment, the antigen-binding domain capable of specifically binding to FAP comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 9. H FAP), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10 L FAP).

[0011] In another embodiment, the antigen binding domain capable of specifically binding to a FAP comprises a heavy chain variable region (V) 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. H FAP), and a light chain variable region (V) 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. L In one embodiment, the antigen-binding domain capable of specifically binding to a FAP comprises (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 15. H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 21 L FAP), (b) a heavy chain variable region (V H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 21 L FAP), (c) a heavy chain variable region (V H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 22 L FAP), or (d) a heavy chain variable region (V H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 25 LMore specifically, the antigen-binding domain capable of specifically binding to FAP comprises: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 15; H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 21 L FAP).

[0012] In one embodiment, the antigen binding domain capable of specifically binding to OX40 comprises or consists of a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

[0013] In a further aspect, the bispecific antigen binding molecule, wherein the antigen binding domains (each) capable of specifically binding to OX40 are: (a) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29; H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32. L OX40), or (b) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37; H OX40), and a light chain variable region (V) 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. L OX40), or (c) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45 HOX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. L OX40), or (d) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53; H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 54, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 56. L OX40) A bispecific antigen-binding molecule is provided, comprising:

[0014] In one embodiment, the antigen binding domain (s) capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29. H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32. L In a further embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37. H OX40), and a light chain variable region (V) 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. LIn another embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45. H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. L In yet another embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53. H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 54, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 56. L OX40).

[0015] In one embodiment, the antigen binding domain (s) capable of specifically binding to OX40 are (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 33 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L OX40), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L OX40), or (iv) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 57 HOX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 58 L OX40)

[0023] In accordance with the present invention, there is provided a bispecific antigen-binding molecule as defined herein above, comprising:

[0016] In one aspect, the antigen binding domain (s) capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 33. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L In another aspect, a bispecific antigen-binding molecule as defined herein above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L In a further aspect, a bispecific antigen-binding molecule as defined herein above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L In a further aspect, a bispecific antigen-binding molecule as defined herein above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 57. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 58 L OX40).

[0017] In certain embodiments, the antigen binding domain (s) capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41. H OX40), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L OX40).

[0018] In another embodiment, the antigen binding domain capable of specifically binding to OX40 comprises: (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 59 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 60 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 61 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40)

[0023] In accordance with the present invention, there is provided a bispecific antigen-binding molecule as defined herein above, comprising:

[0019] In one embodiment, the antigen binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 59. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L In one embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 60. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L In a further embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 61. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40).

[0020] In another embodiment, the bispecific antigen-binding molecule is a humanized or chimeric antibody, particularly a humanized antibody. In a further embodiment, the bispecific antigen-binding molecule comprises an IgG Fc region, particularly 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 Fc receptors and / or effector functions. In one particular embodiment, a bispecific antigen-binding molecule is provided in which the Fc region is of the human IgG1 subclass with amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0021] In another embodiment, a bispecific antigen-binding molecule is provided, wherein the first subunit of the Fc region comprises a knob and the second subunit of the Fc region comprises a hole in a knob-to-hole manner. In particular, a bispecific antigen-binding molecule is provided, wherein the first subunit of the Fc region comprises amino acid substitutions S354C and T366W (numbering according to the Kabat EU index) and the second subunit of the Fc region comprises amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index). In yet another embodiment, the Fc region is derived from a mouse, wherein the first subunit of the Fc region comprises amino acid substitutions K392D and K409D (numbering according to the Kabat EU index) and the second subunit of the Fc region comprises amino acid substitutions E356K and D399K (numbering according to the Kabat EU index). More specifically, provided are bispecific antigen-binding molecules, wherein a first subunit of the Fc region comprises the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index), and a second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0022] In a further aspect, the bispecific antigen-binding molecule comprises: (a) at least two Fab fragments capable of specifically binding to OX40, each attached to the N-terminus of one of the subunits of the Fc region; (b) one crossFab fragment capable of specifically binding to a FAP, fused to the C-terminus of one of the subunits of the Fc region; and (c) an Fc region composed of a first subunit and a second subunit capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0023] Thus, a bispecific antigen-binding molecule (2+1 format) is provided that provides bivalent binding to OX40 and monovalent binding to FAP. In particular, the bispecific antigen-binding molecule comprises one cross-Fab fragment that can specifically bind to FAP, and the VH-C kappa chain of the cross-Fab fragment that can specifically bind to FAP is fused to the C-terminus of one of the subunits of the Fc region. In one specific embodiment, the VH-C kappa chain of the cross-Fab fragment that can specifically bind to FAP is fused to the C-terminus of one of the first subunits of the Fc region, i.e., the knob chain, comprising amino acid substitutions S354C and T366W (numbering according to Kabat EU index).

[0024] In one aspect, there is provided a bispecific antigen binding molecule 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 a FAP fused to the C-terminus of one of the subunits of the Fc region; and (c) An Fc region composed of a first subunit and a second subunit capable of stably associating, wherein a first Fab fragment (aa) is fused to the N-terminus of the first subunit at the C-terminus of the VH-CH1 chain, and a second Fab fragment (ab) is fused to the N-terminus of the second subunit at the C-terminus of the VH-CH1 chain. A bispecific antigen-binding molecule is provided, which consists of:

[0025] Thus, a bispecific antigen binding molecule is provided that provides bivalent binding to OX40 and monovalent binding to a FAP (2+1 format).

[0026] In another aspect, there is provided a bispecific antigen-binding molecule comprising: (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 a FAP fused to the C-terminus of one of the subunits of the Fc region; and (c) An Fc region composed of a first subunit and a second subunit capable of stably associating, 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 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. A bispecific antigen-binding molecule is provided, which consists of:

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

[0028] In one aspect, there is provided a bispecific antigen binding molecule comprising: (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 a FAP fused to the C-terminus of one of the subunits of the Fc region; and (c) an Fc region composed of a first subunit and a second subunit capable of stably associating, wherein a 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 a first Fab fragment (aa), the first Fab fragment (aa) is fused at its C-terminus to the N-terminus of the second subunit of an Fc region comprising amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index), and a third Fab fragment (ac) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit comprising amino acid substitutions S354C and T366W (numbering according to the Kabat EU index); In particular, the VH-Cκ chain of a cross-fab fragment capable of specifically binding to FAP is fused to the C-terminus of the first subunit of an Fc region containing amino acid substitutions S354C and T366W (numbering according to the Kabat EU index).

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

[0030] In one aspect, there is provided a bispecific antigen binding molecule comprising: (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; (ad) a fourth Fab fragment capable of specifically binding to OX40; (b) a cross-Fab fragment capable of specifically binding to a FAP fused to the C-terminus of one of the subunits of the Fc region; and (c) An Fc region composed of a first subunit and a second subunit capable of stably associating, 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 first Fab fragment (aa) is fused at its C-terminus to the N-terminus of the first subunit, the fourth Fab fragment (ad) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the third Fab fragment (ac), and the third Fab fragment (ac) is fused at its C-terminus to the N-terminus of the second subunit. A bispecific antigen-binding molecule is provided, which consists of:

[0031] Thus, a bispecific antigen-binding molecule (4 + 1 format) is provided that provides tetravalent binding to OX40 and monovalent binding to FAP. In one specific embodiment, the VH-C kappa 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 (numbering according to the Kabat EU index).

[0032] In one aspect, a bispecific antigen-binding molecule is provided, wherein each of the two heavy chains comprises two VH-CH1 chains of a Fab fragment capable of specifically binding to OX40, optionally fused to each other by a peptide linker. Thus, in one aspect, the present invention provides a bispecific antigen-binding molecule comprising: (a) two heavy chains, each comprising two VH-CH1 chains of a Fab fragment capable of specifically binding to OX40 and an Fc region subunit, optionally connected to each other by a peptide linker; (b) four light chains, each comprising a VL domain and a C kappa domain of a Fab fragment capable of specifically binding to OX40; and (c) a cross-Fab fragment capable of specifically binding to FAP, comprising a VL-CH1 light chain and a VH-C kappa chain, wherein the VH-C kappa chain is connected to the C-terminus of one of the two heavy chains of (a) optionally by a peptide linker.

[0033] According to another aspect of the present invention, there is provided an isolated nucleic acid encoding the bispecific antigen-binding molecule described hereinabove. The present invention further provides a vector, particularly an expression vector, comprising the isolated nucleic acid of the present invention, and a host cell comprising the isolated nucleic acid or expression vector of the present invention. In some aspects, the host cell is a eukaryotic cell, particularly a mammalian cell. In another aspect, there is provided a method for producing the bispecific antigen-binding molecule described hereinabove, comprising culturing the host cell under conditions suitable for expression of the bispecific antigen-binding molecule and isolating the bispecific antigen-binding molecule. The present invention also encompasses bispecific antigen-binding molecules that specifically bind to OX40 and FAP produced by the method of the present invention.

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

[0035] The present invention also encompasses bispecific antigen-binding molecules or antibodies described herein above for use as pharmaceuticals, as well as pharmaceutical compositions comprising the bispecific antigen-binding molecules.

[0036] In one aspect, (i) Inducing immune stimulation, (ii) in stimulating tumor-specific T cell responses; (iii) inducing apoptosis of tumor cells; (iv) In the treatment of cancer, (v) in slowing the progression of cancer; (vi) in prolonging the survival of patients with cancer; (vii) in the treatment of infectious diseases There is provided a bispecific antigen-binding molecule as described herein above or a pharmaceutical composition of the invention for use.

[0037] In one embodiment, the present invention provides a bispecific antigen-binding molecule as described herein above or a pharmaceutical composition of the present invention for use in the treatment of cancer. In another specific embodiment, the present invention provides a bispecific antigen-binding molecule as described herein above for use in the treatment of cancer, the bispecific antigen-binding molecule being for administration in combination with chemotherapeutic agents, radiation, and / or other agents for use in cancer immunotherapy. In one embodiment, the bispecific agonist OX40 antigen-binding molecule or pharmaceutical composition for use in the treatment of cancer is provided, wherein the bispecific agonist OX40 antigen-binding molecule is for administration in combination with a T cell-activating anti-CD3 bispecific antibody, such as an anti-CEA / anti-CD3 bispecific antibody. In a further embodiment, the bispecific antigen-binding molecule as described herein is for use in the treatment of cancer, wherein the bispecific antigen-binding molecule is for administration in combination with an agent that blocks PD-L1 / PD-1 interaction, such as a PD-L1 antibody, e.g., atezolizumab, or a PD-1 antibody, e.g., nivolumab or pembrolizumab. In another aspect, there is provided a bispecific antigen-binding molecule as described herein above or a pharmaceutical composition of the invention for use in upregulating or prolonging cytotoxic T cell activity.

[0038] In a further aspect, 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 herein above, or a pharmaceutical composition of the invention, thereby inhibiting the growth of the tumor cells. In another aspect, 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 herein above, or a pharmaceutical composition of the invention.

[0039] Also provided are uses of the bispecific antigen-binding molecules described herein above for producing a medicament for treating a disease, particularly for the manufacture of a medicament for the treatment of cancer, in an individual in need thereof, and methods for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising a 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 individual is a mammal, particularly a human. [Brief explanation of the drawings]

[0040] [Figures 1A-1E]Figures 1A-1E show schematic diagrams of bispecific antigen-binding molecules that specifically bind to OX40 and FAP. Figure 1A shows a schematic diagram of a bispecific FAP-OX40 antibody in a 4+1 format as crossFab fragments, with four OX40-binding Fab fragments combined with one FAP (1G1a)-binding moiety, and the VH-C kappa chain fused at the C-terminus of the Fc knob chain (tetravalent for OX40, monovalent for FAP). Figure 1B shows a schematic diagram of a bispecific FAP-OX40 antibody in a 3+1 format, with three OX40-binding Fab fragments combined with one FAP (1G1a)-binding moiety as crossFab fragments, with the VH-C kappa chain fused at the C-terminus of the Fc knob chain (trivalent for OX40, monovalent for FAP). The arms containing the two OX40-binding Fab fragments fused to each other are on the Fc knob chain. Figure 1C shows a schematic diagram of a bispecific FAP-OX40 antibody in a 3+1 format, consisting of three OX40-binding Fab fragments combined with one FAP (1G1a)-binding moiety as crossFab fragments, with the VH-C kappa chain fused to the C-terminus of the Fc knob chain (trivalent for OX40 and monovalent for FAP). An arm containing two OX40-binding Fab fragments fused to each other is located on the Fc hole chain. Figure 1D shows a schematic diagram of a bispecific FAP-OX40 antibody in a 2+1 format, consisting of two OX40-binding Fab fragments combined with one FAP (1G1a)-binding moiety as crossFab fragments, with the VH-C kappa chain fused to the C-terminus of the Fc knob chain (bivalent for OX40 and monovalent for FAP). Figure 1E shows a schematic diagram of the bispecific FAP-OX40 antibody P1AD4524, consisting of four OX40-binding Fab fragments combined with one FAP (4B9)-binding moiety as VH and VL domains in a 4+1 format, where the VL domain is fused at the C-terminus of the Fc knob chain and the VH domain is fused at the C-terminus of the Fc hole chain (tetravalent for OX40, monovalent for FAP). Black dots represent knob-into-hole mutations. [Figures 2A-2F]Figures 2A-2F show cell binding of bispecific antigen-binding molecules containing OX40 clone 49B4 in various formats. The FAP antigen-binding domain H212 is a humanized version of FAP clone 212, referred to herein as FAP(1G1a). Human FAP-negative tumor cells (A549-NLR, Figure 2F), FAP-positive fibroblasts (NIH / 3T3-huFAP-clone 19, Figure 2E), OX40-positive activated PBMCs (activated CD4 and CD8 T cells, Figures 2A and 2C, respectively), and OX40-negative resting PBMCs (resting CD4 and CD8 T cells, Figures 2B and 2D, respectively) were incubated with the indicated serial dilutions of test antibodies and then detected with a fluorescently labeled secondary antibody against human Fcγ. Live cells were gated, and the mean fluorescence intensity of the secondary antibody (baseline corrected by media-only samples) was plotted from duplicates. Error bars indicate SEM. [Figures 3A-3F] Figures 3A-3F show the cell binding of bispecific antigen-binding molecules containing OX40 clone 8H9 in various formats compared to bispecific antigen-binding molecules containing OX40 clone 49B4 in a 4+1 format (P1AE6838). Human FAP-negative tumor cells (A549-NLR) (Figure 3F), FAP-positive fibroblasts (NIH / 3T3-huFAP-clone 19) (Figure 3E), OX40-positive activated PBMCs (activated CD4 and CD8 T cells, Figures 3A and 3C, respectively), and OX40-negative resting PBMCs (resting CD4 and CD8 T cells, Figures 3B and 3D, respectively) were incubated with the indicated serial dilutions of test antibodies and then detected with a fluorescently labeled secondary antibody against human Fcγ. Live cells were gated, and the mean fluorescence intensity of the secondary antibody (baseline corrected by media-only samples) was plotted from duplicates. Error bars indicate SEM. Clone 8H9 bound to OX40-positive cells with subnanomolar affinity and with a strength comparable to that of the trivalent and bivalent antibodies. [Figures 4A-4F]Figures 4A-4F show the cell binding of bispecific antigen binding molecules containing OX40 clone MOXR0916 in various formats compared to bispecific antigen binding molecules containing OX40 clone 49B4 in a 4+1 format (P1AE6838). Human FAP-negative tumor cells (A549-NLR) (Figure 4F), FAP-positive fibroblasts (NIH / 3T3-huFAP-clone 19) (Figure 4E), OX40-positive activated PBMCs (activated CD4 and CD8 T cells, Figures 4A and 4C, respectively), and OX40-negative resting PBMCs (resting CD4 and CD8 T cells, Figures 4B and 4D, respectively) were incubated with the indicated serial dilutions of test antibodies and then detected with a fluorescently labeled secondary antibody against human Fcγ. Live cells were gated, and the mean fluorescence intensity of the secondary antibody (baseline corrected by media-only samples) was plotted from duplicates. Error bars indicate SEM. Clone MOXR0916 bound to OX40-positive cells with nanomolar affinity and with similar potency to the trivalent and bivalent antibodies. [Figures 5A-5F] Figures 5A-5F show the cell binding of bispecific antigen binding molecules containing OX40 clone CLC563 in various formats compared to bispecific antigen binding molecules containing OX40 clone 49B4 in a 4+1 format (P1AE6838). Human FAP-negative tumor cells (A549-NLR) (Figure 5F), FAP-positive fibroblasts (NIH / 3T3-huFAP-clone 19) (Figure 5E), OX40-positive activated PBMCs (activated CD4+ and CD8+ T cells, Figures 5A and 5C, respectively), and OX40-negative resting PBMCs (resting CD4+ and CD8+ T cells, Figures 5B and 5D, respectively) were incubated with the indicated serial dilutions of test antibodies and then detected with a fluorescently labeled secondary antibody against human Fcγ. Live cells were gated, and the mean fluorescence intensity of the secondary antibody (baseline corrected by media-only samples) was plotted from duplicates. Error bars indicate SEM. Clone CLC-563 bound to OX40-positive cells with nanomolar affinity, with potency comparable to that of the trivalent and bivalent antibodies. [Figures 6A-6F]Figures 6A-6F show the cell binding of bispecific antigen binding molecules containing different variants of OX40 clone 49B4 with amino acid mutations in the VH domain in various formats, compared to a bispecific antigen binding molecule (P1AE6838) containing OX40 clone 49B4 in a 4+1 format. Human FAP-negative tumor cells (A549-NLR) (Figure 6F), FAP-positive fibroblasts (NIH / 3T3-huFAP-clone 19) (Figure 6E), OX40-positive activated PBMCs (activated CD4+ and CD8+ T cells, Figures 6A and 6C, respectively), and OX40-negative resting PBMCs (resting CD4+ and CD8+ T cells, Figures 6B and 6D, respectively) were incubated with the indicated serial dilutions of test antibodies, followed by detection with a fluorescently labeled secondary antibody against human Fcγ. Live cells were gated, and the mean fluorescence intensity of the secondary antibody (baseline corrected by media-only samples) was plotted from duplicates. Error bars indicate SEM. All antigen-binding molecules, including the OX40 (49B4) mutant with amino acid mutations, showed slightly improved binding to OX40-positive cells compared to antigen-binding molecules containing clone 49B4. [Figure 7A-7C]Figures 7A-7C show NFκB-mediated luciferase expression activity in the OX40-expressing reporter cell line HeLa_hOx40_NFκB_Luc1. The concentrations of bispecific antigen-binding molecules containing OX40 clone 49B4 in various formats or their controls are plotted against the light emitted units (URLs) measured after incubation and addition of luciferase detection solution. NFκB induction is shown for 4+1, 3+1, or 2+1 formats crosslinked with human FAP-expressing NIH / 3T3 fibroblasts (Figure 7A), with a secondary antibody at a 2:1 ratio (Figure 7B), or with additional crosslinking (w / o) (Figure 7C). An isotype control antibody did not induce any NFκB activation. All OX40-containing constructs induced NFκB activation in a dose-dependent manner. The tetravalent format containing four OX40 Fab fragments already induced specific NFκB activation due to the assembly of the trimeric core OX40 receptor signaling unit in the absence of cross-linking. The same clones in trivalent or bivalent formats were correspondingly less bioactive. The higher the valency of the OX40 antigen-binding domain, the stronger the degree of NFκB activation and the lower the concentration required. Average values ​​of duplicates are shown. Error bars indicate SEM. [Figures 8A-8C]Figures 8A-8C show NFκB-mediated luciferase expression activity in the OX40-expressing reporter cell line HeLa_hOx40_NFκB_Luc1 of different bispecific antigen-binding molecules, including OX40 clone 8H9. The concentrations of bispecific antigen-binding molecules in various formats or their controls are plotted against the light emitted units (URLs) measured after incubation and addition of luciferase detection solution. NFκB induction is shown for NIH / 3T3 fibroblast-expressing human FAP (Figure 8A), 3+1, or 2+1 formats crosslinked with either a 2:1 ratio of secondary antibody (Figure 8B), or without additional crosslinking (Figure 8C). An isotype control antibody did not induce any NFκB activation. All OX40-containing constructs induced NFκB activation in a dose-dependent manner. The tetravalent format containing four OX40 Fab fragments was the most potent, inducing specific NFκB activation even in the absence of cross-linking due to the assembly of the trimeric core OX40 receptor signaling unit. Bispecific antigen binding molecules containing OX40 clone 8H9 in trivalent or bivalent formats were correspondingly less bioactive. Further cross-linking with human FAP-expressing fibroblasts via the FAP-binding moiety or with a secondary cross-linking antibody via the Fc region of the OX40 antigen binding molecule further increased NFκB activation. Clone OX40 (8H9) achieved maximal NFκB activation induction already in the 2+1 format. No further benefit was obtained with trivalents. Average values ​​of duplicates are shown. Error bars indicate SEM. [Figures 9A-9C]Figures 9A-9C show NFκB-mediated luciferase expression activity in the OX40-expressing reporter cell line HeLa_hOx40_NFκB_Luc1 of various bispecific antigen-binding molecules, including the OX40 clone MOXR0916. The concentrations of bispecific antigen-binding molecules in various formats or their controls are plotted against the light emitted units (URLs) measured after incubation and addition of luciferase detection solution. NFκB induction is shown for 3+1 or 2+1 formats crosslinked with human FAP-expressing NIH / 3T3 fibroblasts (Figure 9A), with a secondary antibody at a 2:1 ratio (Figure 9B), or additionally crosslinked w / o (Figure 9C). An isotype control antibody did not induce any NFκB activation. All OX40-containing constructs induced NFκB activation in a dose-dependent manner. The tetravalent format containing four OX40 Fab fragments was the most potent, inducing specific NFκB activation even in the absence of cross-linking due to the assembly of the trimeric core OX40 receptor signaling unit. Bispecific antigen binding molecules containing the OX40 clone MOXR0916 in trivalent or bivalent formats were correspondingly less bioactive. Further cross-linking with human FAP-expressing fibroblasts via the FAP-binding moiety or with a secondary cross-linking antibody via the Fc region of the OX40 antigen binding molecule further increased NFκB activation. Clone OX40 (MOXR0916) achieved maximum NFκB activation induction already in the 2+1 format, with no further benefit observed in the 3+1 format. Average values ​​of duplicates are shown. Error bars indicate SEM. [Figures 10A-10C]Figures 10A-10C show NFκB-mediated luciferase expression activity in the OX40-expressing reporter cell line HeLa_hOx40_NFκB_Luc1 of various bispecific antigen-binding molecules, including the OX40 clone CLC563. The concentrations of bispecific antigen-binding molecules in various formats or their controls are plotted against the light emitted units (URLs) measured after incubation and addition of luciferase detection solution. NFκB induction is shown for 3+1 or 2+1 formats crosslinked with human FAP-expressing NIH / 3T3 fibroblasts (Figure 10A), with a secondary antibody at a 2:1 ratio (Figure 10B), or additionally crosslinked w / o (Figure 10C). An isotype control antibody did not induce any NFκB activation. All OX40-containing constructs induced NFκB activation in a dose-dependent manner. The tetravalent format containing four OX40 Fab fragments was the most potent, inducing specific NFκB activation even in the absence of cross-linking due to the assembly of the trimeric core OX40 receptor signaling unit. Bispecific antigen binding molecules containing the OX40 clone CLC563 in trivalent or bivalent formats were less bioactive. Further cross-linking with human FAP-expressing fibroblasts via the FAP-binding moiety or with a secondary cross-linking antibody via the Fc region of the OX40 antigen binding molecule further increased NFκB activation. Clone OX40 (CLC563) achieved the highest NFκB activation induction in the 3+1 format, which was slightly more potent than the 2+1 format. Average values ​​of duplicates are shown. Error bars indicate SEM. [Figures 11A-11C]Figures 11A-11C show the NFκB-mediated luciferase expression activity in the OX40-expressing reporter cell line HeLa_hOx40_NFκB_Luc1 of different mutants of OX40 clone 49B4 with amino acid mutations in the VH domain in various formats, compared to a bispecific antigen-binding molecule (P1AE6838) containing OX40 clone 49B4 in a 4+1 format. The concentrations of the bispecific antigen-binding molecules in various formats or their controls are blotted against the light emitted units (URLs) measured after incubation and addition of luciferase detection solution. NFκB induction is shown in human FAP-expressing NIH / 3T3 fibroblasts crosslinked (Figure 11A), with a secondary antibody at a 2:1 ratio (Figure 11B), or additionally crosslinked w / o (Figure 11C). The isotype control antibody did not induce any NFκB activation. All amino acid variants induced NKκB activation in a dose-dependent manner comparable to the 4+1 format of the OX40 (49B4) antibody. The tetravalent use of the OX40 antigen-binding domain induced specific NFκB activation due to the assembly of the trimeric core OX40 receptor signaling unit. Further cross-linking with human FAP-expressing fibroblasts via the FAP-binding moiety or with a secondary cross-linking antibody via the Fc region of the OX40 antigen-binding molecule further enhanced NFκB activation, which was already evident at lower concentrations. Mean values ​​of duplicates are shown. Error bars indicate SEM. [Figures 12A-12B]Figures 12A and 12B show the primary T cell bioactivity of the bispecific antigen-binding molecule P1AE6838 compared to various reference molecules (P1AD3690, a non-targeting molecule containing four OX40 (49B4) Fab fragments; P1AD4524, a molecule containing four OX40 (49B4) Fab fragments and the FAP antibody 4B9 as the C-terminal VH / VL; P1AD4353, a molecule containing two OX40 (49B4) Fab fragments and the FAP antibody 4B9; and P1AD3691, a molecule containing two OX40 (49B4) Fab fragments and two FAP (28H1) Fab fragments). Figure 12A shows the number of CD4+ T cells, and Figure 12B shows the CD25 activation marker expression on CD4+ T cells at the endpoint. Increased proliferation and CD25 activation marker expression were observed in a dose-dependent manner using the FAP-targeting tetravalent OX40 antigen-binding molecule. The bivalent molecule had reduced bioactivity compared to the tetravalent, consistent with the fact that 49B4 is an avidity-driven OX40 antibody. The non-targeting OX40 molecule showed minimal activity at the highest tested concentration, while the isotype control showed no activation after baseline correction. Average values ​​of triplicates are shown. Error bars indicate SEM. [Figures 13A-13B] Figures 13A and 13B show the primary T cell bioactivity of bispecific antigen binding molecules containing OX40 (49B4) and FAP (1G1a) in 4+1, 3+1, and 2+1 formats compared to P1AD4524, a molecule containing four OX40 (49B4) Fab fragments and the FAP antibody 4B9 as C-terminal VH / VL. Figure 13A shows the number of CD4+ T cells, and Figure 13B shows CD25 activation marker expression on CD4+ T cells at endpoint. Increased proliferation and CD25 activation marker expression were observed in a dose-dependent manner using the FAP-targeting tetravalent OX40 antigen binding molecule. Trivalent and, to a greater extent, bivalent OX40 antigen binding molecules had reduced bioactivity compared to tetravalent, consistent with the fact that 49B4 is an avidity-driven antibody. The isotype control showed no activation after baseline correction. Average values ​​of three replicates are shown. Error bars indicate SEM. [Figures 14A-14B]Figures 14A and 14B show the primary T cell bioactivity of bispecific antigen binding molecules containing clone OX40 (8H9) in 3+1 and 2+1 formats compared to P1AE6838, a molecule containing four OX40 (49B4) Fab fragments. Figure 14A shows the number of CD4+ T cells, and Figure 14B shows the CD25 activation marker expression on CD4+ T cells at endpoint. Increased proliferation and CD25 activation marker expression were observed in a dose-dependent manner using FAP-targeted tetravalent OX40 antigen binding molecules. Trivalent and bivalent antigen binding molecules containing clone 8H9 produced slightly reduced maximal responses compared to tetravalent clone 49B4 and had comparable subnanomolar EC50s. Isotype controls showed no activation after baseline correction. Average values ​​of triplicates are shown. Error bars indicate SEM. [Figures 15A-15B] Figures 15A and 15B show the primary T cell bioactivity of bispecific antigen binding molecules containing clone OX40 (MOXR0916) in 3+1 and 2+1 formats compared to P1AE6838, a molecule containing four OX40 (49B4) Fab fragments. Figure 15A shows the number of CD4+ T cells, and Figure 15B shows the CD25 activation marker expression on CD4+ T cells at endpoint. Increased proliferation and CD25 activation marker expression were observed in a dose-dependent manner using FAP-targeted tetravalent OX40 antigen binding molecules. Trivalent and bivalent antigen binding molecules containing clone MOXR0916 resulted in similar numbers of CD4+ T cells and reduced maximum CD25 expression compared to tetravalent molecules containing clone 49B4. The reduction in CD4+ T cells at the highest concentration of MOXR0916-containing molecules tested is indicative of activation-induced cell death. The isotype control showed no activation after baseline correction. Average values ​​of triplicates are shown. Error bars indicate SEM. [Figures 16A-16B]Figures 16A and 16B show the primary T cell bioactivity of bispecific antigen binding molecules containing clone OX40 (CLC563) in 3+1 and 2+1 formats compared to P1AE6838, a molecule containing four OX40 (49B4) Fab fragments. Figure 16A shows the number of CD4+ T cells, and Figure 16B shows the CD25 activation marker expression on CD4+ T cells at endpoint. Increased proliferation and CD25 activation marker expression were observed in a dose-dependent manner using FAP-targeted tetravalent OX40 antigen binding molecules. Trivalent and bivalent antigen binding molecules containing clone CLC563 resulted in similar numbers of CD4+ T cells and reduced maximum CD25 expression compared to tetravalent molecules containing clone 49B4. The isotype control showed no activation after baseline correction. Average values ​​of triplicates are shown. Error bars indicate SEM. [Figures 17A-17B] Figures 17A and 17B show the primary T cell bioactivity of various variants of OX40 clone 49B4 with amino acid mutations in the VH domain in various formats, compared with a bispecific antigen binding molecule (P1AE6838) containing OX40 clone 49B4 in a 4+1 format. Figure 17A shows the number of CD8+ T cells, and Figure 17B shows the CD25 activation marker expression on CD4+ T cells at endpoint. Increased proliferation and CD25 activation marker expression were observed in a dose-dependent manner using FAP-targeted tetravalent OX40 antigen binding molecules. The three tetravalent amino acid variants showed activity comparable to that of the parent antibody in terms of CD8+ T cell proliferation and CD25 upregulation on CD4+ T cells. The isotype control showed no activation after baseline correction. Average values ​​of triplicates are shown. Error bars indicate SEM. [Figure 18] FIG. 18 shows a normalized summary of the area under the curve (AUC) of CD25 activation marker expression on CD4+ T cells as measured at endpoint for all antigen-binding molecules. [Figure 19] FIG. 19 shows the serum concentration-time profile and mean curves of the reference molecule P1AD4524 measured in three different female HuFcRn mice (F350, F351 and F353). [Figures 20A-20C] Figures 20A-20C show the equipotential surface area of ​​the Fab region of FAP clone 4B9 contained in the reference compound P1AD4524. The black mesh indicates positively charged patches, and the white mesh indicates negatively charged patches. Figure 20A shows a rear view, Figure 20B shows a front view, and Figure 20C shows the top of the antibody. Figures 20D-20F show the equipotential surface area of ​​the Fab region of FAP clone 1G1a contained in the molecule of the present invention. The black mesh indicates positively charged patches, and the white mesh indicates negatively charged patches. Figure 20D shows a rear view, Figure 20E shows a front view, and Figure 20F shows the top of the Fab. [Figure 21] Figure 21 shows the serum concentration-time profiles and mean curves of the bispecific antigen-binding molecule P1AE6836 containing a C-terminal cross-fab fragment compared to the reference molecule P1AD4524 containing VH and VL domains C-terminally fused to the Fc region, measured in three different female HuFcRn mice (F350, F351, and F353). [Figures 22A-22C] Figures 22A-C show the isoelectric surface area of ​​the Fab region of OX40 clone 49B9 in reference compound P1AD4524. The black mesh indicates positively charged patches, and the white mesh indicates negatively charged patches. Figure 22A shows a posterior view, Figure 22B shows a frontal view, and Figure 22C shows the top of the antibody. [Figures 23A-23C] Figures 23A-C show the equipotential surface area of ​​the Fab region of OX40 clone 8H9. The black mesh indicates positively charged patches, and the white mesh indicates negatively charged patches. Figure 23A shows a posterior view, Figure 23B shows a frontal view, and Figure 23C shows the top of the antibody. [Figures 24A-24C] Figures 24A-C show the equipotential surface area of ​​the Fab region of OX40 clone CLC563. The black mesh indicates positively charged patches, and the white mesh indicates negatively charged patches. Figure 24A shows a posterior view, Figure 24B shows a frontal view, and Figure 24C shows the top of the antibody. [Figures 25A-25C]Figures 25A-C show the equipotential surface area of ​​the Fab region of OX40 clone MOXR0916. The black mesh indicates positively charged patches, and the white mesh indicates negatively charged patches. Figure 25A shows a posterior view, Figure 25B shows a frontal view, and Figure 25C shows the top of the antibody. [Figure 26] Figure 26 is a schematic diagram of the transwell system used in the ARC assay. Madin-Darby canine kidney (MDCK) cells are seeded in the apical compartment of the transwell system. MDCK cells were transfected with human FcRn. [Figures 27A-27C] Figures 27A-27C show pre-existing anti-drug antibody (ADA) reactivity in a panel of human individual plasma samples measured with the assay described in Example 7.1. As described in WO 2017 / 060144, in which the VH and VL domains are C-terminally linked to each heavy chain (Figure 27A), a high incidence with a high signal was observed for the bispecific antigen binding molecule OX40(49B4)xFAP(4B9)(4+1). A lower incidence was detected for bispecific antigen binding molecules in which the VH and VL domains fused to the C-terminus of the Fc domain were replaced by Fab fragments. However, as can be seen for OX40(49B4)xFAP(4B9)(4+1) in Figure 27B and OX40(49B4)xFAP(1G1a)(4+1) in Figure 27C, pre-existing anti-drug antibodies against the Fab fragments still appear to be present. 1G1a is a humanized variant of FAP clone 212 (H212). [Figure 28] Figure 28 compares the pre-existing anti-drug antibody (ADA) reactivity of bispecific antigen binding molecules in a 2+1 format containing various anti-OX40 clones (49B4, 8H9, MOX0916, and CLC-563) in a panel of individual human plasma samples. [Figures 29A-29B]Figure 29A shows that control molecules, i.e., non-targeting tetravalent OX40 (49B4) antigen binding molecule (P1AD3690), FAP (1G1a) antibody (P1AE1689), or germline control antibody (DP47), did not elicit pre-existing anti-drug antibody (ADA) reactivity, but did elicit pre-existing IgG interference, as shown in Figure 29B. Surprisingly, the smaller 2+1 molecule induced a slightly higher incidence than the 3+1 and 4+1 format molecules. [Figures 30A-30C] Figures 30A-30C relate to testing the pre-existing anti-drug antibody (ADA) reactivity of the bispecific antigen-binding molecule OX40(49B4)xFAP(1G1a)(3+1). The molecule containing the CH1 domain with a "free" C-terminal EPKSC induces pre-existing ADA reactivity, as shown in Figure 30A. The respective background signals of buffer measured by performing the assay without drug molecules are shown in Figure 30B, and Figure 30C shows the pre-existing ADA reactivity of the molecule with the background signal subtracted. [Figures 31A-31C] The pre-existing anti-drug antibody (ADA) reactivity in a panel of human individual plasma samples of the bispecific antigen-binding molecule OX40(49B4)xFAP(1G1a)(3+1), as determined in Figure 30C, is also shown in Figure 31A and compared to pre-existing IgG reactivity induced by the bispecific molecule OX40(MOXR0916)xFAP(1G1a)(3+1) containing the EPKSD terminus (Figure 31B) or the bispecific molecule OX40(MOXR0916)xFAP(1G1a)(3+1) containing the EPKSC terminus (Figure 31C). A significant reduction was observed with the EPKSD mutant, whereas the EPKSC mutant completely eliminated pre-existing ADA reactivity. [Figures 32A-32C]Figures 32A to 32C show each molecule set in a 2+1 format. Previous results confirmed that the bispecific antigen-binding molecules OX40(MOXR0916)×FAP(1G1a)(2+1) and P1AF4852 (Figure 32B) with an EPKSC terminus reduced reactivity with pre-existing antibodies in plasma. On the other hand, previous results confirmed that the bispecific antigen-binding molecules OX40(MOXR0916)×FAP(1G1a)(2+1) and P1AF4858 (Figure 32C) with an EPKSC terminus lost reactivity with pre-existing antibodies in plasma compared to the molecule OX40(49B4)×FAP(1G1a)(2+1) with a free C-terminus EPKSC (P1AE6840, Figure 32A). [Figures 33A-33F] Figures 33A-33F confirm that the same effect was observed in three other examples. Pre-existing ADA reactivity in a panel of human individual plasma samples is shown in Figure 33A for OX40(CLC563)xFAP(1G1a)(3+1) with the EPKSCD terminus (P1AF6454), in Figure 33B for OX40(CLC563)xFAP(1G1a)(3+1) with the EPKSCS terminus (P1AF6455), and in Figure 33C for OX40(CLC563)xFAP(1G1a)(4+1) with the EPKSCD terminus (P1AF7205). Figure 33C shows OX40(CLC563)xFAP(1G1a)(4+1) with an EPK SCs terminus (P1AF7217), Figure 33D shows OX40(49B4_K23E_K73E)xFAP(1G1a)(3+1) with an EPK SCs terminus (P1AF6456), and Figure 33F shows OX40(49B4_K23E_K73E)xFAP(1G1a)(3+1) with an EPK SCs terminus (P1AF6457). [Figures 34A-34F]Figures 34A-34F show cell binding of bispecific antigen-binding molecules containing OX40 clone OX40(49B4_K23E_K73E) or OX40(CLC563) in 3+1 and 4+1 formats as D- and S-variants, as indicated. OX40-positive activated PBMCs, gated on activated CD4 T cells (Figures 34A, 34C, 34E) and activated CD8 T cells (Figures 34C, 34D, 34F), respectively, were incubated with the indicated serial dilutions of test antibodies detected by a fluorescently labeled secondary antibody against human Fcγ. Live cells were gated, and the mean fluorescence intensity of the secondary antibody (baseline corrected by media-only samples) was plotted from duplicates. Figure 34A shows the binding of the OX40(CLC563)3+1 construct as the D- and S-variants to activated CD4 cells, and Figure 34B shows the binding of the OX40(CLC563)3+1 construct as the D- and S-variants to activated CD8 T cells. Figures 34C and 34D show the binding of the OX40(CLC563)4+1 construct as the D- and S-variants to activated CD4 cells and activated CD8 cells, respectively. Binding of the OX40(49B4_K23E_K73E)4+1 construct as the D- and S-variants to activated CD4 cells and activated CD8 cells is shown in Figures 34E and 34F, respectively. As control molecules, a non-targeting tetravalent OX40(49B4)4+0 construct (P1AD3690), a tetravalent OX40(49B4)-FAP(4B9)4+1 construct (P1AD4524) and an isotype control were used. [Figures 35A-35F]Figures 35A-35F show NFκB-mediated luciferase expression activity in the OX40-expressing reporter cell line HeLa_hOx40_NFκB_Luc1. Concentrations of bispecific antigen-binding molecules containing OX40 clones OX40(49B4_K23E_K73E) or OX40(CLC563) in 3+1 and 4+1 formats as D and S variants are plotted against the light emitted units (URLs) measured after incubation and addition of luciferase detection solution. (Figure 35A for the OX40(CLC563) 3+1 construct as D- and S-variants, Figure 35C for the OX40(CLC563) 4+1 construct as D- and S-variants, and Figure 35E for the OX40(49B4_K23E_K73E) 4+1 construct as D- and S-variants) crosslinked with human FAP expressing NIH / 3T3 fibroblasts or (Figure 35B Figure 35A shows the induction of NFκB in NIH / 3T3 fibroblasts expressing human FAP (Figure 35B for the OX40(CLC563)3+1 construct as D- and S-variants, Figure 35D for the OX40(CLC563)4+1 construct as D- and S-variants, and Figure 35F for the OX40(49B4_K23E_K73E)4+1 construct as D- and S-variants) without further cross-linking with human FAP. An isotype control antibody did not induce any NFκB activation. All OX40-containing constructs induced NFκB activation in a dose-dependent manner. A tetravalent format containing four OX40 Fab fragments already induced some NFκB activation due to the assembly of the trimeric core OX40 receptor signaling unit in the absence of cross-linking. The S and D variants performed similarly. Averages of duplicates are shown. Error bars indicate SEM. [Figures 36A-36F]Figures 36A-F show the primary T cell bioactivity of bispecific antigen binding molecules containing OX40 clone OX40(49B4_K23E_K73E) or OX40(CLC563) in 3+1 and 4+1 formats as D and S variants as indicated. The bioactivity markers evaluated herein were the expression of the CD25 activation marker of CD4+ T+ cells (Figure 36A for the OX40(CLC563)3+1 construct, Figure 36C for the OX40(CLC563)4+1 construct, and Figure 36E for the OX40(49B4_K23E_K73E)4+1 construct) and CD8+ T cells (Figure 36B for the OX40(CLC563)3+1 construct, Figure 36D for the OX40(CLC563)4+1 construct, and Figure 36F for the OX40(49B4_K23E_K73E)4+1 construct) at the endpoint. Increased proliferation and CD25 activation marker expression were observed in a dose-dependent manner with the FAP-targeted OX40 antigen binding molecules. The non-targeted OX40 molecule showed activity only at the highest tested concentration, while the isotype control showed no activation after baseline correction. No statistically significant differences were observed between the S and D variants. Average values ​​of duplicates are shown. Error bars indicate SEM. [Figures 37A-37F]Figures 37A-F show that co-stimulation with a FAP-targeted OX40 agonist enhances PBMC cytokine secretion induced by CEACAM5 TCB-mediated lysis of tumor cells. PBMCs were co-cultured for 48 hours with MKN45NLR target cells, FAP+NIH / 3T3-huFAP clone 19, CECAM5TCB [2 nM], and bispecific antigen binding molecules containing OX40 clone OX40 (49B4_K23E_K73E) or OX40 (CLC563) in 3+1 and 4+1 formats as D and S variants. The bioactivity markers evaluated herein were the fold increase in GM-CSF (Figure 37A for the OX40(CLC563)3+1 construct, Figure 37C for the OX40(CLC563)4+1 construct, and Figure 37E for the OX40(49B4_K23E_K73E)4+1 construct) and TNF-α (Figure 37B for the OX40(CLC563)3+1 construct, Figure 37D for the OX40(CLC563)4+1 construct, and Figure 37F for the OX40(49B4_K23E_K73E)4+1 construct) in assay supernatants relative to samples treated with TCB alone. Cytokine induction was seen in a dose-dependent manner only for the FAP-bridged OX40 agonist. The non-targeting OX40 control molecule (P1AD3690) and the isotype control showed no activity here. The S-variant shows a trend toward reduced biological activity compared to the D-variant. Average values ​​of triplicates are shown. [Figures 38A-38F]Figures 38A-38F also show that co-stimulation with the FAP-targeting OX40 agonist enhances cytokine secretion in PBMCs induced by CEACAM5 TCB-mediated lysis of tumor cells. PBMCs were co-cultured for 48 hours with MKN45NLR target cells, FAP+NIH / 3T3-huFAP clone 19, CECAM5TCB [2 nM], and bispecific antigen binding molecules containing OX40 clones OX40 (49B4_K23E_K73E) or OX40 (CLC563) in 3+1 and 4+1 formats as D and S variants. The bioactivity markers evaluated herein were the fold increase in IFNγ (Figure 38A for the OX40(CLC563)3+1 construct, Figure 38C for the OX40(CLC563)4+1 construct, and Figure 38E for the OX40(49B4_K23E_K73E)4+1 construct) and IL-2 (Figure 38B for the OX40(CLC563)3+1 construct, Figure 38D for the OX40(CLC563)4+1 construct, and Figure 38F for the OX40(49B4_K23E_K73E)4+1 construct) in assay supernatants relative to samples treated with TCB alone. Cytokine induction was observed in a dose-dependent manner only for the FAP-bridged OX40 agonist. The non-targeting OX40 control molecule (P1AD3690) and the isotype control showed no activity here. The S-variant shows a trend toward reduced biological activity compared to the D-variant. Average values ​​of triplicates are shown. [Figure 39] Figure 39 summarizes the data and shows that co-stimulation with all FAP-targeted OX40 agonists enhances PBMC cytokine secretion induced by CEACAM5 TCB-mediated lysis of tumor cells. The AUCs of the dose-response curves in Figures 37A-F and 38A-F were calculated and normalized to the AUC of the OX40(CLC563) x FAP(1G1a_EPKSCD) 3 + 1 antigen binding molecule (P1AF6454, also known as 3 + 1CLC563 / H212-D). Each symbol represents one cytokine in the boxplot. [Figures 40A-40C]Figures 40A-40C show that co-stimulation with a FAP-targeted OX40 agonist suppresses TGFβ-induced induction of FoxP3 on Treg cells. Human PBMC preparations containing naive CD4+ T cells were cultured in the presence of TGFβ during T cell activation with antibodies against CD28 and CD3. OX40 agonism was provided by serial dilutions of bispecific antigen binding molecules containing OX40 clones OX40 (CLC563) or OX40 (49B4_K23E_K73E) in D and S variants in 3+1 and 4+1 formats. Cross-linking was provided by FAP antigen coated on beads. OX40 agonism prevented Treg induction, visible by a reduction in FoxP3 expression. Viable CD4+CD25+ Treg singlet cells were gated, and the MFI of the αFoxP3 antibody is reported. The FoxP3 MFI of each concentration was corrected by the MFI of the sample without OX40 antibody, and therefore without TGFβ alone. Figure 40A shows the effect of the OX40 (CLC563) 3 + 1 construct, Figure 40B shows the effect of the OX40 (CLC563) 4 + 1 construct, and Figure 40C shows the effect of the OX40 (49B4_K23E_K73E) 4 + 1 construct. The D and S variants of each FAP-targeting OX40 bispecific antigen binding molecule suppressed FoxP3 to a similar extent. The average of three replicates is shown, and error bars represent SEM. [Figure 41] Figure 41 shows single-dose plasma concentration-time profiles in Hu FcRn mice for the OX40(CLC563) x FAP(1G1a_EPKSCD) 3 + 1 antigen binding molecule (P1AE6454) and the OX40(49B4) x FAP(4B9) 4 + 1 antigen binding molecule (P1AD4524). [Figure 42A-42B] Figures 42A and 42B show the release of cytokine IL-6 in human whole blood samples upon incubation with FAPxOX40 bispecific antigen-binding molecules. The median signal for each donor and concentration is shown relative to the signal for Erbitux (registered trademark) as a negative control. Figure 42A shows the median signals for bispecific antibodies C1, C2, and C3 (see Example 8.1.1), and Figure 42B shows the median signals for bispecific antibodies C4, C5, C6, and C7. [Figure 43A-43B] Figures 43A and 43B show the release of cytokine IL-8 in human whole blood samples upon incubation with FAPxOX40 bispecific antigen-binding molecules. The median signal for each donor and concentration is shown relative to the signal for Erbitux (registered trademark) as a negative control. Figure 43A shows the median signals for bispecific antibodies C1, C2, and C3 (see Example 8.1.1), and Figure 43B shows the median signals for bispecific antibodies C4, C5, C6, and C7. [Figure 44A-44B] Figures 44A and 44B show the results of a DC:CD4+ T cell assay assessing the sequence-associated risk of immunogenicity of bispecific FAPxOX40 antibodies P1AF6454 and P1AF6455 compared to keyhole limpet hemocyanin (KLH) and bevacizumab (Avastin®) as positive controls. IFNγ stimulation plotted against stimulation index is shown in Figure 44A, and Figure 44B shows a summary of response rates in percentage of response. [Figure 45A-45B] Figures 45A and 45B relate to the results of a DC:CD4+ T cell assay evaluating P1AD4524 against keyhole limpet hemocyanin (KLH) as a positive control and bevacizumab (Avastin®) and adalimumab (Humira®). IFNγ stimulation plotted against stimulation index is shown in Figure 45A, and Figure 45B shows a summary of the response rate in percent of response. [Figure 46] Figures 46, 47, and 48 show the results of a 4-week immunogenicity study in C57BL / 6 wild-type mice and transgenic C57BL / 6-Tg(hIgG1,k,l) ​​mice, which are immunologically tolerant to human IgG1 antibodies. The immune responses of two individual mouse groups treated with P1AF6455 are shown in Figure 46, those of two individual mouse groups treated with P1AF6454 are shown in Figure 47, and those of two individual mouse groups treated with P1AD4524 are shown in Figure 48. [Figure 47]Figures 46, 47, and 48 show the results of a 4-week immunogenicity study in C57BL / 6 wild-type mice and transgenic C57BL / 6-Tg(hIgG1,k,l) ​​mice, which are immunologically tolerant to human IgG1 antibodies. The immune responses of two individual mouse groups treated with P1AF6455 are shown in Figure 46, those of two individual mouse groups treated with P1AF6454 are shown in Figure 47, and those of two individual mouse groups treated with P1AD4524 are shown in Figure 48. [Figure 48] Figures 46, 47, and 48 show the results of a 4-week immunogenicity study in C57BL / 6 wild-type mice and transgenic C57BL / 6-Tg(hIgG1,k,l) ​​mice, which are immunologically tolerant to human IgG1 antibodies. The immune responses of two individual mouse groups treated with P1AF6455 are shown in Figure 46, those of two individual mouse groups treated with P1AF6454 are shown in Figure 47, and those of two individual mouse groups treated with P1AD4524 are shown in Figure 48. [Figure 49] Figure 49 shows the study design for an efficacy study of bispecific FAPxOX40 antibodies in combination with CEACAM5TCB in MKN45 xenografts in humanized mice (comparison of different OX40 clones). The design and various treatment groups are shown. Compared were bispecific FAPxOX40 antibodies, including antibodies OX40 (49B4_K23E_K73E, referred to herein as 49B4CPV), OX40 (CLC563), OX40 (8H9), and OX40 (49B4). [Figure 50A-50B] Figures 50A and 50B show the results of an efficacy study with the FAPxOX40 bispecific antibody in combination with CEACAM5TCB in MKN45 xenografts in humanized mice. Figure 50A shows the mean tumor volume, or the percent change in tumor volume in individual mice in different treatment groups plotted on the y-axis (Figure 50B). DETAILED DESCRIPTION OF THE INVENTION

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

[0042] As used herein, the term "antigen-binding molecule" refers in the broadest sense 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 term "antigen-binding domain capable of specifically binding to a target cell antigen" or "moiety capable of specifically binding to a target cell antigen" refers to a polypeptide molecule that specifically binds to an antigen. In one embodiment, the antigen-binding domain can activate signal transduction via its target cell antigen. In certain embodiments, the antigen-binding domain can target the entity to which it is bound (e.g., an OX40 agonist antibody) to a target site, such as a specific type of tumor cell or tumor stroma bearing an antigenic determinant. Antigen-binding domains capable of specifically binding to a target cell antigen include antibodies and fragments thereof as further defined herein. Furthermore, antigen-binding domains capable of specifically binding to a target cell antigen include scaffold antigen-binding proteins as further defined herein, such as binding domains based on designed repeat proteins or designed repeat domains (see, e.g., WO 2002 / 020565). In particular, 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] In relation to an antibody or a fragment thereof, the term "antigen-binding domain capable of specific binding to a target cell antigen" refers to a portion of a molecule comprising a region that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain capable of specific antigen binding can be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). Specifically, an antigen-binding domain capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one particular embodiment, the "antigen-binding domain capable of specific antigen binding" is a Fab fragment or a cross-Fab fragment.

[0045] The term "antibody" herein is used in the broadest sense and encompasses 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, so long as they exhibit the desired antigen-binding activity.

[0046] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual antibody in the population is identical and / or binds to the same epitope, except for possible variant antibodies, including, for example, naturally occurring mutations or mutations that arise during production of a monoclonal antibody preparation, of which such variants will generally be present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

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

[0048] The term "monovalent" as used herein means that within an antigen-binding molecule specific for one different antigenic determinant, there is a specific number of binding sites specific for one different antigenic determinant. Thus, the terms "bivalent," "trivalent," "tetravalent," and "hexavalent" refer to the presence of two, three, four, and six binding sites, respectively, specific for a particular antigenic determinant in an antigen-binding molecule. In certain embodiments of the present invention, a bispecific antigen-binding molecule according to the present invention can be monovalent for a particular antigenic determinant (meaning it has only one binding site for that antigenic determinant), or bivalent or tetravalent for a particular antigenic determinant (meaning it has two or four binding sites for that antigenic determinant, respectively). The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody. "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy domain or a heavy chain variable domain) followed by three constant domains (CH1, CH2, and CH3) (also called a heavy chain constant region). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light domain or a light chain variable domain) followed by a light chain constant domain (CL) (also called a light chain constant region). Antibody heavy chains may be divided into one of five types called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Antibody light chains may be assigned to one of two types called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0049] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); 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, Plueckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having extended in vivo half-lives, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments containing two antigen-binding sites that may be bivalent or bispecific; see, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003), and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, Massachusetts; see, e.g., U.S. Pat. No. 6,248,516 B1).Antibody fragments may be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0050] Papain digestion of an intact antibody yields two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to an antibody fragment containing the VL domain and constant domain of the light chain (CL) and the VH domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues in the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment, which contains two antigen-binding sites (two Fab fragments) and part of the Fc region. According to the present invention, the term "Fab fragment" also includes "cross-Fab fragments" or "crossover Fab fragments," as defined below.

[0051] The terms "crossFab fragment" or "xFab fragment" or "crossover Fab fragment" refer to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. Two possible chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the present invention. On the other hand, the variable regions of the Fab heavy and light chains are swapped, i.e., the crossover Fab molecule contains a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). This crossover Fab molecule is called crossFab. (VLVH)On the other hand, when the constant regions of the Fab heavy and light chains are replaced, the crossover Fab molecule contains a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL), and a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1). This crossover Fab molecule is called crossFab. (CLCH1) It is also called.

[0052] A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, in which the antibody domains and linker have one of the following orders from N- to C-terminus: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1, or (d) VL-CH1-linker-VH-CL, and the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules may be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0053] A "crossover single-chain Fab fragment" or "x-scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following orders, from N- to C-terminus: (a) VH-CL-linker-VL-CH1; or (b) VL-CH1-linker-VH-CL, where VH and VL together form an antigen-binding site that specifically binds to a given antigen, and the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules may be further stabilized by the creation of an interchain disulfide bond by the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain, according to the Kabat numbering system).

[0054] A "single-chain variable fragment (scFv)" is a fragment of an antibody heavy chain (V) linked using a short linker peptide of 10 to about 25 amino acids. H ) and light chain (V L ) variable region fusion proteins. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility, and V H N-terminus and V L The scFv antibody fragment may be linked to the C-terminus of a full-length antibody, or vice versa. This protein retains the specificity of the original antibody, although the constant regions have been removed and a linker has been introduced. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments contain a single polypeptide chain characterized by a VH domain (i.e., capable of assembling with a VL domain) or a VL domain (i.e., capable of assembling with a VH domain into a functional antigen-binding site), thereby conferring the antigen-binding properties of a full-length antibody.

[0055] "Scaffold antigen-binding proteins" are known in the art; for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen-binding domains. See, for example, 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 invention, the scaffold antigen binding protein is selected from the group consisting of CTLA-4 (e.g., cytochrome P456), lipocalin (anticalin), protein A-derived molecules such as the Z-domain (affibody), A-domain (avimer / maxibody) of protein A, serum transferrin (transbody); engineered ankyrin repeat proteins (DARPins), variable domains of antibody light or heavy chains (single domain antibodies, sdAb), variable domains of antibody heavy chains (nanobodies, aVH), V NAR Fragments, fibronectin (adnectin), C-type lectin domain (tetranectin); variable domain of novel antigen receptor β-lactamase (V NAR fragments), human gamma-crystallin or ubiquitin (affilin molecules); Kunitz-type domains of human protease inhibitors, microbodies, e.g., proteins from the knottin family, peptide aptamers, and fibronectin (adnectins). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is primarily a cytotoxic T lymphocyte-associated antigen. +It is a CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. Loops corresponding to antibody CDRs may be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as ebibodies (e.g., U.S. Patent No. 7,166,697 B1). Ebibodies are approximately the same size as the isolated variable regions of antibodies (e.g., domain antibodies). For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have a rigid beta-sheet secondary structure with many loops at the open end of a conical structure that can be engineered to bind to different target antigens. Anticalins are 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Patent No. 7,250,297 B1, and U.S. Patent Application Publication No. 20070224633. Affibodies are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. Domains consist of three helical bundles of approximately 58 amino acids. Libraries are created by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and European Patent Application Publication No. 1641818. Avimers are multidomain proteins derived from the A-domain scaffold family. Natural domains of approximately 35 amino acids conform to defined disulfide-bonded structures. Diversity is generated by shuffling the natural variation displayed 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 different target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include transbodies. For further details, see J. Biol. Chem 274, 24066-24073 (1999). Designed ankyrin repeat proteins (DARPins) are derived from ankyrins, a family of proteins that mediate the adhesion of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. Single ankyrin repeats can be engineered to bind different target antigens by randomizing residues in the first α-helix and β-turn of each repeat. 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), and J. Mol. Biol. 369, 1015-1028 (2007), and US Patent Application Publication No. 20040132028 A1. Single-domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domain was derived from the variable domain of a camel-derived antibody heavy chain (nanobody or V). H Furthermore, the term single domain antibody refers to an antibody that contains an autonomous human heavy chain variable domain (aVH) or a shark-derived VH. NARThese include fragments. Fibronectin is a scaffold that can be engineered to bind to antigens. Adnectins consist of a backbone with the native amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the beta sandwich can be engineered to enable Adnectins to specifically recognize therapeutic targets of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent Application Publication No. 20080139791, International Application Publication No. 2005056764, and U.S. Patent No. 6,818,418. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted into the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins, 25-50 amino acids long, containing three to four cysteine ​​bridges. Examples of microproteins include KalataBI, conotoxins, and knottins. Microproteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

[0056] An "antigen-binding molecule that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks the binding of the reference molecule to its antigen by 50% or more in a competitive assay; conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay. An "antigen-binding molecule that does not bind to the same epitope" as a reference molecule refers to an antigen-binding molecule that does not block the binding of the reference molecule to its antigen by 50% or more in a competitive assay; conversely, the reference molecule does not block the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay.

[0057] The term "antigen-binding domain" or "antigen-binding site" refers to a portion of an antigen-binding molecule that comprises a region that specifically binds to and is complementary to part or all of an antigen. When an antigen is large, an antigen-binding molecule may bind only to a specific portion of the antigen, which portion is called an epitope. An antigen-binding domain may be provided, for example, by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0058] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to the site on a polypeptide macromolecule to which an antigen-binding moiety binds (e.g., a contiguous stretch of amino acids or a conformational configuration composed of different regions of noncontiguous amino acids), forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can 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 in the extracellular matrix (ECM). Proteins useful as antigens of the present invention can be any naturally occurring form of the protein from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When referring to a particular protein of the present invention, the term encompasses "full-length," unprocessed proteins and any form of the protein obtained from cellular processing. The term also encompasses naturally occurring variants of the protein, e.g., splice variants or allelic variants.

[0059] "Specific binding" means that the binding is antigen-selective and can be distinguished from unwanted or non-specific 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 known in the art, such as surface plasmon resonance (SPR) technology (analyzed by a BIAcore device) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the 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, as measured, for example, by SPR. In certain embodiments, molecules that bind to an 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~10 -13 M, e.g., 10 -9 M~10 -13 M).

[0060] "Affinity" or "binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its binding partner Y can generally be expressed by the dissociation constant (Kd), which is the ratio of the desorption rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities can involve different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0061] An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) relative to a parent antibody that does not possess such alterations, which alterations improve the affinity of the antibody for antigen.

[0062] As used herein, "target cell antigen" refers to an antigenic determinant displayed on the surface of a target cell, particularly a target cell within a tumor, such as a cancer cell or tumor stromal cell. Thus, the target cell antigen is a tumor-associated antigen. Specifically, the tumor target cell antigen is fibroblast activation protein (FAP).

[0063] The term "fibroblast activation protein (FAP)," also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any naturally occurring FAP derived 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), unless otherwise specified. The term encompasses "full-length," unprocessed FAPs and any form of FAP resulting from processing in cells. The term also encompasses naturally occurring variants of FAPs, 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 760. The amino acid sequence of 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 761. SEQ ID NO: 64 shows the amino acid sequence of His-tagged mouse FAP ECD. SEQ ID NO: 65 shows the amino acid sequence of His-tagged cynomolgus monkey FAP ECD. Preferably, the anti-FAP binding molecules of the invention bind to the extracellular domain of a FAP.

[0064] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th, W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0065] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain, e.g., "complementarity-determining regions" (CDRs), that are hypervariable in sequence and determine antigen-binding specificity. Generally, antibodies contain six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs 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) Antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0066] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR designations can be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0067] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in the following order in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0068] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0069] 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, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0070] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDRs corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have been further modified or altered from those of the original antibody to create the properties according to the invention, particularly in terms of C1q binding and / or Fc receptor (FcR) binding.

[0071] The term "CH1 domain" refers to a portion of an antibody heavy chain polypeptide extending from approximately EU position 118 to EU position 215 (EU numbering system according to Kabat). In one embodiment, the CH1 domain has the amino acid sequence ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKV (SEQ ID NO: 166). Typically, a segment having the amino acid sequence EPKSC (SEQ ID NO: 163) then links the CH1 domain to the hinge region. The inventors have found that a CH1 domain not fused to a hinge region can provide reactivity with pre-existing antibodies (ADAs) in humans that is not present when the variants EPKSCD (SEQ ID NO: 164) or EPKSCS (SEQ ID NO: 165) are present. A CH1 domain with a free C-terminus can be found, for example, in crossfab fragments.

[0072] The term "hinge region" refers to a portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 domains in a wild-type antibody heavy chain (e.g., from about position 216 to about position 230, or from about position 226 to about position 230 according to the EU numbering system of Kabat). The hinge regions of other IgG subclasses can be determined by aligning with the hinge region cysteine ​​residues of the IgG1 subclass sequence. The hinge region is usually a dimeric molecule consisting of two polypeptides with identical amino acid sequences. The hinge region generally contains up to 25 amino acid residues and is flexible, allowing the associated target binding sites to move independently. The hinge region can be subdivided into three domains: the upper, middle, and lower hinge domains (see, e.g., Roux, et al., J. Immunol. 161 (1998) 4083). In one aspect, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 160), wherein X is either S or P. In one aspect, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO: 161), wherein X is either S or P. In one aspect, the hinge region has the amino acid sequence CPXCP (SEQ ID NO: 162), wherein X is either S or P.

[0073] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. An IgG Fc region contains an IgG CH2 domain and an IgG CH3 domain. The "CH2 domain" of a human IgG Fc region typically extends from about amino acid residue 231 to about amino acid residue 340 (EU numbering system according to Kabat). In one embodiment, the CH2 domain has the amino acid sequence APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 153), and is unique in that it is not closely paired with other domains. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native Fc region. It has been speculated that carbohydrates may provide an alternative domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, the carbohydrate chains are attached to the CH2 domain. The CH2 domain of the present invention may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 according to the EU numbering system by Kabat for IgG). In one aspect, the CH3 domain has the amino acid sequence of GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 154).The CH3 region of the present invention may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "protrusion" ("knob") in one chain and an introduced "cavity" ("hole") in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0074] The term "wild-type Fc domain" refers to an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include native human IgG1 Fc regions (non-A and A allotypes), native human IgG2 Fc regions, native human IgG3 Fc regions, and native human IgG4 Fc regions, as well as naturally occurring variants thereof. Wild-type Fc regions are set forth in SEQ ID NO: 155 (IgG1, Caucasian allotype), SEQ ID NO: 156 (IgG1, Afro-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 "wild-type" (human) Fc domain amino acid sequence by at least one "amino acid mutation." In one embodiment, the variant Fc region has at least one amino acid mutation, e.g., about 1 to about 10 amino acid mutations, compared to a native Fc region, and in one embodiment, about 1 to about 5 amino acid mutations in the native Fc region. In one aspect, the (variant) Fc region has at least about 95% homology to the wild-type Fc region.

[0075] The "knob-into-hole" technique is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In a further specific embodiment, the Fc domain subunit containing the knob modification further comprises the amino acid substitution S354C, and the Fc domain subunit containing the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0076] A "region equivalent to the Fc region of an immunoglobulin" is intended to include allelic variants of naturally occurring immunoglobulin Fc regions and variants having alterations that result in substitutions, additions, or deletions, but that do not substantially reduce the ability of the immunoglobulin to mediate effector function (e.g., antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N- or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art to have minimal effect on activity (see, e.g., Bowie, JU et al., Science 247:1306-10 (1990)).

[0077] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0078] Fc receptor binding-dependent effector functions can be mediated by the interaction of the Fc region of an antibody with Fc receptors (FcRs), which are dedicated cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of red blood cells and other cellular targets (e.g., tumor cells) coated with the corresponding antibodies by antibody-dependent cellular cytotoxicity (ADCC) (see, e.g., Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are called FcγRs. Fc receptor binding is described, for example, in 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.

[0079] Cross-linking of receptors to the Fc region of IgG antibodies (FcγR) triggers a wide variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as control of immune complex clearance and antibody production. In humans, three classes of FcγR have been characterized, these are: FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modifications within the Fc region at at least one of amino acid residues E233-G236, P238, D265, N297, A327, and P329 (numbered according to the EU index of Kabat) reduce binding to FcγRI. Substitution of IgG2 residues at positions 233-236 with IgG1 and IgG4 reduced binding to FcγRI by 10-3 times and ablated human mononuclear cell responses to antibody-sensitized erythrocytes (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624). FcγRII (CD32) binds complexed IgG with moderate to low affinity and is widely expressed. The receptor can be divided into two subtypes: FcγRIIA and FcγRIIB. FcγRIIA is found primarily on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcγRIIB appears to play a role in inhibitory processes and has been found on B cells, macrophages, as well as mast cells and eosinophils. On B cells, FcγRIIB appears to function in further suppressing immunoglobulin production and isotype switching, for example, to the IgE class. On macrophages, FcγRIIB plays a role in inhibiting phagocytosis, as mediated by FcγRIIA. On eosinophils and mast cells, the B form may help suppress the activation of these cells by binding IgE to its respective receptor. Decreased binding to FcγRIIA has been found, for example, for antibodies comprising an IgG Fc region with mutations in at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to the EU index of Kabat). FcγRIII (CD16) binds IgG with moderate to low affinity and exists as two species. FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Reduced binding to FcγRIIIA has been found, for example, for antibodies containing an IgG Fc region with a mutation at least in one of the following amino acid residues: E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (numbering according to the EU index of Kabat).

[0080] Mapping of the binding sites on human IgG1 for Fc receptors, the mutation sites described above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al. J. Biol. Chem. 276 (2001) 6591-6604.

[0081] The term "ADCC" or "antibody-dependent cellular cytotoxicity" refers to the lysis of target cells by the antibodies reported herein, a function mediated by Fc receptor binding, in the presence of effector cells. The ability of an antibody to trigger the initial steps in mediating ADCC is investigated by measuring antibody binding to cells expressing Fcγ receptors, such as recombinantly expressing FcγRI and / or FcγRIIA or NK cells (which inherently express FcγRIIIA). Specifically, binding to FcγR on NK cells is measured.

[0082] An "activating Fc receptor" is an Fc receptor that, upon ligation by the Fc region of an antibody, triggers a signaling event that stimulates the receptor-containing cell to exert effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0083] As used herein, the term "OX40," unless otherwise specified, refers to any naturally occurring 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," unprocessed OX40, as well as any form of OX40 resulting from processing within a cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants. The amino acid sequence of an exemplary human OX40 is set forth in SEQ ID NO: 1 (Uniprot P43489, version 112), and the amino acid sequence of an exemplary mouse OX40 is set forth in SEQ ID NO: 66 (Uniprot P47741, version 101).

[0084] As used herein, the term "OX40 agonist" includes any moiety that agonizes the OX40 / OX40L interaction. OX40 as used in this context preferably refers to human OX40, and therefore, an OX40 agonist is preferably an agonist of human OX40. Typically, the moiety is an agonistic OX40 antibody or antibody fragment, particularly a Fab fragment.

[0085] The terms "anti-OX40 antibody," "anti-OX40," "OX40 antibody," and "antibody that specifically binds to OX40" refer to an antibody that can bind to OX40 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic when targeting OX40. In one aspect, the extent of binding of an anti-OX40 antibody to an unrelated, non-OX40 protein is less than about 10% of the binding of the antibody to OX40, as measured, for example, by flow cytometry (FACS). In certain embodiments, an antibody that binds to OX40 has an affinity of ≦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~10 -13 M, e.g. 10 -8 M~10- 10 Dissociation constant (K D )

[0086] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable, non-immunogenic linker peptides include, for example, (G4S) n ,(SG4) n or G4 (SG4) nis a peptide linker, wherein "n" is generally a number from 1 to 10, typically from 2 to 4, in particular 2, i.e., the peptide is selected from the group consisting of GGGGS (SEQ ID NO: 67), GGGGSGGGGS (SEQ ID NO: 68), SGGGGSGGGG (SEQ ID NO: 69) and GGGGSGGGGSGGGG (SEQ ID NO: 70), but also includes the sequences GSPGSSSSGS (SEQ ID NO: 71), (GS) (SEQ ID NO: 72), (GS) (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). Peptide linkers 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).

[0087] The term "amino acid" as used in this application refers to the group of naturally occurring carboxy α-amino acids, including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (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).

[0088] By "fused" or "linked" is meant that the components (e.g., antibody heavy chain and Fab fragment) are joined by peptide bonds, either directly or via one or more peptide linkers.

[0089] "Percent amino acid sequence identity" to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, etc. It can also be achieved using SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, with sequence B, or with sequence B (alternatively, it can be written as a given amino acid sequence A having or containing a certain percent amino acid sequence identity with or against sequence B for a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A differs from the length of amino acid sequence B, the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0090] In certain embodiments, amino acid sequence variants of the bispecific antigen-binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the TNF ligand trimer-containing antigen-binding molecule. Amino acid sequence variants of the TNF ligand trimer-containing antigen-binding molecule can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, as long as the final construct possesses the desired characteristics (e.g., antigen binding). Target sites for substitutional mutagenesis include HVRs and framework regions (FRs). Conservative substitutions are provided in Table B under the heading "Preferred Substitutions" and are further described below with reference to amino acid side chain classes (1) to (6). Amino acid substitutions may be introduced into the molecule of interest and the products screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC). [Table A]

[0091] Amino acids can be classified according to general side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0092] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0093] The term "amino acid sequence variant" includes substantial variants in which amino acid substitutions are present in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing will have altered (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antigen-binding molecule and / or will substantially retain the specific biological properties of the parent antigen-binding molecule. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, phage-displayed, and screened for a specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antigen complex may be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted or excluded as candidates for substitution. Variants may be screened to determine whether they possess desired properties.

[0094] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of a terminal insertion is a bispecific antigen-binding molecule of the present invention having an N-terminal methionyl residue. Other insertional variants of the molecule include N- or C-terminal fusions to polypeptides that increase the serum half-life of the bispecific antigen-binding molecule.

[0095] In certain embodiments, the bispecific antigen-binding molecules provided herein are modified to increase or decrease the degree of glycosylation of the antibody. Glycosylation variants of the molecule can be conveniently obtained by altering the amino acid sequence to create or remove one or more glycosylation sites. In cases where the antigen-binding molecule contains an Fc region, the carbohydrate attached to the Fc region can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally linked to Asn297 in the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications may be performed within the TNF family ligand trimer-containing antigen-binding molecule to generate variants with specific improved properties. In one aspect, a bispecific antigen-binding molecule or antibody variant of the present invention is provided that has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. Such fucosylation variants may have improved ADCC function; see, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) or U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). In another aspect, a bispecific antigen-binding molecule or antibody variant of the present invention is provided that contains bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region are bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, e.g., WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Pat. App. Pub. No. 2005 / 0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.Such 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.).

[0096] In certain embodiments, it may be desirable to create cysteine-engineered variants of the bispecific antigen-binding molecules of the present invention, e.g., "thioMAbs," in which one or more residues of the molecule are replaced with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the molecule. By replacing these residues with cysteine, reactive thiol groups are located at accessible sites on the antibody, which may be used to conjugate the antibody to other moieties (e.g., drug moieties or linker-drug moieties) and create immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antigen-binding molecules may be created, for example, as described in U.S. Pat. No. 7,521,541.

[0097] The terms "nucleic acid" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed 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. Nucleic acid molecules are often described by their base sequence, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, and both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, the mRNA may be chemically modified to increase the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2101823 B1).

[0098] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location than that normally contained within a cell that contains the nucleic acid molecule.

[0099] An "isolated nucleic acid encoding a bispecific antigen-binding molecule or antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of a bispecific antigen-binding molecule or antibody, including nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules are present in one or more locations within a host cell.

[0100] A nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention is intended to be identical to the reference sequence, except that the nucleotide sequence of the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the 5'-terminal position or 3'-terminal position of the reference nucleotide sequence, or anywhere between the 5'-terminal position and the 3'-terminal position, either individually between residues in the reference sequence or in one or more consecutive groups within the reference sequence. In practical terms, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0101] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide that contains a specific sequence of nucleic acid elements that enable transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector contains, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, expression cassettes of the invention contain a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention, or a fragment thereof.

[0102] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and direct the expression of an operably associated specific gene in a target cell. This term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which the vector is introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows for the stable transcription of large amounts of mRNA. Once the expression vector is inside the target cell, the protein encoded by the ribonucleic acid molecule or gene is produced by the cellular transcriptional and / or translational machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antibody of the present invention, or a fragment thereof.

[0103] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and progeny derived from the host cell, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to produce the bispecific antigen-binding molecules of the present invention. Host cells include cultured cells, e.g., cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 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, but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0104] An "effective amount" of an agent refers to the amount necessary to cause a physiological change in a cell or tissue to which the agent is administered.

[0105] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount effective, at the necessary dosage and for the necessary period of time, to achieve a desired therapeutic or preventative result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0106] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0107] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained in the preparation is effective and that does not contain any additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition will be administered.

[0108] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0109] The term "package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products, including information regarding the indications, uses, dosage, administration, concomitant therapy, contraindications and / or warnings regarding such therapeutic products.

[0110] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course in the individual being treated, and may be performed prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, remission or palliation of symptoms, and recovery or improved prognosis. In some embodiments, the molecules of the invention are used to delay the onset of disease or slow the progression of the disease.

[0111] The term "cancer", as used herein, includes lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, cancer of the central nervous system, This refers to proliferative disorders such as central nervous system (CNS) neoplasms, spinal axis tumors, brain stem gliomas, 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 a combination of one or more of the above cancers).

[0112] The term "chemotherapeutic agent" as used herein refers to a chemical compound useful in the treatment of cancer. In one aspect, the chemotherapeutic agent is an antimetabolite. In one aspect, 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 particular aspect, the antimetabolite is capecitabine or gemcitabine. In another aspect, the antimetabolite is fluorouracil. In one aspect, the chemotherapeutic agent is an agent that affects microtubule formation. In one aspect, the agent that affects microtubule formation is selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, vinorelbine, taxotere, etoposide, and teniposide. In another embodiment, the chemotherapeutic agent is an alkylating agent such as cyclophosphamide. In one embodiment, the chemotherapeutic agent is a cytotoxic antibiotic such as a topoisomerase II inhibitor. In one embodiment, the topoisomerase II inhibitor is doxorubicin.

[0113] Bispecific antibodies of the present invention The present invention provides a novel bispecific antigen-binding molecule comprising a new anti-FAP antibody (clone 212). This bispecific antigen-binding molecule comprising the novel anti-FAP antibody has particularly advantageous properties (e.g., productivity, stability, binding affinity, biological activity, targeting efficiency, reduced internalization, excellent pharmacokinetic (PK) properties (e.g., improved clearance), reduced toxicity, a wider dosage range that can be administered to patients, and thereby potentially enhanced efficacy). Furthermore, the bispecific antigen-binding molecule is prepared in an advantageous format depending on the OX40 antibody contained therein.

[0114] Exemplary Bispecific Antigen-Binding Molecules In one aspect, the present invention provides a bispecific antigen-binding molecule characterized by targeted agonistic binding to OX40. In particular, the bispecific antigen-binding molecule is an OX40 agonist targeted to FAP. In another specific aspect, the bispecific antigen-binding molecule of the present invention comprises an Fc region composed of a first subunit and a second subunit that can stably associate and that contain a mutation that reduces effector function. The use of an Fc region that contains a mutation that reduces or abolishes effector function prevents nonspecific agonism through cross-linking via Fc receptors and enhances OX40. + The bispecific antigen-binding molecules described herein have the advantage over conventional antibodies capable of specifically binding to OX40 in that they selectively induce an immune response in target cells that are typically close to the tumor, i.e., in the tumor stroma.

[0115] Thus, the bispecific antigen-binding molecule is characterized by FAP-targeted agonistic binding to OX40. In the presence of FAP-expressing cells, the bispecific antigen-binding molecule can induce NFκB activation in human OX40-positive NFκB reporter cells.

[0116] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) at least two antigen-binding domains capable of specifically binding to OX40; (b) an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), the heavy chain variable region (V) comprising: (i) CDR-H1 comprising the 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 the amino acid sequence of SEQ ID NO: 5. H a light chain variable region (V FAP) comprising (iv) a 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, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; L an antigen-binding domain comprising an FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule comprising:

[0117] In one embodiment, the antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP) comprises a heavy chain variable region (VH) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (ii) a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5. H and a light chain variable region (V FAP) comprising (iv) a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, (v) a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 8. L FAP) and

[0118] In one embodiment, the Fc region comprises one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antibody to an Fc receptor.

[0119] In a further embodiment, the antigen-binding domain capable of specifically binding to a FAP comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:9. H FAP) and a light chain variable region (V) comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 10. L In one embodiment, the antigen-binding domain capable of specifically binding to FAP comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 9. H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10 L FAP).

[0120] In another embodiment, the antigen-binding domain capable of specifically binding to a FAP comprises a heavy chain variable region (V) 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. HFAP) and a light chain variable region (V) 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. L and an antigen-binding domain comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, the antigen-binding domain capable of specifically binding to FAP comprises: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 15. H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 21 L FAP), (b) a heavy chain variable region (V H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 21 L FAP), (c) a heavy chain variable region (V H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 22 L FAP), or (d) a heavy chain variable region (V H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 25 L More specifically, the antigen-binding domain capable of specifically binding to FAP comprises: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 15; H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 21 L FAP).

[0121] In one embodiment, an antigen binding domain capable of specifically binding to OX40 comprises or consists of a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

[0122] In a further aspect, the antigen binding domain capable of specifically binding to OX40 comprises: (a) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29; HOX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32. L OX40), or (b) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37; H OX40), and a light chain variable region (V) 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. L OX40), or (c) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45 H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. L OX40), or (d) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53; H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 54, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 56. L OX40) A bispecific antigen-binding molecule is provided, comprising:

[0123] In one embodiment, an antigen binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VH1) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29. H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32. L In a further embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37. H OX40), and a light chain variable region (V) 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. L In another embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45. H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. L In yet another embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53. HOX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 54, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 56. L In one particular embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37. H OX40), and a light chain variable region (V) 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. L OX40).

[0124] In one embodiment, the antigen binding domain capable of specifically binding to OX40 comprises: (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 33 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L OX40), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L OX40), or (iv) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 57 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 58 L OX40)

[0023] In accordance with the present invention, there is provided a bispecific antigen-binding molecule as defined herein above, comprising:

[0125] In one embodiment, the antigen binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 33. H OX40), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L In another aspect, a bispecific antigen-binding molecule as defined herein above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41. H OX40), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L In a further aspect, a bispecific antigen-binding molecule as defined herein above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49. H OX40), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L In a further aspect, a bispecific antigen-binding molecule as defined herein above is provided, wherein the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 57. H OX40), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 58 L In one particular embodiment, the antigen-binding domains capable of specifically binding to OX40 each comprise a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41. H OX40), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L OX40).

[0126] In another embodiment, the antigen binding domain capable of specifically binding to OX40 comprises: (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 59 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 60 HOX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 61 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40)

[0023] In accordance with the present invention, there is provided a bispecific antigen-binding molecule as defined herein above, comprising:

[0127] In one embodiment, the antigen binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 59. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L In one embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 60. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L In a further embodiment, the antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 61. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L In one particular embodiment, the antigen binding domains capable of specifically binding to OX40 each comprise a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 60. H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40).

[0128] Bispecific antigen-binding molecules that bind to OX40 and FAP In another aspect, (a) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29; HOX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32. L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), the heavy chain variable region (V) comprising: (i) CDR-H1 comprising the 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 the amino acid sequence of SEQ ID NO: 5. H a light chain variable region (V FAP) comprising (iv) a 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, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; L an antigen-binding domain comprising an FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0129] In a further aspect, (a) a heavy chain variable region (V) 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; H OX40), and a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34 L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) at least one antigen-binding domain capable of specifically binding to a FAP, the heavy chain variable region (V) 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; HFAP) and a light chain variable region (V) 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. L an antigen-binding domain comprising FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0130] In certain embodiments, (a) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; H OX40), and a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34. L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15; H a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of: L at least one antigen-binding domain capable of specifically binding to a FAP, including a FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0131] In another aspect, (a) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37; H OX40), and a light chain variable region (V) 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. L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), the heavy chain variable region (V) comprising: (i) CDR-H1 comprising the 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 the amino acid sequence of SEQ ID NO: 5. H a light chain variable region (V FAP) comprising (iv) a 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, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; L an antigen-binding domain comprising an FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0132] In a further aspect, (a) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41; H A light chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 42 and SEQ ID NO: 43. L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) at least one antigen-binding domain capable of specifically binding to a FAP, the heavy chain variable region (V) 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; H FAP) and a light chain variable region (V) 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. L an antigen-binding domain comprising an FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0133] In certain embodiments, (a) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41; H A light chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 42 and SEQ ID NO: 43. L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15; H a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of: L at least one antigen-binding domain capable of specifically binding to a FAP, including a FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0134] In another aspect, (a) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), the heavy chain variable region (V) comprising: (i) CDR-H1 comprising the 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 the amino acid sequence of SEQ ID NO: 5. Ha light chain variable region (V FAP) comprising (iv) a 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, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; L an antigen-binding domain comprising an FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0135] In a further aspect, (a) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49; H A light chain variable region (V) comprising an amino acid sequence selected from the group consisting of: L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) at least one antigen-binding domain capable of specifically binding to a FAP, the heavy chain variable region (V) 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; H FAP) and a light chain variable region (V) 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. L an antigen-binding domain comprising an FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0136] In certain embodiments, (a) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49; H A light chain variable region (V) comprising an amino acid sequence selected from the group consisting of: L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15; H a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of: L at least one antigen-binding domain capable of specifically binding to FAP, including FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0137] In yet another aspect, (a) a heavy chain variable region (V) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53; H OX40), and a light chain variable region (V) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 54, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 56. L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP), the heavy chain variable region (V) comprising: (i) CDR-H1 comprising the 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 the amino acid sequence of SEQ ID NO: 5. H a light chain variable region (V FAP) comprising (iv) a 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, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; L an antigen-binding domain comprising an FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0138] In a further aspect, (a) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57; H A light chain variable region (V) comprising an amino acid sequence selected from the group consisting of: L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) at least one antigen-binding domain capable of specifically binding to a FAP, the heavy chain variable region (V) 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; H FAP) and a light chain variable region (V) 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. L an antigen-binding domain comprising an FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0139] In certain embodiments, (a) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57; H A light chain variable region (V) comprising an amino acid sequence selected from the group consisting of: L OX40), and at least two antigen-binding domains capable of specifically binding to OX40; (b) a heavy chain variable region (V) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15; H a light chain variable region (V) comprising an amino acid sequence selected from the group consisting of: L at least one antigen-binding domain capable of specifically binding to FAP, including FAP; (c) an Fc region composed of first and second subunits capable of stably associating; A bispecific antigen-binding molecule is provided, comprising:

[0140] Bispecific antigen-binding molecules that are bivalent for binding to OX40 and monovalent for binding to FAP (2+1 format) In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) two Fab fragments capable of specifically binding to OX40; (b) a heavy chain variable region (V) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (ii) a 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) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5. H a light chain variable region (V FAP) comprising (iv) a 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, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; L one cross-Fab fragment capable of specifically binding to FAP, comprising a FAP; (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating; The present invention provides a bispecific antigen-binding molecule comprising:

[0141] Thus, bispecific antigen-binding molecules are provided that bivalently bind to OX40 and monovalently bind to FAP.

[0142] In one aspect, (a) two heavy chains each comprising a VH domain and a CH1 domain of a Fab fragment capable of specifically binding to OX40, and an Fc region subunit; (b) two light chains each comprising a VL domain and a CL domain of a Fab fragment capable of specifically binding to OX40; (c) a crossFab fragment capable of specifically binding to FAP, the crossFab fragment comprising a VL-CH1 chain and a VH-CL chain, wherein the VH-CL chain is linked to the C-terminus of one of the two heavy chains of (a); A bispecific antigen-binding molecule is provided, comprising:

[0143] In one embodiment, the VH-CL (VH-C kappa) chain is linked to the C-terminus of an Fc knob heavy chain. In one embodiment, the VH-C kappa chain is linked to the C-terminus of an Fc knob heavy chain comprising the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index).

[0144] In another aspect, (a) two heavy chains each comprising a VH domain and a CH1 domain of a Fab fragment capable of specifically binding to OX40, and an Fc region subunit; (b) two light chains each comprising a VL domain and a CL domain of a Fab fragment capable of specifically binding to OX40; (c) a cross-Fab fragment capable of specifically binding to FAP, the cross-Fab fragment 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 of (a); A bispecific antigen-binding molecule is provided, comprising:

[0145] In one embodiment, the VL-CH1 chain is linked to the C-terminus of an Fc knob heavy chain. In one embodiment, the VL-CH1 chain is linked to the C-terminus of an Fc knob heavy chain comprising the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index).

[0146] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) two light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:94, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:90; or (c) a bispecific antigen-binding molecule comprising two light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 96, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 90. In one aspect, 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, one 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, one 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, one 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.

[0147] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) two light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:93, one light chain comprising an amino acid sequence 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:93, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:94, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:98; or (c) a bispecific antigen-binding molecule comprising two light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:93, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:96, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:98. In one aspect, 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, one 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, one 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, one 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.

[0148] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) two light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 100, one light chain comprising an amino acid sequence 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 100, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 94, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 102; or (c) a bispecific antigen-binding molecule comprising two light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 100, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 96, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 102. In one aspect, the invention provides a bispecific antigen-binding molecule comprising: (a) two light chains, each comprising the amino acid sequence of SEQ ID NO: 100, one 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, one 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, one 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.

[0149] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) two light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 104, one light chain comprising an amino acid sequence 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 104; one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 94; a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 106; or (c) a bispecific antigen-binding molecule comprising two light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 104, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 96, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 106. In one aspect, the invention provides a bispecific antigen-binding molecule comprising: (a) two light chains, each comprising the amino acid sequence of SEQ ID NO: 104, one 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, one 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, one 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.

[0150] Bispecific antigen-binding molecules that are trivalent for binding to OX40 and monovalent for binding to FAP (3+1 format) In another aspect, (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 a FAP fused to the C-terminus of one of the subunits of the Fc region; and (c) an Fc region composed of first and second subunits capable of stably associating, 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 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. A bispecific antigen-binding molecule is provided, which consists of:

[0151] In one aspect, 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 a FAP fused to the C-terminus of one of the subunits of the Fc region; and (c) An Fc region composed of a first and a second subunit capable of stably associating, 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 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 another aspect, (a) a heavy chain comprising a VH-CH1 chain of a first Fab fragment capable of specifically binding to OX40, optionally fused at its N-terminus to a VH-CH1 chain of a second Fab fragment capable of specifically binding to OX40 via a peptide linker, and an Fc region subunit; (b) a heavy chain comprising a VH-CH1 domain of a Fab fragment capable of specifically binding to OX40, an Fc region subunit, and a VH-CL chain of a Fab fragment capable of specifically binding to FAP, optionally fused to the C-terminus of the Fc region subunit via a peptide linker; (c) three light chains each comprising a VL domain and a CL domain of a Fab fragment capable of specifically binding to OX40; (d) a light chain comprising a VL domain and a CH1 domain of a Fab fragment capable of specifically binding to a FAP; A bispecific antigen-binding molecule is provided, comprising:

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

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

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

[0156] In one particular embodiment, the peptide linker is selected from GGGGS (SEQ ID NO:67), GGGSGGGGS (SEQ ID NO:68), SGGGGSGGGG (SEQ ID NO:69), GGGGSGGGGSGGGG (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). Particularly interesting peptide linkers 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).

[0157] In one aspect, (a) a first heavy chain comprising an amino acid sequence 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 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 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 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 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 94; or (c) a bispecific antigen-binding molecule is provided, comprising: 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: 96. In one aspect, a bispecific antigen-binding molecule is provided, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 86, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 90, three light chains each comprising the amino acid sequence of SEQ ID NO: 87, 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: 86, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 90, three light chains each comprising the amino acid sequence of SEQ ID NO: 87, 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: 86, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 90, three light chains each comprising the amino acid sequence of SEQ ID NO: 87, and a light chain comprising the amino acid sequence of SEQ ID NO: 96.

[0158] In one aspect, (a) a first heavy chain comprising an amino acid sequence 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 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 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 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 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:94; or (c) a bispecific antigen-binding molecule is provided, comprising: a first heavy chain comprising an amino acid sequence 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:96. In one aspect, a bispecific antigen-binding molecule is provided, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 97, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 95, three light chains each comprising the amino acid sequence of SEQ ID NO: 93, 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: 97, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 95, three light chains each comprising the amino acid sequence of SEQ ID NO: 93, 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: 97, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 95, three light chains each comprising the amino acid sequence of SEQ ID NO: 93, and a light chain comprising the amino acid sequence of SEQ ID NO: 96,

[0159] In another aspect, (a) a first heavy chain comprising an amino acid sequence 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 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 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 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 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:94; or (c) a bispecific antigen-binding molecule is provided, comprising: a first heavy chain comprising an amino acid sequence 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:96. In one aspect, 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:96.

[0160] In another aspect, (a) a first heavy chain comprising an amino acid sequence 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 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 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 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 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 94; or (c) a bispecific antigen-binding molecule is provided, comprising: a first heavy chain comprising an amino acid sequence 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 96. In one aspect, a bispecific antigen-binding molecule is provided, comprising: (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 103, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 105, three light chains each comprising the amino acid sequence of SEQ ID NO: 104, 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: 103, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 105, three light chains each comprising the amino acid sequence of SEQ ID NO: 104, 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: 103, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 105, three light chains each comprising the amino acid sequence of SEQ ID NO: 104, and a light chain comprising the amino acid sequence of SEQ ID NO: 96.

[0161] Bispecific antigen-binding molecules that are tetravalent for binding to OX40 and monovalent for binding to target cell antigens (4+1 format) In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) four antigen-binding domains capable of specifically binding to OX40; (b) one antigen-binding domain capable of specifically binding to FAP, the light chain variable region (V) comprising: (i) CDR-H1 comprising the 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 the amino acid sequence of SEQ ID NO: 5; and (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; (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. L two antigen-binding domains containing FAP; (c) an Fc domain composed of a first and a second subunit capable of stably associating; and

[0162] Thus, a bispecific antigen-binding molecule is provided that binds tetravalently to OX40 and monovalently to FAP.

[0163] In one embodiment, a bispecific antigen-binding molecule is provided in which four antigen-binding domains capable of specifically binding to OX40 are Fab fragments, two of which are fused to each other in the heavy chain, optionally via a peptide linker. In a specific embodiment, the antigen-binding molecule comprises two heavy chains, each comprising a VHCH1-peptide linker-VHCH1 fragment. In a specific embodiment, the peptide linker has the amino acid sequence of SEQ ID NO: 68.

[0164] In one aspect, 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; (ad) a fourth Fab fragment capable of specifically binding to OX40; (b) a cross-Fab fragment capable of specifically binding to a FAP fused to the C-terminus of one of the subunits of the Fc region; and (c) An Fc region composed of a first and a second subunit capable of stably associating, 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 is also fused at its C-terminus to the N-terminus of the first subunit, and the fourth Fab fragment (ad) is fused at the C-terminus of the VH-CH1 chain to the N-terminus of the VH-CH1 chain of the third Fab fragment (ac) and is also fused at its C-terminus to the N-terminus of the second subunit. It consists of:

[0165] In one aspect, (a) four light chains, each of which contains a VL domain and a CL domain of a Fab fragment capable of specifically binding to OX40; (b) two heavy chains, each of which comprises a VH-CH1 domain of a Fab fragment capable of specifically binding to OX40 fused to the N-terminus of the VH-CH1 domain of a second Fab fragment capable of bispecifically binding to OX40, and an Fc region subunit; (c) a cross-fab fragment in which the VH-CL domain is linked to the C-terminus of one heavy chain via a peptide linker; A bispecific antigen-binding molecule is provided, which consists of:

[0166] In one aspect, (a) four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 89; (b) four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 94, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 89; (d) a bispecific antigen-binding molecule is provided, comprising four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 96, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 89. In one aspect, a bispecific antigen-binding molecule is provided, comprising: (a) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 86, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 89; (b) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 86, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 89; or (c) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 86, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 89.

[0167] In another aspect, (a) four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:93, one light chain comprising an amino acid sequence 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:95; (b) four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:93, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:94, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:95; or (c) a bispecific antigen-binding molecule is provided, comprising four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:93, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:96, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:95. In one aspect, a bispecific antigen-binding molecule is provided, comprising: (a) four light chains each comprising the amino acid sequence of SEQ ID NO: 93, one 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: 92, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 95; (b) four light chains each comprising the amino acid sequence of SEQ ID NO: 93, one 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: 92, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 95; or (c) four light chains each comprising the amino acid sequence of SEQ ID NO: 93, one 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: 92, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 95.

[0168] In a further aspect, (a) four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 108; (b) four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:94, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:108; or (c) a bispecific antigen-binding molecule is provided, comprising four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 96, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 108. In a further aspect, a bispecific antigen-binding molecule is provided, comprising: (a) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 107, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 108; or (b) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 107, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 108; or (c) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 107, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 108.

[0169] In a further aspect, (a) four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence 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 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 94, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 110; or (c) a bispecific antigen-binding molecule is provided, comprising four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 96, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 110. In one aspect, a bispecific antigen-binding molecule is provided, comprising: (a) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 109, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 110; or (b) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 109, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 110; or (c) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 109, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 110.

[0170] In a further aspect, (a) four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence 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 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 112; (c) four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:94, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:112; (c) a bispecific antigen-binding molecule is provided, comprising four light chains, each comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 87, one light chain comprising an amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 96, a first heavy chain comprising an amino acid sequence 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 at least about 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 112. In a further aspect, a bispecific antigen-binding molecule is provided, comprising: (a) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 111, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 112; or (b) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 111, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 112; or (c) four light chains, each comprising the amino acid sequence of SEQ ID NO: 87, one 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: 111, and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 112.

[0171] Fc domain modifications that reduce Fc receptor binding and / or effector function The bispecific antigen-binding molecules of the present invention further comprise an Fc domain composed of a first subunit and a second subunit capable of stably associating.

[0172] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody presented herein, thereby creating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0173] The Fc domain confers desirable pharmacokinetic properties to the bispecific antibodies of the invention, including a long serum half-life and a desirable tissue-to-blood distribution ratio, which contribute to favorable accumulation in target tissues. However, at the same time, it may cause undesirable targeting of the bispecific antibodies of the invention to cells expressing Fc receptors rather than cells containing the desired antigen. Thus, in specific embodiments, the Fc domain of the bispecific antibodies of the invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to native IgG Fc domains, particularly IgG1 Fc domains or IgG4 domains. More specifically, the Fc domain is an IgG1 Fc domain.

[0174] In one such embodiment, the Fc domain (or a bispecific antigen-binding molecule comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to an Fc receptor and / or less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function of a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the invention comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or a bispecific antigen-binding molecule of the invention comprising the Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a particular embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In one embodiment, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or a bispecific antigen-binding molecule of the invention comprising said Fc domain) exhibits a binding affinity for FcRn that is greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90%, compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule of the invention comprising a native IgG1 Fc domain).

[0175] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function compared to a non-engineered Fc domain. In certain embodiments, the Fc domain of a bispecific antigen-binding molecule of the present invention comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In another embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least two-fold, at least five-fold, or at least ten-fold. In one embodiment, a bispecific antigen-binding molecule of the present invention comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity to an Fc receptor compared to a bispecific antibody of the present invention comprising a non-engineered Fc domain. In certain embodiments, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a particular embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more particularly human FcγRIIB. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, and most particularly human FcγRIIIa. Preferably, binding to these respective receptors is reduced. In some embodiments, binding affinity to complement components (specifically, binding affinity to C1q) is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn (i.e., preservation of the binding affinity of the Fc domain to the receptor) is achieved when the Fc domain (or a bispecific antigen-binding molecule of the invention comprising the Fc domain) exhibits a binding affinity to FcRn that is greater than about 70% of the binding affinity of an unengineered form of the Fc domain (or a bispecific antigen-binding molecule of the invention comprising this unengineered form of Fc).The Fc domain, or a bispecific antigen-binding molecule of the present invention comprising the Fc domain, may exhibit greater than about 80%, or even greater than about 90%, of such affinity. In certain embodiments, the Fc domain of a bispecific antigen-binding molecule of the present invention is engineered to have reduced effector function compared to an unengineered Fc domain. Reduced effector function includes, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0176] Antibodies with reduced effector function include those containing one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). Certain antibody variants have been described with improved or reduced binding to FcRs (e.g., U.S. Patent No. 6,737,056, WO 2004 / 056312, and Shields, R.L. et al., J. Biol. Chem. 276 (2001), pp. 6591-6604).

[0177] In one aspect, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331, and P329. In some aspects, the Fc domain comprises amino acid substitutions L234A and L235A ("LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and further comprises an 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 the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor binding of a human IgG1 Fc domain, as described in PCT Application WO 2012 / 130831A1, which also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function. Such antibodies are IgG1s with the mutations L234A and L235A, or the mutations L234A, L235A, and P329G (numbering according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0178] In one aspect, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising the amino acid substitutions L235E, S228P, and P329G. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition, vol. 38, pp. 84-91 (2010)).

[0179] Antibodies with increased half-life and improved binding to neonatal Fc receptors are responsible for the transfer of mature IgG to the fetus (Guyer, R.L. et al., J. Immunol. 117 (1976) pp. 587-593 and Kim, J.K. et al., J. Immunol. 24 (1994) pp. 2429-2434) and are described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having 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, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan, AR, and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0180] Binding to Fc receptors can be easily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare) and Fc receptors that can be obtained by recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of an Fc domain or a cell-activating bispecific antigen-binding molecule comprising an Fc domain to an Fc receptor can be evaluated using a cell line known to express a particular Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor). The effector function of an Fc domain or a bispecific antigen-binding molecule of the present invention comprising an Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362, Hellstrom et al., Proc Natl Acad Sci USA 83, 7059-7063 (1986), and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985), U.S. Patent No. 5,821,337, Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0181] The following sections describe preferred embodiments of bispecific antigen-binding molecules of the present invention that contain Fc domain modifications that 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 capable of specifically binding to OX40; (b) an antigen-binding domain capable of specifically binding to a FAP; and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of an antibody to an Fc receptor, particularly an Fcγ receptor. In another embodiment, the present invention relates to a bispecific antigen-binding molecule comprising: (a) at least two antigen-binding domains capable of specifically binding to OX40; (b) an antigen-binding domain capable of specifically binding to a FAP; and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of an antibody to an Fc receptor, particularly an Fcγ receptor. In a particular embodiment, the Fc domain is an Fc domain of the human IgG1 subclass, with the amino acid mutations L234A, L235A and P329G (numbering according to the Kabat EU index).

[0182] Fc domain modifications that promote heterodimerization The bispecific antigen-binding molecules of the present invention comprise different antigen-binding sites fused to one or the other of the two subunits of the Fc domain, and therefore the two subunits of the Fc domain may be contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. To increase the yield and purity of the bispecific antigen-binding molecules of the present invention during recombinant production, it is advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecules of the present invention that promote the desired association of the polypeptides.

[0183] Therefore, in a specific embodiment, the present invention relates to a bispecific antigen-binding molecule comprising: (a) at least two antigen-binding domains capable of specifically binding to OX40; (b) an antigen-binding domain capable of specifically binding to FAP; and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, wherein the Fc domain comprises a modification that promotes the association of the first and second subunits of the Fc domain. The longest protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0184] In a specific embodiment, the modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Thus, the present invention relates to a bispecific antigen-binding molecule comprising (a) at least two antigen-binding domains capable of specifically binding to OX40, (b) an antigen-binding domain capable of specifically binding to a FAP, and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating, wherein the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole according to the knob-into-hole method. In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0185] Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Complementary cavities of identical or similar size to the protrusions are created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0186] Thus, in one embodiment, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen-binding molecule of the present invention, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be repositioned within a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, into which the protrusion in the CH3 domain of the first subunit can be repositioned. The protrusion and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, the second subunit of the Fc domain further comprises a threonine residue at position 366 replaced with a serine residue (T366S) and a leucine residue at position 368 replaced with an alanine residue (L368A).

[0187] In yet a further embodiment, the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (Kabat EU numbering).

[0188] In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, as described, for example, in PCT Application WO 2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favorable.

[0189] The C-terminus of the heavy chain of a bispecific antibody as reported herein may be a complete C-terminus ending in the amino acid residue PGK. The C-terminus of the heavy chain may also be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. In one preferred embodiment, the C-terminus of the heavy chain is a truncated C-terminal ending PG. In one embodiment of all embodiments reported herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the Kabat EU index). In all embodiments described herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, numbering according to the Kabat EU index).

[0190] Modifications within the Fab domain In one aspect, 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 comprising a first subunit and a second subunit capable of stably associating, wherein either the variable domains VH and VL or the constant domains CH1 and CL are exchanged in one of the Fab fragments. The bispecific antibody is prepared according to the crossmab technique.

[0191] Multispecific antibodies with domain replacement / swap in one binding arm (CrossMab VH-VL or CrossMab CH-CL) are described in WO 2009 / 080252 and Schaefer, W. et al., PNAS, 108 (2011) 11187-1191. These multispecific antibodies clearly reduce by-products caused by mismatches between a light chain for one antigen and an incorrect heavy chain for a second antigen (compared to approaches without such domain replacement).

[0192] In one aspect, 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 composed of a first subunit and a second subunit capable of stably associating, wherein in one of the Fab fragments, the constant domains CL and CH1 are substituted with each other such that the CH1 domain is part of the light chain and the CL domain is part of the heavy chain. More specifically, in a second Fab fragment capable of specifically binding to a target cell antigen, the constant domains CL and CH1 are substituted with each other such that the CH1 domain is part of the light chain and the CL domain is part of the heavy chain.

[0193] In a specific aspect, 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 a FAP, wherein in the Fab fragment capable of specifically binding to a FAP, the constant domains CL and CH1 are substituted for each other such that the CH1 domain is part of the light chain and the CL (Ckappa) domain is part of the heavy chain.

[0194] Thus, in one aspect, the present invention provides 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 the Fc region; and (b) a crossFab fragment capable of specifically binding to a FAP fused to the C-terminus of one of the subunits of the Fc region.

[0195] In another embodiment, to further improve correct pairing, a bispecific antigen-binding molecule comprising (a) at least two Fab fragments capable of specifically binding to OX40, (b) a crossFab fragment capable of specifically binding to FAP, and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating may contain differently charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the crossed or non-crossed CH1 and CL domains. In a specific embodiment, the present invention relates to a bispecific antigen-binding molecule in which, in one of the CL domains, the amino acid at position 123 (EU numbering) is substituted with arginine (R), the amino acid at position 124 (EU numbering) is substituted with lysine (K), and in one of the CH1 domains, the amino acid at position 147 (EU numbering) and / or position 213 (EU numbering) is substituted with glutamic acid (E).

[0196] Exemplary Antibodies of the Invention In one aspect, the present invention provides new antibodies and antibody fragments that specifically bind to FAP. These antibodies bind to epitopes distinct from the known FAP antibodies 4B9 or 28H1 and are therefore particularly suitable for incorporation into bispecific antigen-binding molecules that can be used in combination with other FAP-targeting molecules. The new antibodies can be produced in large quantities and at high titers, and exhibit high thermal stability (aggregation temperature T agg ) and is further characterized as binding with high affinity to human FAP as measured by Biacore assay.

[0197] In one embodiment, an antibody that specifically binds to FAP (clone 212) comprises a heavy chain variable region (VH) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (ii) a 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) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5. Ha light chain variable region (V FAP) comprising (iv) a 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, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; L Antibodies are provided, including antibodies against the α- and β-actin-binding domains of the α- and β

[0198] In one embodiment, there is provided a humanized antibody that specifically binds to a FAP, the humanized antibody comprising a heavy chain variable region (VH) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (ii) a 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) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5. H a light chain variable region (V FAP) comprising (iv) a 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, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; L Humanized antibodies are provided, including the FAP.

[0199] In another embodiment, an antibody that competes for binding with an antibody that specifically binds to a FAP, comprising any one of a heavy chain variable region (V) 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. H FAP), and any light chain variable region (V) 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. L Antibodies are provided that include either a phosphodiesterase inhibitor (MPI) or a phosphodiesterase inhibitor (FAP).

[0200] In one aspect, an antibody is provided that competes for binding with an antibody that specifically binds to FAP, the antibody comprising 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.

[0201] In a further embodiment, there is provided an antibody that specifically binds to a FAP, (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 15; H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 21 L FAP), (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 16 H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 21 L FAP), (c) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 16 H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 22 L FAP), or (d) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 19 H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 25 L FAP) An antibody that specifically binds to a FAP is provided, comprising:

[0202] In a further aspect, an antibody capable of specifically binding to a FAP, comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 15. H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 21 L Antibodies are provided, including antibodies against the α- and β-actin-binding domains of the α- and β

[0203] In another aspect, the present invention provides new 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 have improved PK properties compared to 49B4, and are believed to bind to human OX40 with high affinity as measured by Biacore assay.

[0204] Thus, a humanized antibody that specifically binds to OX40, (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 59 HOX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40), (b) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 60 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40), (c) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 61 H OX40) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L OX40) A humanized antibody is provided, comprising:

[0205] Polynucleotides The present invention further provides an isolated nucleic acid encoding a bispecific antigen-binding molecule or fragment thereof described herein, or an isolated nucleic acid encoding an antibody described herein.

[0206] The isolated polynucleotides encoding the bispecific antigen-binding molecules of the present invention may be expressed as a single polynucleotide encoding the complete antigen-binding molecule, or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by co-expressed polynucleotides may associate, for example, via disulfide bonds or other means, to form a functional antigen-binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a separate polynucleotide derived from the heavy chain portion of the immunoglobulin. When co-expressed, the heavy chain polypeptide associates with the light chain polypeptide to form an immunoglobulin.

[0207] In some aspects, the isolated polynucleotide encodes a polypeptide comprised in a bispecific molecule of the invention as described herein.

[0208] In one aspect, the present invention provides a heavy chain variable region (V) comprising: (a) at least two antigen-binding domains capable of specifically binding to OX40; and (b) a heavy chain variable region (V) comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (ii) a 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) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5. H and (iv) an antigen-binding domain having specific binding ability to FAP, the antigen-binding domain comprising a FAP, and a light chain variable region (V) comprising a 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. L The present invention relates to an isolated polynucleotide encoding a bispecific antigen-binding molecule comprising: (a) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; (b) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating with each other.

[0209] In certain 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 may be single-stranded or double-stranded.

[0210] Recombination Method The bispecific antigen-binding molecules of the present invention can be obtained, for example, by recombinant production. For recombinant production, one or more polynucleotides encoding the bispecific antigen-binding molecule or polypeptide fragments thereof are provided. One or more polynucleotides encoding the bispecific antigen-binding molecule are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides may be easily isolated and sequenced using conventional procedures. In one aspect of the present invention, a vector, preferably an expression vector, is provided, comprising one or more polynucleotides of the present invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the bispecific antigen-binding molecule (fragment) along with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in 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). An expression vector may be part of a plasmid, a virus, or a nucleic acid fragment. An expression vector contains an expression cassette into which a polynucleotide encoding a bispecific antigen-binding molecule or a polypeptide fragment thereof (i.e., a coding region) is cloned, operably associated with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons translated into amino acids. A "stop codon" (TAG, TGA, or TAA), although not translated into an amino acid, may be considered to be part of the coding region, although any flanking sequences, if present, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region.Two or more coding regions may be present 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 two or more coding regions; for example, the vectors of the present invention may encode one or more polypeptides, which are separated into final proteins by post- or co-translational proteolytic cleavage. Furthermore, vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions, either fused or unfused to the polynucleotide encoding the bispecific antigen-binding molecule of the present invention, or a polypeptide fragment thereof, or a variant or derivative 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 refers to the association of a coding region for a gene product (e.g., a polypeptide) with one or more regulatory sequences in such a manner that expression of the gene product is under the influence or control of the regulatory sequence(s). Two DNA segments (e.g., a polypeptide coding region and its associated promoter) are "operably associated" if the introduction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA segments does not interfere with the ability of expression control sequences to direct expression of the gene product or to transcribe the DNA template. Thus, a promoter region may be operably associated with a polypeptide-encoding nucleic acid if the promoter is capable of effecting transcription of the nucleic acid. The promoter may also be a cell-specific promoter that directs substantial transcription of the DNA only in a given cell. Other transcription control elements besides a promoter, such as enhancers, operators, and transcription termination signals, may be operably associated with a polynucleotide to direct cell-specific transcription.

[0211] Suitable promoters and other transcription control regions are disclosed herein. Various transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoters and enhancer segments from cytomegalovirus (e.g., in combination with the immediate early promoter, intron-A), Simian Virus 40 (e.g., early promoters), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit α-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Further suitable transcription control regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from viral systems (particularly, internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also contain other features such as, for example, an origin of replication and / or chromosomal integration elements, e.g., retroviral long terminal repeats (LTRs) or adeno-associated viral (AAV) inverted terminal repeats (ITRs).

[0212] Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretory or signal peptides to direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if secretion of a bispecific antigen-binding molecule or a polypeptide fragment thereof is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid encoding the bispecific antigen-binding molecule of the present invention or a polypeptide fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein as the growing protein chain begins to exit through the rough endoplasmic reticulum. Those skilled in the art know that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of that sequence that retains the ability to direct the cleavage of an operably associated polypeptide is used. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0213] DNA encoding short protein sequences that can be used to facilitate subsequent purification (e.g., a histidine tag) or to assist in labeling the fusion protein may be included within or at the end of the polynucleotide encoding the bispecific antigen-binding molecule of the present invention, or a polypeptide fragment thereof.

[0214] In a further aspect of the present invention, host cells are provided comprising one or more polynucleotides of the present invention. In certain aspects, host cells are provided comprising one or more vectors of the present invention. The polynucleotides and vectors may incorporate any of the features described herein in connection with the polynucleotides and vectors, respectively, alone or in combination. In one aspect, the host cell comprises (e.g., is transformed or transfected with) a vector comprising a polynucleotide encoding (a portion of) the bispecific antigen-binding molecule of the present invention. As used herein, the term "host cell" refers to any type of cell line that can be engineered to produce the fusion protein of the present invention or a fragment thereof. Suitable host cells for replicating and supporting the expression of antigen-binding molecules are well known in the art. Such cells may be transfected or transduced with a particular expression vector, if appropriate, and large quantities of the vector-containing cells may be grown to inoculate a large-scale fermenter, thereby obtaining sufficient quantities of the antigen-binding molecule for clinical use. Suitable host cells include prokaryotic microorganisms (e.g., Escherichia coli) or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides may be produced in bacteria, particularly if glycosylation is not required. After expression, the polypeptide may be isolated from the bacterial cell paste in appropriate fractions and further purified. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce polypeptides with partially or completely human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006).

[0215] Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified and may be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40 (COS-7) transformed monkey kidney CV1 line; human embryonic kidney lines (e.g., 293 or 293T cells as described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM as described in Mather, Biol Reprod 23, 243-251 (1980)). 4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human hepatocytes (HepG2), mouse mammary tumor cells (MMT060562), TRI cells (e.g., as described in Mathematica, Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5 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, NS0, P3X63, and Sp2 / 0.For a review of specific mammalian host cells suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few, but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocytic cell (e.g., Y0, NS0, Sp20 cell). Standard techniques for expressing foreign genes in these systems are known in the art. Cells that express a polypeptide containing either an immunoglobulin heavy chain or a light chain can be engineered to express the other immunoglobulin chain, such that the expressed product is an immunoglobulin having both a heavy and a light chain.

[0216] In one aspect, there is provided a method for producing a bispecific antigen-binding molecule of the invention, or a polypeptide fragment thereof, comprising culturing a host cell comprising a polynucleotide encoding a bispecific antigen-binding molecule of the invention, or a polypeptide fragment thereof, as provided herein, under conditions suitable for expressing the bispecific antigen-binding molecule of the invention, or a polypeptide fragment thereof, and recovering the bispecific antigen-binding molecule of the invention, or a polypeptide fragment thereof, from the host cell (or host cell culture medium).

[0217] Bispecific molecules of the invention prepared as described herein can be purified by techniques well known in the art, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and size-exclusion chromatography. The actual conditions used to purify 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, an antibody, ligand, receptor, or antigen to which the bispecific antigen-binding molecule binds can be used. For example, a matrix containing Protein A or Protein G can be used to affinity purify the fusion proteins of the invention. Sequential Protein A or G affinity chromatography and size-exclusion chromatography can be used to isolate antigen-binding molecules, essentially as described in the Examples. The purity of a bispecific antigen-binding molecule or a fragment thereof can be measured by any of a variety of well-known analytical techniques, including gel electrophoresis, high-pressure liquid chromatography, and the like. For example, bispecific antigen-binding molecules expressed as described in the Examples were shown to be intact and properly assembled, as demonstrated by reducing or non-reducing SDS-PAGE.

[0218] Assay The antigen-binding molecules provided herein can be characterized for their binding properties and / or biological activity by various assays known in the art, particularly those described in more detail in the Examples.

[0219] 1. Binding Assay The binding of the bispecific antigen-binding molecules provided herein to corresponding target-expressing cells can be evaluated, for example, by using a mouse fibroblast cell line expressing human fibroblast activation protein (FAP) and flow cytometry (FACS) analysis.The binding of the bispecific antigen-binding molecules provided herein to OX40 can be determined by using activated human PBMCs as described in Example 3.1.

[0220] 2. Activity Assay The bispecific antigen-binding molecule of the present invention is tested for biological activity.Biological activity can include the efficacy and specificity of the bispecific antigen-binding molecule.Efficacy and specificity are demonstrated by assays that demonstrate agonistic signal transduction through OX40 receptor upon target antigen binding.Furthermore, the stimulation of OX40 signal transduction is measured through the induced NFκB activation in human OX40-positive NFB reporter cells, as described in Example 4.1.

[0221] Pharmaceutical Compositions, Formulations, and Routes of Administration In a further aspect, the present invention provides pharmaceutical compositions comprising any of the bispecific antigen-binding molecules provided herein, for example, for use in any of the following methods of treatment. In one aspect, the pharmaceutical composition comprises any of the bispecific antigen-binding molecules provided herein and at least one pharmaceutically acceptable excipient. In another aspect, 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.

[0222] 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" generally refers to molecular moieties and compositions that are non-toxic to recipients at the dosages and concentrations used, i.e., do not cause adverse allergic or other untoward reactions, when administered to animals (e.g., humans), as appropriate. The preparation of pharmaceutical compositions containing at least one bispecific antigen-binding molecule according to the present invention and, optionally, additional active ingredients, will be known to those skilled in the art in light of the present disclosure, as exemplified by "Remington's Pharmaceutical Sciences," 18th Edition (Mack Printing Company, 1990), incorporated herein by reference. In particular, the composition is a lyophilized formulation or an aqueous solution. As used herein, "pharmaceutically acceptable excipients" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, salts, stabilizers, and combinations thereof, as would be known to one of skill in the art.

[0223] Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the bispecific antigen-binding molecules of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solutions may contain formulating agents, such as suspending agents, stabilizing agents, and dispersing agents. Alternatively, the bispecific antigen-binding molecules may be in powder form for constitution with a suitable vehicle, such as pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antigen-binding molecules of the present invention in the required amount in an appropriate solvent, with various other ingredients, as required, as listed below. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered liquid medium. The liquid medium should be appropriately buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose prior to injection. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be recognized that endotoxin contamination should be minimized to a safe level, e.g., less than 0.5 ng / mg protein.Suitable pharmaceutically acceptable additives include, but are not limited to, buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins Examples of suitable suspensions include proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., 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 (e.g., sodium carboxymethylcellulose, sorbitol, dextran, etc.). Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions. Additionally, suspensions of the active compound may be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleat or triglycerides, or liposomes.

[0224] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may also be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, for example, films or microcapsules. In certain embodiments, sustained absorption of injectable compositions may be brought about by the use in the compositions of agents delaying absorption (e.g., aluminum monostearate, gelatin, or combinations thereof).

[0225] Exemplary pharmaceutically acceptable additives of the present invention further include interstitial drug dispersing agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

[0226] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations containing a histidine acetate buffer.

[0227] In addition to the above-mentioned compositions, antigen-binding molecules can also be formulated as depot preparations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the fusion protein can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, such as a sparingly soluble salt.

[0228] Pharmaceutical compositions containing the bispecific antigen-binding molecules of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, additives, or auxiliary agents that facilitate the processing of proteins into pharmaceutically usable preparations. The appropriate formulation will vary depending on the selected route of administration.

[0229] The bispecific antigen-binding molecule may be formulated in the composition in a free acid or base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or free base. Pharmaceutically acceptable salts include acid addition salts formed with free amino groups of the proteinaceous composition, or with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, or from organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0230] The compositions of the present invention may contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other, and such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0231] Formulations to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0232] Therapeutic methods and compositions Any of the bispecific antigen-binding molecules provided herein can be used in therapeutic methods.For use in therapeutic methods, the bispecific antigen-binding molecules of the present invention can be formulated, dosed, and administered in a manner consistent with good medical practice.Factors to be considered in this regard include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the drug, the method of administration, the administration schedule, and other factors known to medical professionals.

[0233] In one aspect, there is provided a bispecific antigen-binding molecule of the invention for use as a medicament.

[0234] In further aspects, the bispecific antigen-binding molecules of the invention are provided for use in (i) inducing immune stimulation, (ii) stimulating tumor-specific T cell responses, (iii) causing apoptosis of tumor cells, (iv) in the treatment of cancer, (v) delaying cancer progression, (vi) prolonging survival of patients with cancer, and (vii) in the treatment of infectious diseases. In a particular aspect, the bispecific antigen-binding molecules of the invention are provided for use in the treatment of disease, particularly for use in the treatment of cancer.

[0235] In certain aspects, bispecific antigen-binding molecules of the present invention are provided for use in methods of treatment. In one aspect, the present invention provides a bispecific antigen-binding molecule as described herein for use in treating a disease in an individual in need thereof. In certain aspects, the present invention provides a bispecific antigen-binding molecule for use in a method of treating an individual having a disease, the method comprising administering a therapeutically effective amount of the bispecific antigen-binding molecule to the individual. In certain aspects, the disease being treated is cancer. The subject, patient, or "individual" in need of treatment is typically a mammal, more particularly a human.

[0236] In one aspect, there is provided a method for i) inducing immune stimulation, (ii) stimulating a tumor-specific T cell response, (iii) causing apoptosis of tumor cells, (iv) treating cancer, (v) delaying the progression of cancer, (vi) prolonging survival of a patient suffering from cancer, or (vii) treating an infectious disease, comprising administering to an individual in need thereof a therapeutically effective amount of a bispecific antigen-binding molecule of the invention.

[0237] In a further aspect, the present invention provides use of a bispecific antigen-binding molecule of the present invention in the manufacture or preparation of a medicament for treating a disease in an individual in need thereof. In one aspect, the medicament is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the medicament to an individual having the disease. In certain aspects, the disease to be treated is a proliferative disorder, particularly 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, anal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other examples of cancer include carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Other cell proliferative disorders treatable using the bispecific antigen-binding molecules or antibodies of the present invention include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included. In certain embodiments, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain tumor, and head and neck cancer. Those skilled in the art will readily appreciate that in many cases, the bispecific antigen-binding molecules or antibodies of the present invention may not provide a cure, but may provide an effect. In some aspects, physiological changes that have some effect are also considered therapeutically beneficial. Thus, in some embodiments, the amount of a bispecific antigen-binding molecule or antibody of the invention that produces a physiological change is considered an "effective amount," or a "therapeutically effective amount."

[0238] 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) for preventing or treating a disease depends on the type of disease being 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 prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the bispecific antigen-binding molecule, and the discretion of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient in the composition and the appropriate dose for each individual subject. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0239] The bispecific antigen-binding molecules of the present invention are suitably administered to patients once or over a continuous treatment regimen. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the bispecific antigen-binding molecule may be an initial candidate dosage for administration to a patient, whether by one or more individual administrations or continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment is usually continued until a desired suppression of disease symptoms occurs. One exemplary dose of the bispecific antigen-binding molecule of the present invention ranges from about 0.005 mg / kg to about 10 mg / kg. In other examples, dosages may also include about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight, about 500 mg / kg body weight, to about 1000 mg / kg body weight, or more, or any range derivable therebetween. Examples of ranges derivable from the numbers recited herein include about 0.1 mg / kg / body weight to about 20 mg / kg / body weight, about 5 μg / kg / body weight to about 1 mg / kg / body weight, etc., may be administered based on the numbers recited above. Thus, the patient may be administered one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof). Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., the patient receives about two to about 20 doses, or, for example, about six doses of the fusion protein). In certain embodiments, the bispecific antigen-binding molecule is administered every three weeks. An initial high loading dose may be followed by one or more smaller doses. However, other dosing regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0240] The bispecific antigen-binding molecules of the present invention are generally used in an amount effective to achieve the intended purpose. For use in treating or preventing a disease state, the bispecific antigen-binding molecules of the present invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determining a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. For systemic administration, a therapeutically effective dosage can be initially estimated from in vitro assays, such as cell culture assays. The IC as determined in cell culture can then be used to determine the therapeutically effective amount. 50 The dose may be formulated in an animal model to achieve a blood concentration range including: (i) a dose of 100 mg / kg / day or more; (ii) a dose of 100 mg / kg / day or more; (iii) a dose of 100 mg / kg / day or more; (iv) a dose of 100 mg / kg / day or more; (v) a dose of 100 mg / kg / day or more; (vi ...

[0241] Dosage amount and interval can be individually adjusted to provide plasma levels of the bispecific antigen-binding molecules of the present invention sufficient to maintain therapeutic efficacy. Useful patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.1 to 1 mg / kg / day. Therapeutically effective plasma concentrations may be achieved by administering multiple doses each day. 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 molecules or antibodies of the present invention may not be related to plasma concentration. One of skill in the art can optimize a therapeutically effective local dose without undue experimentation.

[0242] The therapeutically effective dose of the bispecific antigen-binding molecules of the present invention described herein generally provides a therapeutic effect without causing substantial toxicity. The toxicity and therapeutic efficacy of the fusion protein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal experiments, LD 50 (the dose that is lethal to 50% of the population) and ED 50The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Bispecific antigen-binding molecules exhibiting large therapeutic indices are preferred. In one embodiment, the bispecific antigen-binding molecules or antibodies of the present invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating an administration range appropriate for human use. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on various factors, such as the dosage used, the route of administration utilized, and the condition of the subject. The exact formulation, route of administration, and dosage can be chosen by the individual physician in consideration of the patient's condition (see, for example, Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).

[0243] The attending physician of a patient treated with a fusion protein of the invention will know how and when to discontinue, interrupt, or adjust administration due to toxicity, organ failure, etc. Conversely, the attending physician will also know to adjust treatment to higher levels if the clinical response is inadequate (without causing toxicity). The magnitude of the dose administered in the management of the disorder of interest will vary with the severity of the condition being treated, the route of administration, etc. The severity of the condition may, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps dosing frequency, will also vary according to the age, weight, and response of the individual patient.

[0244] Other drugs and treatments The bispecific antigen-binding molecules of the present invention may be administered in combination with one or more other agents. For example, the bispecific antigen-binding molecules 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 a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredients appropriate for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is another anti-cancer agent, such as a microtubule-disrupting agent...

Claims

1. (aa) a first Fab fragment capable of specifically binding to OX40; (ab) a second Fab fragment capable of specifically binding to OX40; and (ac) a third Fab fragment capable of specifically binding to OX40; and (b) a cross-Fab fragment capable of specifically binding to fibroblast activation protein (FAP) fused to the C-terminus of one of the subunits of the Fc region, A heavy chain variable region (V Ｈ FAP) and a light chain variable region (V Ｌ FAP); or A heavy chain variable region (V Ｈ FAP) and a light chain variable region (V Ｌ FAP); or A heavy chain variable region (V Ｈ FAP) and a light chain variable region (V Ｌ FAP); or A heavy chain variable region (V Ｈ FAP) and a light chain variable region (V Ｌ FAP) A cross-Fab fragment comprising: (c) an Fc region composed of a first subunit and a second subunit capable of stably associating, 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 first Fab fragment (aa) is fused at its C-terminus to the N-terminus of the first subunit, the third Fab fragment (ac) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit, and the Fc region comprises one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor and / or an effector function; A bispecific antigen-binding molecule comprising:

2. The antigen-binding domain capable of specifically binding to FAP comprises a heavy chain variable region (V Ｈ FAP) and a light chain variable region (V Ｌ 2. The bispecific antigen-binding molecule of claim 1 , comprising:

3. An antigen-binding domain capable of specifically binding to OX40, (i) a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:27, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:28, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:29; Ｈ OX40), and a light chain variable region (V OX40) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

32. Ｌ OX40), or (ii) a heavy chain variable region (VH1) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37; Ｈ OX40), and a light chain variable region (V OX40) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

40. Ｌ OX40), or (iii) a heavy chain variable region (VH) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; Ｈ OX40), and a light chain variable region (V OX40) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

48. Ｌ OX40), or (iv) a heavy chain variable region (VH) comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:51, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:52, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:53; Ｈ OX40), and a light chain variable region (V OX40) comprising (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:54, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:55, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

56. Ｌ OX40) 3. The bispecific antigen-binding molecule of claim 1 or 2, comprising:

4. An antigen-binding domain capable of specifically binding to OX40, (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 (V Ｈ OX40) and a light chain variable region (V Ｌ OX40), or (ii) a heavy chain variable region (V Ｈ OX40) and a light chain variable region (V Ｌ OX40), or (iii) a heavy chain variable region (V Ｈ OX40) and a light chain variable region (V Ｌ OX40), or (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57 (V Ｈ OX40) and a light chain variable region (V Ｌ OX40) The bispecific antigen-binding molecule of any one of claims 1 to 3, comprising:

5. An antigen-binding domain capable of specifically binding to OX40, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:59 Ｈ OX40) and a light chain variable region (V Ｌ OX40), or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60 (V Ｈ OX40) and a light chain variable region (V Ｌ OX40), or (iii) a heavy chain variable region (V Ｈ OX40) and a light chain variable region (V Ｌ OX40).

6. The antigen-binding domain capable of specifically binding to OX40 comprises a heavy chain variable region (V Ｈ OX40) and a light chain variable region (V Ｌ OX40).

7. The bispecific antigen-binding molecule of any one of claims 1 to 6, wherein the Fc region is an IgG Fc region.

8. The bispecific antigen-binding molecule of claim 7 , wherein the Fc region is an IgG1 Fc region or an IgG4 Fc region.

9. 9. The bispecific antigen-binding molecule of any one of claims 1 to 8, wherein the Fc region is of the human IgG1 subclass with the amino acid mutations L234A, L235A and P329G (numbering according to the Kabat EU index).

10. (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO:97, a second heavy chain comprising the amino acid sequence of SEQ ID NO:95, three light chains each comprising the amino acid sequence of SEQ ID NO:93, 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:97, a second heavy chain comprising the amino acid sequence of SEQ ID NO:95, three light chains each comprising the amino acid sequence of SEQ ID NO:93, 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:97, a second heavy chain comprising the amino acid sequence of SEQ ID NO:95, three light chains each comprising the amino acid sequence of SEQ ID NO:93, and a light chain comprising the amino acid sequence of SEQ ID NO:

96.

2. The bispecific antigen-binding molecule of claim 1 , comprising:

11. (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 86, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 90, three light chains each comprising the amino acid sequence of SEQ ID NO: 87, 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: 86, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 90, three light chains each comprising the amino acid sequence of SEQ ID NO: 87, 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: 86, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 90, three light chains each comprising the amino acid sequence of SEQ ID NO: 87, and a light chain comprising the amino acid sequence of SEQ ID NO:

96.

2. The bispecific antigen-binding molecule of claim 1 , comprising:

12. An isolated nucleic acid encoding a bispecific antigen-binding molecule according to any one of claims 1 to 11.

13. An expression vector comprising the isolated nucleic acid of claim 12.

14. 14. A host cell comprising the isolated nucleic acid of claim 12 or the expression vector of claim 13.

15. 12. A method for producing a bispecific antigen-binding molecule according to any one of claims 1 to 11, comprising culturing a host cell according to claim 14 under conditions suitable for expression of the bispecific antigen-binding molecule and isolating the bispecific antigen-binding molecule.

16. A pharmaceutical composition comprising the bispecific antigen-binding molecule of any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

17. 17. The pharmaceutical composition of claim 16, further comprising an additional therapeutic agent.

18. A bispecific antigen-binding molecule according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 16 for use as a medicament.

19. (i) inducing immune stimulation, (ii) in stimulating tumor-specific T cell responses; (iii) inducing apoptosis of tumor cells; (iv) in the treatment of cancer, (v) in slowing the progression of cancer, (vi) prolonging survival of patients with cancer; (vii) in the treatment of infectious diseases A bispecific antigen-binding molecule according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 16 for use.

20. A bispecific antigen-binding molecule according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 16 for use in the treatment of cancer.

21. 17. The bispecific antigen-binding molecule of any one of claims 1 to 11 or the pharmaceutical composition of claim 16 for use in the treatment of cancer, wherein said bispecific antigen-binding molecule or pharmaceutical composition is for administration in combination with chemotherapeutic agents, radiation and / or other agents for use in cancer immunotherapy.

22. 17. The bispecific antigen-binding molecule of any one of claims 1 to 11 or the pharmaceutical composition of claim 16, for use in the treatment of cancer, wherein the bispecific agonist OX40 antigen-binding molecule is for administration in combination with a T-cell activating anti-CD3 bispecific antibody.

23. Use of a bispecific antigen-binding molecule according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 16 in the manufacture of a medicament for the treatment of cancer.

24. 17. A medicament for treating an individual having cancer, comprising an effective amount of a bispecific antigen-binding molecule according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 16.

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