Agonist LTBR antibodies and bispecific antibodies containing them

Abstract

The present invention relates to novel antibodies that bind to the lymphotoxin beta receptor (LTBR) and bispecific antigen-binding molecules comprising these novel LTBR antibodies and an antigen-binding domain that binds to a tumor-associated antigen, in particular fibroblast activation protein (FAP), methods for producing these molecules, and methods of using them.

Description

[Technical Field]

[0001] The present invention relates to novel antibodies that bind to lymphotoxin beta receptors (LTBRs), and bispecific antigen-binding molecules comprising these novel LTBR antibodies and an antigen-binding domain that binds to tumor-associated antigens, particularly fibroblast-activating proteins (FAPs), as well as methods for producing these molecules and methods for using them. [Background technology]

[0002] In recent years, the landscape of cancer treatment has changed dramatically since the development and approval of cancer immunotherapies, such as drugs that block the immune checkpoints PD-1 / PD-L1 and CTLA-4. These cancer immunotherapies have emerged as new standard treatments, either alone or in combination with other therapies, because they can achieve sustained responses in cancer indications such as melanoma, non-small cell lung cancer, and bladder cancer. However, only a portion of patients (<30%) experience sustained benefits from these treatments, and the vast majority of patients relapse due to primary or acquired resistance mechanisms. Furthermore, several common cancer indications (such as colorectal cancer and pancreatic cancer) are mostly refractory to these immunomodulatory agents. Therefore, there is an urgent medical need to develop therapies that address resistance mechanisms and enhance the response to cancer immunotherapy, including checkpoint inhibitors.

[0003] Clinical data have shown that patients who do not respond to or do poorly respond to checkpoint inhibitors exhibit a non-T-cell inflammatory immunophenotype characterized by the absence of cytotoxic T cells or their localization limited to the tumor stroma. Therefore, novel therapies aimed at increasing immune infiltration would be highly useful in improving response rates to checkpoint inhibitors and expanding their clinical benefits.

[0004] The TNFR superfamily consists of 19 ligands and 29 receptors, which are structurally similar but involved in many diverse physiological functions. Generally, members of this superfamily can induce cell death (e.g., DR5-TRAIL, Fas-FasL) or promote survival and inflammation (e.g., TNF-TNFR). Lymphotoxin beta receptor (LTBR) is a member of the TNFR superfamily belonging to this second category and is expressed by various cells, including stromal cells of the tumor microenvironment, myeloid cells, and epithelial-derived tumor cells. Ligand-receptor interactions within the TNFR superfamily can be monovalent or polyvalent. For example, OX40 and its ligand (OX40L) form a monovalent ligand-receptor pair, while LTBR, ​​LTα, LTβ, and LIGHT exhibit polyvalent interactions, forming a complex network of interconnected pathways. Activation of LTBR by lymphotoxin α1β1 (LTα1β1) or LIGHT ligand binding induces receptor oligomerization and signal transduction via classical and non-classical NFκB pathways, leading to upregulation of inflammatory and developmental genes such as adhesion molecules (ICAM and VCAM), chemoattractants (CXCL9, 10, 11), and lymphoid tissue organizing chemokines (CCL21, CCL19, CXCL13) (Lu and Browning., Front Immunol. 2014, 5:47, doi: 10.3389 / fimmu. 2014.00047). This pathway is essential for the development and maintenance of secondary lymphoid organs, as demonstrated by the phenotype of ltbr knockout mice, which lack development of lymph nodes and Peyer's patches (Fuetterer et al, Immunity 1998, 9(1), 59-70, doi:10.1016 / s1074-7613(00)80588-9). Furthermore, it is also key to the development and maintenance of high endothelial venules (HEVs) (Browning et al, Immunity 2005, 23(5), 539-550, doi:10.1016 / j.immuni.2005.10.002). In many solid tumors, multicellular aggregates resembling secondary lymphoid organs, consisting of T cells, activated dendritic cells (DCs), B cells, and HEVs, are histologically detected.Clinical evidence has shown that the presence of such ectopic lymphoid organs, also known as tertiary lymphoid tissue-like structures (TLSs), and HEVs correlate with improved prognosis in several tumor indications (Dieu-Nosjean et al, Immunol. Rev. 2016, 271(1), 260-275, doi:10.1111 / imr.12405). TLSs and HEVs are thought to form in response to signals from the same molecules involved in lymph node development, such as activation of the LTBR pathway. Therefore, LTBR activation may promote TLS formation in the tumor microenvironment and induce an anti-tumor immune response.

[0005] Indeed, preclinical evidence supports the hypothesis that LTBR activation increases immune infiltration, enhances response to checkpoint inhibitors, and induces TLS formation. LTBR activation with agonist antibodies or targeted ligands increased T cell infiltration in several mouse tumor models (Lukashev et al, Cancer Res. 2006, 66(19), 9617-9624, doi:10.1158 / 0008-5472.CAN-06-0217). As a result, combination therapy with checkpoint inhibitors was more effective when combined with LTBR agonists (Allen et al, Sci Transl Med. 2017, 9(385), eaak9679, doi: 10.1126 / scitranslmed.aak9679; Johansson-Percival et al, Cell Rep. 2017, 13(12), 2687-2698, doi: 10.1016 / j.celrep. 2015.12.004; and Tang et al, Cancer Cell 2016, 29(3), 285-296, doi: 10.1016 / j.ccell. 2016.02.004). Furthermore, evidence of TLS formation and HEV development in response to such agonists has been reported.

[0006] In addition to the important role of LTBR in the development of TLS and HEV, LTBR activation has been shown to induce cell death in certain cancer cell lines (Browning et al, j Exp Med 1996, 183(3), 867-878, doi:10.1084 / jem.183.3.867). A Phase I trial evaluated the safety and tolerability of a humanized LTBR agonist antibody (hCBE11) in patients with advanced solid tumors (ClinicalTrials.gov, NCT00105170). The trial was interrupted and subsequently terminated before patient enrollment was completed. This suggests the possibility of significant safety issues associated with widespread LTBR agonism in humans.

[0007] Given the significant therapeutic potential of LTBR agonists for improving cancer immunotherapy, there is a need to provide agonist LTBR antibodies with superior pharmacological profiles, or bispecific antibodies with favorable safety profiles that activate LTBR only in the tumor microenvironment and not in other LTBR-expressing tissues. [Overview of the project]

[0008] This invention relates to novel antibodies, particularly agonist hu LTBR antibodies, that specifically bind to the human lymphotoxin beta receptor (LTBR). These antibodies can bind to human LTBR and cynomolgus monkey LTBR with an affinity difference of less than twofold. Some of the novel agonist hu LTBR antibodies can also bind to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR. This invention also relates to multispecific antibodies, including these novel agonist antibodies.

[0009] Therefore, this specification provides agonistrin phototoxin beta receptor (LTBR) antibodies that specifically bind to human LTBR and cynomolgus monkey LTBR, ​​wherein the antibodies are (i) Heavy chain complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 29. H(LTBR), and a light chain variable region (V) comprising a complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32 L LTBR); or (ii) a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45 H (LTBR), and a light chain variable region (V) comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48 L LTBR); or (iii) a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77 H (LTBR), and a light chain variable region (V) comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80 L LTBR); or (iv) a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85 H (LTBR), and a light chain variable region (V) comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88 L LTBR); or (v) a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 37 H (LTBR), and a light chain variable region (V) comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 40L LTBR), or (vi) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56. L LTBR), or (vii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 61 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64. L LTBR); or (viii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 67, CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 69 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 72. L LTBR) Includes.

[0010] In one embodiment, the agonist LTBR antibody described herein is provided, wherein the agonist antibody has the following characteristics: (a) It binds to human LTBR and cynomolgus monkey LTBR with an affinity difference of less than twofold; or (b) When measured by ELISA, EC < 4 nM 50 When bound to the human LTBR extracellular domain and measured by ELISA, the EC was less than 5 nM. 50 Binds to the extracellular domain of cynomolgus monkey LTBR; or (c) requiring crosslinking for its agonist activity in order to activate human LTBR; or (d) requiring crosslinking for its agonist activity in order to induce upregulation of ICAM in human umbilical vein endothelial cells or cancer-associated fibroblasts; or (e) Inhibit the interaction between human LTBR and its human ligands, lymphotoxin α1β2 and LIGHT. It has at least one of the following.

[0011] In one embodiment, (i) Heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 33 H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 34. L LTBR), or (ii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 50. L LTBR), or (iii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 82. L LTBR), or (iv) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 90. L LTBR), or (v) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 98. LLTBR), (vi) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 42. L LTBR), (vii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 58. L LTBR), or (viii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 65 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 66. L LTBR), or (ix) Heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 73 H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 74. L LTBR) An agonist LTBR antibody containing [the specified component] is provided.

[0012] In one embodiment, the agonist LTBR antibody described herein is (i) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82L LTBR), or (iv) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or (v) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR), or (vi) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L LTBR), or (vii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 58 L LTBR), or (viii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO. 65 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 66 L LTBR), (ix) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 73 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 74 L LTBR) Includes.

[0013] In one embodiment, the agonist LTBR antibody described herein is provided, wherein the agonist LTBR antibody further specifically binds to mouse LTBR. In one embodiment, the agonist LTBR antibody has an EC of less than 1 nM when measured by ELISA. 50 It then binds to the mouse LTBR extracellular domain.

[0014] In one embodiment, an agonist LTBR antibody that binds more specifically to mouse LTBR is: A heavy chain variable region (V) comprising a complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 29 H LTBR), and a light chain variable region (V) comprising a complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 32 L LTBR), or A heavy chain variable region (V) comprising a CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 45 H LTBR), and a light chain variable region (V) comprising a CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 48 L LTBR), or A heavy chain variable region (V) comprising a CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 77 H LTBR), and a light chain variable region (V) comprising a CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 80 L LTBR), or A heavy chain variable region (V) comprising a CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 85 H LTBR), and a light chain variable region (V) comprising a CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and a CDR-L3 containing the amino acid sequence of SEQ ID NO: 88 L LTBR) is included.

[0015] In one aspect, an agonist LTBR antibody that binds more specifically to mouse LTBR is Heavy chain variable region (V H LTBR) containing the amino acid sequence of SEQ ID NO: 33 and light chain variable region (V L LTBR) containing the amino acid sequence of SEQ ID NO: 34, or Heavy chain variable region (V H LTBR) containing the amino acid sequence of SEQ ID NO: 49 and light chain variable region (V L LTBR) containing the amino acid sequence of SEQ ID NO: 50, or Heavy chain variable region (V H LTBR) containing the amino acid sequence of SEQ ID NO: 81 and light chain variable region (V L LTBR) containing the amino acid sequence of SEQ ID NO: 82, or Heavy chain variable region (V H LTBR) containing the amino acid sequence of SEQ ID NO: 89 and light chain variable region (V L LTBR) containing the amino acid sequence of SEQ ID NO: 90, or Heavy chain variable region (V H LTBR) containing the amino acid sequence of SEQ ID NO: 97 and light chain variable region (V L LTBR) is included.

[0016] In a particular embodiment, the agonist LTBR antibody described herein binds to the epitope region of SEQ ID NO: 351 on human LTBR. In one embodiment, such an agonist LTBR antibody comprises a heavy chain variable region (V H LTBR) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 29, and a light chain variable region (V L LTBR) comprising light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. In one embodiment, it comprises a heavy chain variable region (V H LTBR) containing the amino acid sequence of SEQ ID NO: 33 and a light chain variable region (V L LTBR) containing the amino acid sequence of SEQ ID NO: 34.

[0017] The present invention also relates to agonist LTBR antibodies that are multispecific and specific antibodies, including the agonist LTBR antibodies described herein. In a particular embodiment, a bispecific agonist LTBR antibody is provided. In any of the embodiments described herein, the agonist LTBR antibody preferably comprises an Fc domain of human origin, particularly a human IgG subclass, more specifically a human IgG1 subclass. In one embodiment, the agonist LTBR antibody comprises an Fc domain of a human IgG1 subclass that includes one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor. In one embodiment, the agonist LTBR antibody comprises an Fc domain of a human IgG1 subclass having the amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

[0018] In one embodiment, the agonist LTBR antibody is a bispecific antibody that specifically binds to LTBR and tumor-associated antigens (TAAs).

[0019] In one embodiment, a bispecific antibody capable of specifically binding to lymphotoxin beta receptor (LTBR) and fibroblast-activating protein (FAP) is provided, and thus an agonist LTBR antibody is provided, which combines an antigen-binding domain that specifically binds to FAP with at least one antigen-binding domain that can agonist-bound to LTBR, ​​particularly hu LTBR, ​​where LTBR-mediated activation is provided by crosslinking via binding to FAP expressed on tumor stromal cells. In contrast to conventional non-targeted agonist LTBR antibodies, by targeting tumor-associated targets (TAAs) such as FAP, LTBR agonism can be restricted to the tumor microenvironment (tumor endothelium and cancer-associated fibroblasts), thereby reducing potential side effects.

[0020] Fibroblast-activating protein (FAP) is a serine protease highly expressed on the cell surface of cancer-associated stromal cells and fibroblast reticular cells of secondary lymphoid organs, but its expression in other normal tissues is very limited. Because FAP is widely found in various cancer indications, it can be used as a target site for drugs that accumulate in the tumor stroma.

[0021] Therefore, in one embodiment, a bispecific agonist LTBR antibody, (a) A first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (b) A second antigen-binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antibody to the Fc receptor. A bispecific agonist LTBR antibody containing the above is provided.

[0022] A bispecific antibody has an Fc domain comprising a first subunit and a second subunit containing one or more amino acid substitutions that reduce the antibody's binding affinity and / or effector function to the Fc receptor. This decrosslinks the Fc receptor, and tumor-specific activation is achieved through crosslinking via the binding of an antigen-binding domain that specifically binds to FAP upon binding to a tumor-associated target. A bispecific antibody that activates LTBR only when bound to FAP is provided. Thus, a bispecific antibody that activates LTBR in the tumor stroma is provided.

[0023] In one embodiment, a bispecific agonist LTBR antibody, (a) A first Fab fragment that specifically binds to fibroblast-activating protein (FAP), (b) A second Fab fragment that specifically binds to the lymphotoxin beta receptor (LTBR), and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. A bispecific agonist LTBR antibody containing the above is provided.

[0024] In one embodiment, a bispecific agonist LTBR antibody is provided, comprising a third antigen-binding domain that specifically binds to LTBR, ​​namely (a) a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (b) a second antigen-binding domain and a third antigen-binding domain that specifically bind to lymphotoxin beta receptor (LTBR), and (c) an Fc domain composed of a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor.

[0025] In one particular embodiment, the third antigen-binding domain that specifically binds to LTBR is identical to the second antigen-binding domain that specifically binds to LTBR, ​​meaning that the second and third antigen-binding domains that specifically bind to the lymphotoxin beta receptor (LTBR) are the same. In one embodiment, the second and third antigen-binding domains that specifically bind to LTBR are Fab fragments that specifically bind to LTBR. In one embodiment, the Fab fragment that specifically binds to LTBR is a crossfab fragment. In a further embodiment, the first antigen-binding domain that specifically binds to FAP is a Fab fragment.

[0026] In one embodiment, the bispecific agonist LTBR antibody disclosed herein has a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (i) Heavy chain complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 3, CDR-H2 containing the amino acid sequence of SEQ ID NO: 4, and heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 5 HFAP), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 8. L FAP), or (ii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21 H The light chain variable region (V) includes FAP), as well as CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24. L FAP), or (iii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 13 H FAP), and the light chain variable region (V) including CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 16. L FAP) It contains a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP).

[0027] In one embodiment, the first antigen-binding domain that specifically binds to FAP includes (i) a heavy chain complementarity-determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 3, CDR-H2 containing the amino acid sequence of SEQ ID NO: 4, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 5, and a heavy chain variable region (V H FAP), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 8. L FAP), or (ii) a heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21.H FAP), and a light chain variable region (V containing a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 L FAP). In a particular embodiment, the first antigen-binding domain that specifically binds to FAP comprises: (i) a heavy chain complementarity determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5, a heavy chain variable region (V H FAP), and a light chain complementarity determining region (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain variable region (V L FAP).

[0028] In one embodiment, the first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region (V having an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 9 H FAP), and a light chain variable region (V having an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 10 L FAP), or the first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region (V having an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 25 H FAP), and a light chain variable region (V having an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 26 L FAP), or the first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region (V having an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 17 H FAP), and a light chain variable region (V having an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 18 L FAP). In one embodiment, the first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region (V having the amino acid sequence of SEQ ID NO: 9 H FAP), and a light chain variable region (V having the amino acid sequence of SEQ ID NO: 10L The first antigen-binding domain that comprises FAP or specifically binds to FAP is a heavy-chain variable region (V H FAP) and a light-chain variable region (V L FAP) that comprises an amino acid sequence of SEQ ID NO: 26. The first antigen-binding domain that comprises FAP or specifically binds to FAP is a heavy-chain variable region (V H FAP), and a light-chain variable region (V L FAP). In a particular embodiment, the first antigen-binding domain that specifically binds to FAP is a heavy-chain variable region (V H FAP) and a light-chain variable region (V L FAP) that comprises an amino acid sequence of SEQ ID NO: 10. In particular, the first antigen-binding domain that specifically binds to FAP is a heavy-chain variable region (V H FAP), and a light-chain variable region (V L FAP), and is a Fab fragment.

[0029] In another embodiment, the bispecific agonist LTBR antibody disclosed herein is a second antigen-binding domain that specifically binds to LTBR, (i) a heavy-chain variable region (V H LTBR) that comprises a heavy-chain complementarity-determining region (CDR-H1) that comprises an amino acid sequence of SEQ ID NO: 27, a CDR-H2 that comprises an amino acid sequence of SEQ ID NO: 28, and a CDR-H3 that comprises an amino acid sequence of SEQ ID NO: 29, and a light-chain variable region (V L LTBR) that comprises a light-chain complementarity-determining region (iv) CDR-L1 that comprises an amino acid sequence of SEQ ID NO: 30, a CDR-L2 that comprises an amino acid sequence of SEQ ID NO: 31, and a CDR-L3 that comprises an amino acid sequence of SEQ ID NO: 32; or (ii) a heavy-chain variable region (V HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR); or (iii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L LTBR); or (iv) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L LTBR); or (v) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 37 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 40. L LTBR), or (vi) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56. LLTBR), or (vii) a heavy chain variable region (V) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61 H LTBR), and a light chain variable region (V) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 63, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 64 L LTBR); or (viii) a heavy chain variable region (V) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69 H LTBR), and a light chain variable region (V) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 70, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 71, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 72 L LTBR) comprising a second antigen-binding domain that specifically binds to LTBR.

[0030] In one aspect, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR are (i) a heavy chain variable region (V) comprising a heavy chain complementarity determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 27, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29 <00> H LTBR), and a light chain variable region (V) comprising a light chain complementarity determining region (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32 L LTBR); or (ii) a heavy chain variable region (V) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45 HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR); or (iii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L LTBR); or (iv) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L LTBR); or (v) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 37 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 40. L LTBR), or (vi) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56. LLTBR), or (vii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 61 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64. L LTBR); or (viii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 67, CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 69 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 72. L LTBR) Includes.

[0031] In one embodiment, a bispecific agonist LTBR antibody disclosed herein is provided, wherein a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR is, (i) Heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 33 H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 34. L LTBR), or (ii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 50. L LTBR), or (iii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 81 (V HLight chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 82. L LTBR), or (iv) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 90. L LTBR), or (v) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 98. L LTBR), (vi) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 42. L LTBR), (vii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 58. L LTBR), or (viii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 65 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 66. L LTBR), or (ix) Heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 73 H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 74. L LTBR) Includes.

[0032] In one embodiment, the bispecific agonist LTBR antibody disclosed herein comprises a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR, (i) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or (iv) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or (v) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR), or (vi) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L LTBR), or (vii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 58 L LTBR), or (viii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO. 65 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 66 L LTBR), (ix) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 73 (V HLight chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 74 L LTBR) It includes a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR.

[0033] In another embodiment, a bispecific agonist LTBR antibody disclosed herein is provided, wherein a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR is provided. The heavy chain complementarity-determining region (CDR-H1) contains the amino acid sequence of SEQ ID NO: 27, the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 28, and the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L LTBR) Includes.

[0034] In one embodiment, a bispecific agonist LTBR antibody comprises a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR. Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR) It includes a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR.

[0035] In another embodiment, a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR is: (i) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR) Includes.

[0036] In a particular embodiment, the second antigen-binding domain (and optionally the third antigen-binding domain) that specifically binds to LTBR is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L Includes LTBR). In another particular embodiment, the second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 41. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L It includes LTBR). In yet another specific embodiment, the second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 49. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L Includes LTBR.

[0037] Therefore, in a particular embodiment, the bispecific agonist LTBR antibody described herein has (a) a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 9. HFAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 L (b) a first antigen-binding domain that specifically binds to FAP, including (f) the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L The antibody comprises (a) a heavy chain variable region (V) that specifically binds to LTBR. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 L (b) a first antigen-binding domain that specifically binds to FAP, including (f) the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L The molecule comprises (a) a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 9. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 L (b) a first antigen-binding domain that specifically binds to FAP, including (f) the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L It includes a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to the LTBR, ​​which is the LTBR.

[0038] In all of the embodiments described herein, the bispecific agonist LTBR antibody comprises an Fc domain consisting of a first subunit and a second subunit, each containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor. In one embodiment, the Fc domain consisting of the first and second subunits is an IgG Fc domain. In a particular embodiment, the Fc domain consisting of the first and second subunits is an IgG1 Fc domain or an IgG4 Fc domain. In a particular embodiment, the Fc domain is from a human IgG1 subclass having amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

[0039] In another embodiment, a bispecific agonist LTBR antibody as previously defined herein is provided, wherein, according to the knob-into-hole method, a first subunit of the Fc region comprises a knob and a second subunit of the Fc region comprises a hole. In particular, a bispecific antigen-binding molecule is provided, wherein (i) the first subunit of the Fc region comprises amino acid substitutions S354C and T366W (numbered according to the Kabat EU index) and the second subunit of the Fc region comprises amino acid substitutions Y349C, T366S and Y407V (numbered according to the Kabat EU index), or (ii) the first subunit of the Fc region comprises amino acid substitutions K392D and K409D (numbered according to the Kabat EU index) and the second subunit of the Fc region comprises amino acid substitutions E356K and D399K (numbered according to the Kabat EU index). More specifically, the provided antibody is a bispecific agonist LTBR antibody in which the first subunit of the Fc region contains amino acid substitutions S354C and T366W (numbered according to the Kabat EU index), and the second subunit of the Fc domain contains amino acid substitutions Y349C, T366S and Y407V (numbered according to the Kabat EU index).

[0040] In a further embodiment, a bispecific agonist LTBR antibody as defined herein, (a) A first Fab fragment that specifically binds to FAP, (b) A second Fab fragment that specifically binds to LTBR, ​​and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antibody to the Fc receptor. A bispecific agonist LTBR antibody is provided, comprising the following: In one embodiment, the second Fab fragment that specifically binds to LTBR is a crossFab fragment. Thus, a bispecific agonist LTBR antibody is provided that provides monovalent binding to LTBR and monovalent binding to FAP.

[0041] In another embodiment, (a) A first Fab fragment that specifically binds to FAP, (b) A second Fab fragment and a third Fab fragment that specifically bind to LTBR, ​​and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antibody to the Fc receptor. A bispecific agonist LTBR antibody is provided, comprising a first Fab fragment that specifically binds to FAP, with its N-terminus fused to the C-terminus of one of the Fc domain subunits, and a second and third Fab fragment that specifically bind to LTBR, ​​each fused to the N-terminus of one of the Fc domain subunits at its C-terminus.

[0042] Therefore, a bispecific agonist LTBR antibody is provided that provides a divalent binding to LTBR and a monovalent binding to FAP.

[0043] According to another aspect of the present invention, one or more isolated polynucleotides encoding the agonist LTBR antibody or bispecific agonist LTBR antibody described herein are provided. The present invention further provides vectors, particularly expression vectors, comprising the isolated polynucleotides of the present invention, and host cells comprising the isolated nucleic acids or expression vectors of the present invention. In some aspects, the host cells are eukaryotic cells, particularly mammalian cells. In another aspect, a method for producing the agonist LTBR antibody or bispecific agonist LTBR antibody described herein is provided, comprising culturing such host cells under conditions suitable for the expression of the agonist LTBR antibody or bispecific agonist LTBR antibody, and isolating the agonist LTBR antibody or bispecific agonist LTBR antibody. The present invention also encompasses the agonist LTBR antibody or bispecific agonist LTBR antibody produced by the method of the present invention.

[0044] The present invention further provides a pharmaceutical composition comprising an agonist LTBR antibody or a bispecific agonist LTBR antibody as described herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises an additional therapeutic agent.

[0045] The present invention also includes agonist LTBR antibodies or bispecific agonist LTBR antibodies as described herein, or pharmaceutical compositions comprising bispecific agonist LTBR antibodies for use as pharmaceuticals.

[0046] In one embodiment, the bispecific agonist LTBR antibody described herein or the pharmaceutical composition of the present invention is provided for use in (a) in inducing ICAM upregulation on endothelial cells or cancer-associated fibroblasts, or (b) in enhancing T cell adhesion.

[0047] In certain embodiments, the agonist LTBR antibody or bispecific agonist LTBR antibody described herein, or the pharmaceutical composition of the present invention, is provided for use in the treatment of cancer. In another particular embodiment, the present invention provides the agonist LTBR antibody or bispecific agonist LTBR antibody described herein, for use in the treatment of cancer, wherein the agonist LTBR antibody or bispecific agonist LTBR antibody is administered in combination with a chemotherapeutic agent, radiotherapy, and / or other agent for use in cancer immunotherapy. In one embodiment, the agonist LTBR antibody or bispecific agonist LTBR antibody described herein is for use in the treatment of cancer, and the agonist LTBR antibody or bispecific agonist LTBR antibody is for administration in combination with an agent that blocks PD-L1 / PD-1 interaction. In another embodiment, the agonist LTBR antibody or bispecific agonist LTBR antibody described herein, or the pharmaceutical composition of the present invention, is provided for use in the upregulation or extension of cytotoxic T cell activity.

[0048] In a further embodiment, the present invention provides a method for inhibiting the proliferation of tumor cells in an individual, comprising administering an effective amount of the agonist LTBR antibody or bispecific agonist LTBR antibody described herein, or the pharmaceutical composition of the present invention, to the individual to inhibit the proliferation of tumor cells. In another embodiment, the present invention provides a method for treating or delaying cancer in an individual, comprising administering an effective amount of the agonist LTBR antibody or bispecific agonist LTBR antibody described herein, or the pharmaceutical composition of the present invention, to the individual.

[0049] Also provided is the use of the bispecific antigen-binding molecules described herein for the manufacture of a pharmacopoeia for the treatment of a disease in an individual requiring treatment of the disease, specifically for the manufacture of a pharmacopoeia for the treatment of cancer, and a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising the agonist LTBR antibody of the present invention or a bispecific agonist LTBR antibody in a pharmaceutically acceptable form. In certain embodiments, the disease is cancer. In any of the above embodiments, the individual is a mammal, in particular a human. [Brief explanation of the drawing]

[0050] [Figure 1A-G] Figures 1A–1G show schematic diagrams of recombinant soluble proteins (receptors, ligands, and tool proteins) used as phage display and immunization antigens for generating anti-LTBR antibodies. Figure 1A shows a schematic diagram of a so-called biotinylated Fc depleter (Fc depleter kh NC avi biotinylated, P1AA0981). This consists of a knob-into-hole (kh) Fc domain used as a preclearing agent to prevent clones from binding to the Fc domain. Figure 1B shows a schematic diagram of human lymphotoxin α1β2 single-chain avi his (P1AE1235). Figure 1C shows a schematic diagram of mouse lymphotoxin α1β2 single-chain avi his (P1AE1236). Figure 1D shows a schematic diagram of monomeric huLTBR ectodomain ECD (S28-M227) as Fc-fusion avi biotinylated (P1AE2835). Figure 1E shows a schematic diagram of monomeric muLTBR ectodomain ECD(S28-L223)Fc-fusion avi biotinylation (P1AE4410). Figure 1F shows a schematic diagram of monomeric cynoLTBR ectodomain ECD(S28-M227)Fc-fusion avi biotinylation (P1AE4411). Figure 1G shows a schematic diagram of monomeric N-terminal human LTBR (ECD full length) hu IgG1 Fc-fusion kih HRYF avi (P1AE1217). [Figure 2A-F]Figures 2A-2F show schematic diagrams of additional antigens used for screening and clonal characterization of anti-LTBR antibodies and bispecific FAP-LTBR antibodies. Figure 2A shows a schematic diagram of dimeric hu LTBR-Fc fusion biotinylation (P1AE2401). Figure 2B shows a schematic diagram of N-terminal and C-terminal extension-Fc fusion wt kih HRYF-Avi-His biotinylation (P1AE7979) of human LTBR ECD (S28-M227). Figure 2C shows a schematic diagram of monomeric N-terminal cyno LTbR (ECD full length) hu IgG1 Fc fusion kih HRYF avi biotinylation (P1AE2656). Figure 2D shows a schematic diagram of dimeric mouse LTBR-Fc disulfide-linked homodimer biotinylation (P1AE2655). Figure 2E shows a schematic diagram of human FAP-Avi-His biotinylation (P1AA5347). Figure 2F shows a schematic diagram of mouse FAP-Avi-His biotinylation (P1AD9907). [Figure 3] Figure 3 shows the identification of Fab clone FAPltbr.P218.076 (P1AE5929 after IgG conversion) as a cross-reactive antibody in sandwich ELISA, and specific binding to human LTBR (P1AE2835) and mouse LTBR (P1AE4410) was determined. This antibody originates from phage display. Since the antigen used for phage display is an Fc-tagged fusion protein, a human Fc fragment (P1AA0981) was used to confirm specific binding to LTBR and exclude Fc binding. The Y-axis represents OD450-900. [Figure 4] Figure 4 shows the inhibition of the LTBR-lymphotoxin α1β2 interaction by anti-LTBR IgG antibodies. The selected anti-LTBR IgG was titrated at 3-fold dilutions starting from 100 nM (15 μg / ml). [Figure 5] Figure 5 shows the inhibition of the LTBR-LIGHT interaction by anti-LTBR IgG antibodies. The selected anti-LTBR IgG was titrated at a 3-fold dilution starting at 100 nM (15 μg / ml). [Figure 6]Figure 6 shows the results for various anti-LTBR IgG assays, both in the absence and in the presence of a crosslinking antibody (linker), as determined by the HeLa NFκB luc reporter assay described in Example 1.7. The concentration of anti-LTBR IgG is plotted against the emitted light units (RLU) measured after incubation and the addition of the luciferase detection solution. [Figure 7] Figure 7 shows the results of evaluation of cell surface LTBR binding of anti-LTBR IgG (FACS in recombinant human LTBR CHO cells), plotted as the median fluorescence intensity against concentration. Anti-LTBR IgG was used in a titration with a 3-fold dilution starting at 20 μg / ml. [Figure 8] Figure 8 shows an epitope binning heatmap based on the binding value (percentage) of each of the eight selected anti-LTBR IgGs. [Figure 9A-E] Figures 9A-9E show schematic diagrams of anti-LTBR antibodies and bispecific FAP-LTBR antibodies. Figure 9A shows a schematic diagram of a monospecific anti-LTBR human IgG1 PG LALA antibody. Figure 9B shows a schematic diagram of a 1+1 anti-LTBR / anti-FAP bispecific antibody as a human IgG1 PG LALA crossMab having a cross-V domain in the anti-LTBR Fab arm and a charge in the CH1 / Ck domain of the anti-Fab arm. Figure 9C shows a schematic diagram of a 1+1 anti-LTBR / anti-FAP bispecific antibody as a human IgG1 PG LALA crossMab having a cross-CH1 / Ck domain in the anti-FAP Fab arm. Figure 9D shows a schematic diagram of a 2+1 anti-LTBR / anti-FAP bispecific antibody as a human IgG1 PG LALA crossMab having a cross-CH1 / Ck domain in the anti-FAP Fab arm and a second anti-LTBR Fab fragment fused to the C-terminus of the Fc domain. Figure 9E shows a schematic diagram of a 2+1 anti-LTBR / anti-FAP bispecific antibody as a human IgG1 PG LALA crossMab, having crossed V domains in both anti-LTBR Fab arms and a charge in the CH1 / Ck domain of the anti-Fab arm fused to the C-terminus of the Fc domain. [Figure 10]Figure 10 shows the inhibition of the LTBR-lymphotoxin α1β2 interaction by anti-FAP / anti-LTBR bispecific antibodies. The selected anti-FAP / anti-LTBR bispecific antibodies were titrated at 3-fold dilutions starting from 100 nM (15 μg / ml). [Figure 11] Figure 11 shows the inhibition of LTBR-LIGHT interaction by anti-FAP / anti-LTBR bispecific antibodies. The selected anti-FAP / anti-LTBR bispecific antibodies were titrated at 3-fold dilutions starting from 100 nM (15 μg / ml). [Figure 12A-C] Figures 12A and 12B show schematic diagrams of mouse surrogate bispecific FAP-LTBR antibodies. Figure 12A shows a schematic diagram of a 1+1 anti-LTBR / anti-FAP bispecific antibody as a mouse IgG1 DA PG crossMab, having a cross-V domain in the anti-Fab arm and a charge in the CH1 / Ck domain of the anti-muLTBR Fab arm. Figure 12B shows a schematic diagram of a 2+1 anti-LTBR / anti-FAP bispecific antibody as a mouse IgG1 DA PG crossMab, having a cross-CH1 / Ck domain of an anti-FAP Fab fragment fused to the C-terminus of the Fc domain and having a charge in the CH1 / Ck domains of both anti-LTBR Fabs. Figure 12C shows a schematic diagram of a 2+1 anti-LTBR / non-targeted (DP47) bispecific antibody as a mouse IgG1 DA PG crossmab, having a cross-CH1 / Ck domain of a non-targeted (DP47) Fab fragment fused to the C-terminus of the Fc domain and having a charge in the CH1 / Ck domains of both anti-LTBR Fabs. [Figure 13A-D] Figures 13A–13D show that anti-LTBR agonist antibodies upregulate ICAM on endothelial cells in a crosslinking-dependent manner. The median fluorescence intensity of ICAM on human umbilical vein endothelial cells (HUVECs) treated with anti-human LTBR agonist antibodies is shown in the presence (Figures 13A and 13C) or absence (Figures 13B and 13D) of Fc crosslinking antibodies (anti-Fc crosslinking agents). Data are normalized to the activity of the anti-LTBR antibody CBE11 (P1AE1873). [Figure 14A-D]Figures 14A–14D demonstrate that anti-LTBR agonist antibodies upregulate ICAM on cancer-associated fibroblasts (CAFs) in a crosslinking-dependent manner. Median fluorescence intensities of ICAM on immortalized CAFs treated with anti-human LTBR agonist antibodies are shown in the presence (Figures 14A and 14C) or absence (Figures 14B and 14D) of anti-Fc crosslinking agents. Data are normalized to the activity of the anti-LTBR antibody CBE11 (P1AE1873). [Figure 15A-C] Figures 15A–15F show the binding of the FAP-LTBR bispecific antigen-binding molecule to human (Figures 15A and 15D), cynomolgus monkey (Figures 15B and 15E), and mouse LTBR (Figures 15C and 15F) on CHO-K1 cells modified to overexpress human, cynomolgus monkey, or mouse LTBR. The median fluorescence intensity of anti-human Fc secondary antibodies detecting the bound FAP-LTBR bispecific molecule on cells overexpressing human LTBR (Figures 15A and 15D), cynomolgus monkey LTBR (Figures 15B and 15E), or mouse LTBR (Figures 15C and 15F) is shown. Data are normalized to baseline. [Figure 15D-F] Figures 15A–15F show the binding of the FAP-LTBR bispecific antigen-binding molecule to human (Figures 15A and 15D), cynomolgus monkey (Figures 15B and 15E), and mouse LTBR (Figures 15C and 15F) on CHO-K1 cells modified to overexpress human, cynomolgus monkey, or mouse LTBR. The median fluorescence intensity of anti-human Fc secondary antibodies detecting the bound FAP-LTBR bispecific molecule on cells overexpressing human LTBR (Figures 15A and 15D), cynomolgus monkey LTBR (Figures 15B and 15E), or mouse LTBR (Figures 15C and 15F) is shown. Data are normalized to baseline. [Figure 16A-D]Figures 16A–16D demonstrate that the FAP-LTBR bispecific antigen-binding molecule upregulates ICAM on cancer-associated fibroblasts in a FAP-dependent manner. Median fluorescence intensities of ICAM on immortalized CAFs expressing LTBR and FAP endogenously (Figures 16A and 16C, hTERT CAF) or immortalized CAFs expressing LTBR only (Figures 16B and 16D, hTERT CAF_delFAP) are shown. Data are normalized to ICAM expression in the untreated control. [Figure 17A-D] Figures 17A–17D demonstrate that the FAP-LTBR bispecific molecule upregulates ICAM on endothelial cells in a FAP-dependent manner. The median fluorescence intensity of ICAM on CD31+ HUVEC cells co-cultured with FAP-overexpressing NIH-3T3 cells (Figures 17A and 17C) or FAP-nonexpressing NIH-3T3 cells (Figures 17B and 17D) is shown. Data are normalized to untreated control ICAM expression, and EC50 values ​​are indicated in the notes. [Figure 18A-D] Figures 18A-18F demonstrate that the FAP-LTBR bispecific antigen-binding molecule induces chemoattractant secretion by endothelial cells in a FAP-dependent manner. Supernatants of HUVEC cells co-cultured with NIH-3T3 cells overexpressing FAP (Figures 18A, 18C, and 18E) or NIH-3T3 cells not expressing FAP (Figures 18B, 18D, and 18F) were treated with the FAP-LTBR bispecific molecule for 48 hours, and the concentrations of various chemokines (CXCL9, CXCL10, and CXCL11) were measured using Bio-plex. [Figure 18E-F] Figures 18A-18F demonstrate that the FAP-LTBR bispecific antigen-binding molecule induces chemoattractant secretion by endothelial cells in a FAP-dependent manner. Supernatants of HUVEC cells co-cultured with NIH-3T3 cells overexpressing FAP (Figures 18A, 18C, and 18E) or NIH-3T3 cells not expressing FAP (Figures 18B, 18D, and 18F) were treated with the FAP-LTBR bispecific molecule for 48 hours, and the concentrations of various chemokines (CXCL9, CXCL10, and CXCL11) were measured using Bio-plex. [Figure 19]Figure 19 shows increased T cell adhesion on endothelium stimulated with the FAP-LTBR bispecific antigen-binding molecule. It shows the area of ​​labeled T cells adhered to HUVEC cultures stimulated with FAP-LTBR bispecific antibody (2nM) in the presence (black bars) or absence (white bars) of FAP. TNFα (0.5 ng / mL) is used as a positive control. [Figure 20A-B] Figures 20A and 20B demonstrate that the FAP-LTBR bispecific antibody surrogate upregulation of adhesion molecules on mouse fibroblasts in a FAP-dependent manner in vitro. The median fluorescence intensity of VCAM on NIH-3T3 cells treated with the FAP-LTBR antibody surrogate or anti-mouse LTBR agonist antibody (5G11) (Figure 20A) or NIH-3T3 FAP-overexpressing cells (Figure 20B) is shown. Data are normalized to baseline VCAM expression in the untreated control. [Figure 21A-B] Figures 21A and 21B demonstrate that the FAP-LTBR bispecific antibody surrogate molecule upregulates adhesion molecules on endothelial cells in a FAP-dependent manner in vitro. The median fluorescence intensity of ICAM on CD31+ HUVEC cells co-cultured with NIH-3T3 cells (Figure 21A) or NIH-3T3 cells overexpressing FAP (Figure 21B) is shown. [Figure 22] Figure 22 shows the study design of an in vivo mouse study to evaluate the safety, efficacy, and pharmacodynamic profiles of a FAP-LTBR bispecific antibody alone or in combination with an anti-PD-L1 antibody in a subcutaneous MC38-hu CEA tumor model. The timeline indicates the treatment and sacrifice times, and the table details the treatment groups, doses, and schedule of this study. [Figure 23A-B]Figure 23A shows that FAP-LTBR surrogate molecules inhibit tumor growth and improve the response to aPD-L1 treatment in a subcutaneous tumor model of MC38-huCEA in monotherapy. Figure 23A plots tumor growth curves for mice treated with vehicle, P1AF4664, P1AF4674, aPD-L1, P1AF4664+aPD-L1, and P1AF4674+aPD-L1. Comparing tumor volumes at day 12 of treatment, statistically significantly larger volumes were observed in vehicle vs. P1AF4764, vehicle vs. P1AF4664+aPD-L1, vehicle vs. P1AF4674+aPD-L1, and aPD-L1 vs. P1AF4674+aPD-L1. Statistical analysis was performed using one-way ANOVA and Holm-Sidak multiple comparison tests (*p<0.05, **p<0.01, ***p<0.001). (Figure 23B). [Figure 23C-H] Individual tumor growth curves for mice treated with vehicle, P1AF4664, P1AF4674, aPD-L1, P1AF4664+aPD-L1, and P1AF4674+aPD-L1 are shown in Figures 23C-23H. [Figure 24A-B] Figures 24A and 24B demonstrate that FAP-LTBR substitute molecules were able to induce endothelial activation in an MC38-huCEA subcutaneous tumor model. Flow cytometry analysis of tumor single-cell suspensions was performed on days 9 and 16 after the start of treatment. Endothelial cells were gated as CD45-, huCEA-, CD31+, and podoplanin-, and the median fluorescence intensity of ICAM (Figures 24A and 24B) was quantified. The data indicate that treatment with P1AF4664 and P1AF4674 increased ICAM on tumor endothelial cells throughout the treatment period. Statistical analysis was performed using one-way ANOVA and Holm-Sidak multiple comparison tests (*p<0.05, **p<0.01, ***p<0.001). [Figure 25]Figure 25 shows the study design of an in vivo mouse study to evaluate the safety, efficacy, and pharmacodynamic profiles of a FAP-LTBR bispecific antibody alone or in combination with an anti-PD-L1 antibody in a subcutaneous KPC4662-huCEA tumor model. The timeline includes treatment and sacrifice times, and the table details the treatment groups, doses, and schedules of this KPC4662-huCEA trial protocol. [Figure 26A-C] Figures 26A-26C demonstrate that FAP-LTBR surrogate molecules induce hyperendothelial venules in a KPC4662-huCEA subcutaneous tumor model. A significant increase in Meca79-positive vessels as a proportion of all vessels was recorded in the treatment group compared to the vehicle group. Statistical analysis was performed using one-way ANOVA and Holm-Sidak multiple comparison tests (*p<0.05, **p<0.01, ***p<0.001) (Figure 26A). Representative immunofluorescence staining for CD31 (all vessels) and Meca79 (HEV) in P1AF4664 treatment samples 6 days after the start of treatment is shown in Figures 26B and 26C, respectively. [Figure 27] Figure 27 shows the study design of an in vivo mouse study to evaluate the safety, efficacy, and pharmacodynamic profiles of LTBR bispecific antigen-binding molecules alone or in combination with an anti-PD-L1 antibody in an orthotopic EMT6 tumor model. The timeline indicates the treatment and sacrifice times, and the table details the treatment groups, dosages, and schedules of this EMT6 study protocol. The vehicle and mouse surrogate LTBR bispecific antigen-binding molecules P1AG5459 and P1AG5461 (non-targeted control) were injected at all of the above time points (d0, d2, d5, d7, etc.) unless otherwise specified. Unless otherwise specified, PDL1 was injected only at the time points indicated by empty arrows. [Figure 28A-B]Figures 28A and 28B show that the mouse surrogate FAP-LTBR bispecific antibody (P1AG5459) inhibits tumor growth in an EMT6 orthotopic tumor model and improves the response to aPD-L1 therapy when used as monotherapy. The median tumor growth curves for vehicle, P1AG5459, P1AG5461, aPD-L1, and P1AG5459+aPD-L1 treated mice are plotted in Figure 28A. A comparison of tumor volume at 22 days post-treatment is plotted in Figure 28B, showing statistically significantly larger volumes in vehicle vs. P1AG5459, vehicle vs. P1AG5459+aPD-L1, P1AG5459 vs. P1AG5461, and P1AG5461 vs. P1AG5459+aPD-L1. Statistical analysis was performed using one-way ANOVA and Holm-Sidak multiple comparison tests (*p<0.05, **p<0.01, ***p<0.001). [Figure 29A-C] Figures 29A-29C demonstrate that the mouse surrogate LTBR bispecific antibody P1AG5459 was able to induce endothelial activation and HEV differentiation in an FAP-dependent manner in an EMT6 orthotopic tumor model. Flow cytometry analysis of tumor single-cell suspension was performed 13 days after the start of treatment. Endothelial cells were gated as CD45-, CD31+, and podoplanin-, and the median fluorescence intensity of ICAM (Figure 29A) and VCAM (Figure 29B), or the percentage of Meca79+ endothelial cells (Figure 29C) was quantified. The data show that treatment with P1AG5459 alone or in combination with aPD-L1 increased ICAM and VCAM on tumor endothelial cells and increased the frequency of Meca79+ endothelial cells, rather than the non-targeted control P1AG5461 (Figure 29C). The effects on endothelial activation and HEV differentiation were more pronounced in responsive mice (□) than in non-responsive mice (●). [Figure 30A] Figure 30A shows that treatment with P1AG5459 alone or in combination with aPD-L1 increases the amount of Meca79+ endothelial cells quantified by immunofluorescence imaging 13 days after the start of treatment, while P1AG5461 does not. An example image shows the major vascular structures (shown by CD31 staining) and the localization of Meca79+ endothelium. [Figure 30B-D]Figures 30B, 30C, and 30D show the quantification of CD8 T cells (Figure 30B), CD4 T cells (Figure 30C), and B220+ B cells (Figure 30D) infiltration into EMT6 orthotopic tumors, measured by immunofluorescence imaging 13 days after the start of treatment. P1AG5459, alone or in combination with aPD-L1, and aPD-L1 alone, increase the amount of T and B cells in the tumor to some extent, but P1AG5461 does not. In the case of P1AG5459 monotherapy, a clear correlation can be observed between responsive mice (□) and non-responsive mice (●). [Figure 31A-C] Figures 31A-31C demonstrate that the mouse surrogate FAP-LTBR bispecific antibody (P1AG5459) induces upregulation of chemokines in an EMT6 orthotopic tumor model. The concentrations of chemokines CXCL13 (Figure 31A), CCL5 (Figure 31B), and CCL10 (Figure 31C) were measured in tumor lysates collected 13 days after the start of treatment. Tumors treated with P1AG5459 tended to have higher levels of CXCL13, CCL5, and CXCL10. Responsive tumors (□) tended to show higher concentrations of chemokines. [Figure 32A] Figures 32A-32C show that tumor growth inhibition mediated by the mouse surrogate FAP-LTBR bispecific antibody (P1AG5459) in the EMT6 orthotopic tumor model is mediated by CD8, CD4 T cells, and B cells. The median tumor growth curve is plotted in Figure 32A. A comparison of tumor volumes at day 15 is plotted in Figure 32B, showing that in the absence of CD8 T cells, there is no difference in tumor volume between vehicle-treated mice and P1AG5459-treated mice. A comparison of tumor volumes at day 20 is plotted in Figure 32C, showing that in the absence of CD4 T cells or CD20+ expressing B cells, there is no difference in tumor volume between vehicle-treated mice and P1AG5459-treated mice. [Figure 32B-C]Figures 32A-32C show that tumor growth inhibition mediated by the mouse surrogate FAP-LTBR bispecific antibody (P1AG5459) in the EMT6 orthotopic tumor model is mediated by CD8, CD4 T cells, and B cells. The median tumor growth curve is plotted in Figure 32A. A comparison of tumor volumes at day 15 is plotted in Figure 32B, showing that in the absence of CD8 T cells, there is no difference in tumor volume between vehicle-treated mice and P1AG5459-treated mice. A comparison of tumor volumes at day 20 is plotted in Figure 32C, showing that in the absence of CD4 T cells or CD20+ expressing B cells, there is no difference in tumor volume between vehicle-treated mice and P1AG5459-treated mice. [Figure 33] Figure 33 shows representative immunofluorescence images of orthotopic colorectal cancer tumors treated with vehicle or mouse surrogate FAP-LTBR bispecific antibody (P1AG5459). Tumors are identified by Ki67 staining of proliferating cells, and tumor boundaries are marked by dashed lines. CD31 marks blood vessels, pNAD / Meca79 marks high endothelial venules, and CD8 marks CD8 T cells. Representative images show that P1AG5459 induces HEV differentiation and CD8 invasion in tumors, but not in surrounding normal colon tissue. [Figure 34] Figure 34 shows the development of tertiary lymphoid tissue-like structures (TLS) in orthotopic CRC-carrying mice treated with P1AG5459. Ki67 staining of the entire section allowed for the identification of the tumor margin, marked by the dashed line. B220 (B cell) staining of the entire section highlighted B cell aggregates, which are magnified in subsequent images. In the magnified images, the cellular composition of the TLS is characterized in more detail, including pNAD / Meca79+ high endothelial venules, CD11c+ bone marrow cells, germinal center-like B220+ Ki67+ proliferating cells, and CD8 and CD4 T cells, some of which co-express the activation marker PD1 and the stem cell marker TCF1. [Figure 35A-B]Figures 35A and 35B compare FAP-LTBR bispecific antigen-binding molecules containing different LTBR antigen-binding domains in their ability to upregulate ICAM on endothelial cells in a FAP-dependent manner. The median fluorescence intensity of ICAM on CD31+ HUVEC cells co-cultured with FAP-overexpressing NIH-3T3 cells (Figure 35A) or FAP-nonexpressing NIH-3T3 cells (Figure 35B) is shown. Data are normalized to untreated control ICAM expression. [Figure 36A-C] Figures 36A–36C compare the ability of FAP-LTBR bispecific antigen-binding molecules containing different LTBR antigen-binding domains to bind to human (Figure 36A), cynomolgus monkey (Figure 36B), and mouse LTBR (Figure 36C) on CHO-K1 cells engineered to overexpress human, cynomolgus monkey, or mouse LTBR. The median fluorescence intensity of anti-human Fc secondary antibodies detecting bound FAP-LTBR bispecific molecules on cells overexpressing human LTBR (Figure 36A), cynomolgus monkey LTBR (Figure 36B), or mouse LTBR (Figure 36C) is shown. Data are normalized to baseline. [Figure 37A-E]Figures 37A-37E show schematic diagrams of anti-LTBR IgG antibodies and bispecific FAP-LTBR antibodies. Figure 37A shows a schematic diagram of a monospecific anti-LTBR human IgG1 PG LALA antibody (BHA10, P1AH0119). Figure 37B shows a schematic diagram of a bispecific 2+1 anti-LTBR / anti-FAP(4B9) antibody as human IgG1 PG LALA having two N-terminal Fab arms specific to LTBR(CBE11) and C-terminal anti-FAP VL and VH domains of clone 4B9 (P1AE1079). Figure 37C shows a schematic diagram of a 1+1 anti-LTBR(BHA10) / anti-FAP(4B9) bispecific antibody (P1AH5884) as human IgG1 PG LALA crossMab having cross-VL / VH domains on the anti-LTBR Fab arms and a charge in the CH1 / Ck domain of the anti-Fab arms. Figure 37D shows a schematic diagram of the 2+1 anti-LTBR(BHA10) / anti-FAP(4B9) bispecific antibody (P1AH5885) as a human IgG1 PG LALA crossMab, having cross-V domains in both anti-LTBR Fab arms and a charge in the CH1 / Ck domain of the anti-Fab arm fused to the C-terminus of the Fc domain (Fc knob chain). Figure 37E shows a schematic diagram of the 2+1 anti-LTBR(P1AE9459) / anti-FAP(212) bispecific antibody (P1AH5886) as a human IgG1 PG LALA crossMab, having cross-V domains in both anti-LTBR Fab arms and a charge in the CH1 / Ck domain of the anti-Fab arm fused to the C-terminus of the Fc domain (Fc knob chain). [Figure 38A]Figures 38A–38D show the deuteration difference maps between bound human LTBR and uncomplexed LTBR. The deuteration difference maps show the primary sequence of human LTBR (P1AH2680) and compare the deuteration difference between human LTBR + antibody / ligand and LTBR without antibody / ligand for five different labeling times: 15 seconds, 1 minute, 10 minutes, 60 minutes, and 300 minutes. The bars above the sequence represent the peptide. The boxes indicate the recommended epitopes for each antibody / ligand. Below, the proposed epitopes are mapped to the alphafold model AF-P36941-F1-model_v2 from S28 to M227 (black region). The proposed epitopes on human LTBR for P1AE9459 (Figure 38A), P1AE1873 (Figure 38B), P1AH0119 (Figure 38C), and P1AE1235 (Figure 38D, ligand) are shown. [Figure 38B] Figures 38A–38D show the deuteration difference maps between bound human LTBR and uncomplexed LTBR. The deuteration difference maps show the primary sequence of human LTBR (P1AH2680) and compare the deuteration difference between human LTBR + antibody / ligand and LTBR without antibody / ligand for five different labeling times: 15 seconds, 1 minute, 10 minutes, 60 minutes, and 300 minutes. The bars above the sequence represent the peptide. The boxes indicate the recommended epitopes for each antibody / ligand. Below, the proposed epitopes are mapped to the alphafold model AF-P36941-F1-model_v2 from S28 to M227 (black region). The proposed epitopes on human LTBR for P1AE9459 (Figure 38A), P1AE1873 (Figure 38B), P1AH0119 (Figure 38C), and P1AE1235 (Figure 38D, ligand) are shown. [Figure 38C]Figures 38A–38D show the deuteration difference maps between bound human LTBR and uncomplexed LTBR. The deuteration difference maps show the primary sequence of human LTBR (P1AH2680) and compare the deuteration difference between human LTBR + antibody / ligand and LTBR without antibody / ligand for five different labeling times: 15 seconds, 1 minute, 10 minutes, 60 minutes, and 300 minutes. The bars above the sequence represent the peptide. The boxes indicate the recommended epitopes for each antibody / ligand. Below, the proposed epitopes are mapped to the alphafold model AF-P36941-F1-model_v2 from S28 to M227 (black region). The proposed epitopes on human LTBR for P1AE9459 (Figure 38A), P1AE1873 (Figure 38B), P1AH0119 (Figure 38C), and P1AE1235 (Figure 38D, ligand) are shown. [Figure 38D] Figures 38A–38D show the deuteration difference maps between bound human LTBR and uncomplexed LTBR. The deuteration difference maps show the primary sequence of human LTBR (P1AH2680) and compare the deuteration difference between human LTBR + antibody / ligand and LTBR without antibody / ligand for five different labeling times: 15 seconds, 1 minute, 10 minutes, 60 minutes, and 300 minutes. The bars above the sequence represent the peptide. The boxes indicate the recommended epitopes for each antibody / ligand. Below, the proposed epitopes are mapped to the alphafold model AF-P36941-F1-model_v2 from S28 to M227 (black region). The proposed epitopes on human LTBR for P1AE9459 (Figure 38A), P1AE1873 (Figure 38B), P1AH0119 (Figure 38C), and P1AE1235 (Figure 38D, ligand) are shown. [Modes for carrying out the invention]

[0051] definition Unless otherwise defined, technical and scientific terms used herein have the same meanings as they are commonly used in the art to which this invention pertains. For the purposes of interpreting this specification, the following definitions apply, and wherever appropriate, a term used in the singular also includes the plural, and conversely, a term used in the plural also includes the singular.

[0052] As used herein, the terms “antigen-binding molecule” and “antibody” are interchangeable and, in their broadest sense, refer to molecules that specifically bind to an antigenic determinant. Examples of antigen-binding molecules include antibodies, bispecific or multispecific antibodies, antibody fragments, and scaffold antigen-binding proteins. The term “antibody” as used herein is in its broadest sense and encompasses, but is not limited to, a wide range of antibody structures, including monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0053] As used herein, the terms “antigen-binding domain capable of specifically binding to a target cell antigen” or “a portion capable of specifically binding to a target cell antigen” refer to a polypeptide molecule that specifically binds to an antigen. In one embodiment, an antigen-binding domain can activate signal transduction via its target cell antigen. In certain embodiments, an antigen-binding domain can guide the entity to which it binds (e.g., an LTBR agonist antibody) to a target site, such as a specific type of tumor cell or tumor stroma having 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 design repeat proteins or design repeat domains (see, for example, International Publication No. 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-activating protein (FAP). In relation to an antibody or a fragment thereof, the term “antigen-binding domain having specific binding ability to target cell antigens” refers to a portion of a molecule containing a region that specifically binds to and is complementary to some or all of the antigen. Antigen-binding domains capable of specific antigen binding may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Specifically, an antigen-binding domain capable of specific antigen binding includes the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH) of an antibody. In another embodiment, an “antigen-binding domain capable of specifically binding to target cell antigens” may also be a Fab fragment or a cross-Fab fragment.

[0054] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous collection of antibodies, i.e., the individual antibodies in the collection are identical and / or bind to the same epitope, except for possible variant antibodies, including, for example, naturally occurring mutations or mutations that occur during the production of monoclonal antibody preparations, such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on an antigen.

[0055] As used herein, the term “monospecific” antibody means an antibody having one or more binding sites that each bind to the same epitope of the same antigen. The term “bispecific” means that an antigen-binding molecule can specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule comprises two antigen-binding sites, each of which is specific to a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells. Furthermore, the bispecific antigen-binding molecules described herein may also form part of a multispecific antibody.

[0056] As used in this application, the term "~valent" indicates that an antigen-binding molecule that is specific to one antigenic determinant contains a specific number of binding sites that are specific to that antigenic determinant. Therefore, the terms "divalent," "tetravalent," and "hexavalent" indicate that an antigen-binding molecule contains two, four, and six binding sites, respectively, that are specific to a particular antigenic determinant. In certain aspects of the present invention, the bispecific antigen-binding molecule according to the present invention may be monovalent to a particular antigenic determinant (meaning it has only one binding site for that antigenic determinant), or it may be divalent or tetravalent to a particular antigenic determinant (meaning it has two or four binding sites, respectively, for that antigenic determinant).

[0057] 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 naturally occurring antibody. “Natural antibody” refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, a natural IgG class antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two disulfide-linked 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 chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody may be assigned to one of five types called α(IgA), δ(IgD), ε(IgE), γ(IgG), or μ(IgM), some of which may be further classified into subtypes such as γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), α1(IgA1), and α2(IgA2). The light chain of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain.

[0058] An "antibody fragment" refers to a molecule other than an intact antibody, containing a portion of an intact antibody that binds to an 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 specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For an overview of scFv fragments, see, for example, Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994). See also International Publication No. 93 / 16185 and U.S. Patents No. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting extended in vivo half-lives, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments having two antigen-binding sites that may be bivalent or bispecific. See, for example, EP404097; International Publication No. 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). A single-domain antibody is an antibody fragment that includes 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, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).Antibody fragments can 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., Escherichia coli or phages), as described herein.

[0059] Papain digestion of an intact antibody yields two identical antigen-binding fragments, called “Fab” fragments, each containing a heavy-chain variable domain and a light-chain variable domain, as well as a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term “Fab fragment” refers to a light-chain fragment containing the VL domain and constant domain of the light chain (CL), and an antibody fragment containing the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxyl terminus of the heavy-chain CH1 domain, including one or more cysteines derived from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue of the constant domain has a free thiol group. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. According to the present invention, the term “Fab fragment” also includes “crossosFab fragments” or “crossover Fab fragments” as defined below.

[0060] The terms "cross-Fab fragment," "xFab fragment," or "crossover Fab fragment" refer to Fab fragments in which either the variable or constant regions of the heavy and light chains are exchanged. Two possible chain compositions of the crossover Fab molecule are possible and are included in the bispecific antibodies of the present invention. In one case, the variable regions of the Fab heavy and light chains are exchanged, i.e., the crossover Fab molecule includes 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)It is also called [another name]. On the other hand, when the constant regions of the Fab heavy chain and light chain are exchanged, the crossover Fab molecule includes a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL), and a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1). This crossover Fab molecule is called CrossFab. (CLCH1) It is also called [another name].

[0061] A "single-chain Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences from the N-terminus to the 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 domain and the CH1 domain. In addition, these single-chain Fab molecules may be further stabilized by the formation of interchain disulfide bonds through the insertion of cysteine ​​residues (for example, at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering).

[0062] A "crossover single-chain Fab fragment" or "x-scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences from N-terminus to C-terminus: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; VH and VL together form an antigen-binding site that specifically binds to a certain 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 interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., position 44 of the variable heavy chain and position 100 of the variable light chain according to Kabat numbering).

[0063] A "single-chain variable fragment (scFv)" is a linkage of the antibody's heavy chain (V) using a short linker peptide of 10 to approximately 25 amino acids. H ) and light chain (V L It is a fusion protein of the variable region of ). The linker is usually rich in glycine for flexibility and also rich in serine or threonine for solubility, V H The N-terminus of V L It can be linked to the C-terminus and vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, the antibody fragment contains a single-chain polypeptide characteristic of the VH domain (i.e., capable of assembling together with the VL domain), or characteristic of the VL domain (i.e., capable of assembling together with the VH domain at a functional antigen-binding site), thereby conferring the antigen-binding properties of a full-length antibody.

[0064] "Scaffold antigen-binding proteins" are known in this art, and for example, fibronectin and engineered ankyrin repeat proteins (DARPin) 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 aspect of the present invention, the scaffold antigen-binding protein is CTLA-4 (epibody), lipocalin (anticalin), protein A-derived molecules, such as the Z-domain (afibody), A-domain (avimer / maxibody), serum transferrin (transbody); designed ankyrin repeat protein (DARPin), variable domain of antibody light or heavy chain (single-domain antibody, sdAb), variable domain of antibody heavy chain (nanobody, aVH), V NAR Fragment, fibronectin (adonectin), C-type lectin domain (tetranectin); variable domain (V) of novel antigen receptor beta-lactamase NAR Fragments), human γ-crystallin or ubiquitin (affiliin molecules); selected from the group consisting of Knitz-type domains and microbodies of human protease inhibitors, such as Nottin family proteins, peptide aptamers and fibronectin (adonectin). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is mainly CD4 +CTLA-4 is a CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig folding. The loop corresponding to the antibody's CDR may be substituted with heterologous sequences to give different binding properties. CTLA-4 molecules designed to have different binding specificities are also known as organisms (e.g., U.S. Patent No. 7166697B1). Organisms are approximately the same size as the isolated variable region of an antibody (e.g., a domain antibody). For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipokalin is a family of extracellular proteins that carry small hydrophobic molecules such as steroids, pyrine, retinoids, and lipids. Lipokalin has a rigid beta-sheet secondary structure with many loops at the open end of a conical structure, which can be manipulated to bind to different target antigens. Antikarin is 160-180 amino acids in size and is derived from lipokalin. For further details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Patent No. 7,250297 B1, and U.S. Patent Application Publication No. 20070224633. Affibodies are scaffolds derived from protein A of Staphylococcus aureus that can be manipulated to bind to antigens. The domain consists 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 EP1641818A1. Avimers are multi-domain proteins derived from the A-domain scaffold family. The native domain of approximately 35 amino acids fits into a defined disulfide-bonded structure. Diversity is created by shuffling the native variations shown 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 manipulated to bind to different target antigens by inserting peptide sequences into a permissible surface loop. An example of a manipulated transferrin scaffold is a transbody. For further details, see J. Biol. Chem 274, 24066-24073 (1999). The engineered ankyrin repeat protein (DARPin) is derived from ankyrin, a family of proteins that mediate the attachment of intrinsic membrane proteins of the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha helices and a beta-turn. A single ankyrin repeat can be manipulated to bind to different target antigens by randomizing residues in the first alpha helix and beta-turn of each repeat. Its 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 U.S. Patent Application Publication No. 20040132028A1. A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of a camel-derived antibody heavy chain (nanobody or V). H (H fragment). Furthermore, the term single-domain antibody refers to autonomous human heavy chain variable domains (aVH) or shark-derived V NARThis includes fragments. Fibronectin is a scaffold that can be manipulated to bind to antigens. Adnectin consists of a scaffold with the native amino acid sequence of the 10th domain of 15 repeat units of human fibronectin type III (FN3). Three loops at one end of the beta-sandwich can be manipulated so that adnectin can specifically recognize the therapeutic target of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent Application Publication No. 20080139791, International Publication No. 2005056764, and U.S. Patent No. 6818418B1. 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). The microbodies are derived from naturally occurring microproteins 25–50 amino acid long containing 3–4 cysteine ​​crosslinks, examples of which include KalataBI, conotoxin, and Nottin. The microproteins have loops that can be manipulated to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on the manipulated Nottin domain, see International Publication No. 2008098796.

[0065] An antibody that "binds to the same epitope as the 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, and conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay. An antibody that "does not bind to the same epitope as the 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, and 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.

[0066] The term "antigen-binding domain" or "antigen-binding site" refers to a portion of an antibody that specifically binds to part or all of an antigen and contains a region complementary to part or all of the antigen. If the antigen is large, the antibody may bind to only a specific portion of the antigen, which is called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0067] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope,” and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds, forming an antigen-antigen complex (e.g., a contiguous extension of amino acids or a conformational structure composed of discontinuous amino acids from different regions). Useful antigenic determinants may be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, proteins useful as antigens herein may be any native form of 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. Where a particular protein is referred to herein, the term includes not only “full-length,” unprocessed proteins but also any type of protein resulting from intracellular processing. The term also includes naturally occurring protein variants, e.g., splice variants or allele variants.

[0068] "Specific binding" means that the binding is selective for the antigen and can be distinguished from undesirable or nonspecific interactions. The binding ability of an antigen-binding molecule to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art (e.g., surface plasmon resonance (SPR) technique (analysis using a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and classical binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the binding of the antigen-binding molecule to the antigen, as measured, for example, by SPR. In certain embodiments, the molecule that binds to the antigen has 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, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) is the answer.

[0069] "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 pair (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of the binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constants (koff and kon, respectively). Thus, equivalent affinities may include different rate constants, as long as the ratio of the rate constants is the same. Affinity can be measured by common methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0070] An "affinity-mature" antibody is an antibody that has one or more modifications in one or more hypervariable regions (HVRs) compared to an unmodified parent antibody, and such modifications result in an improved affinity of the antibody for the antigen.

[0071] As used herein, "target cell antigen" refers to the antigenic determinants presented on the surface of target cells, particularly target cells within a tumor, such as cancer cells or tumor stromal cells. Therefore, the target cell antigen is a tumor-associated antigen. Specifically, the "tumor-associated antigen" or TAA is a fibroblast-activating protein (FAP).

[0072] The term “fibroblast-activating protein (FAP),” also known as prolyl endopeptidase FAP or seplacase (EC3.4.21), refers, unless otherwise specified, to any natural 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). This term encompasses “full-length” unprocessed FAP, as well as any form of FAP resulting from intracellular processing. This term also encompasses naturally occurring variants of FAP, such as splice variants or allele variants. In one embodiment, the antigen-binding molecule of the present invention can specifically bind 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 Avi-His tagged human FAP is shown in SEQ ID NO: 264. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 126, SEQ ID NO: 282) or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. SEQ ID NO: 265 shows the amino acids of Avi-His tagged mouse FAP. Preferably, the anti-FAP binding molecule of the present invention binds to the extracellular domain of FAP.

[0073] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain involved in the binding of antigen-binding molecules 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 (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th, WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0074] As used herein, the terms “hypervariable region” or “HVR” refer to each of the regions of an antibody variable domain that are hypervariable within a sequence and determine antigen-binding specificity, such as “complementarity-determining regions” (CDRs).

[0075] Generally, an antibody contains six CDRs, three located in the VH (CDR-H1, CDR-H2, CDR-H3) and three located in the VL (CDR-L1, CDR-L2, CDR-L3). Examples of CDRs used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact sites 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)).

[0076] Unless otherwise specified, the designation of the CDR shall be determined in accordance with Kabat et al. above. Those skilled in the art will understand that the designation of the CDR may also be determined in accordance with Chothia, McCallum, or any other scientifically recognized nomenclature above.

[0077] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0078] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining portion of the heavy chain and / or light chain originates from a different source or species.

[0079] The "class" of an antibody refers to the type of constant domain or constant region in its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes) IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0080] A “humanized” antibody refers to a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the HVR (e.g., CDR) corresponds to a non-human antibody and all or substantially all of the FR corresponds to a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. A “humanized” antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. Other forms of “humanized antibodies” as encompassed in the present invention are those in which the constant region has been further modified or altered from the constant region of the original antibody to produce the properties according to the present invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0081] The term "CH1 domain" refers to the portion of the antibody heavy chain polypeptide extending approximately from EU position 118 to EU position 215 (Kabat's EU numbering system). In one embodiment, the CH1 domain has the amino acid sequence ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 283). Typically, a segment having the amino acid sequence EPKSC (SEQ ID NO: 284) follows to ligate the CH1 domain to the hinge region; however, in the case of a CH1 domain with a free C-terminus (e.g., a crossfab fragment), this segment may also consist of the amino acid sequence EPKSCD (SEQ ID NO: 285) or EPKSCS (SEQ ID NO: 286).

[0082] The term "hinge region" refers to the portion of the antibody heavy chain polypeptide that binds the CH1 and CH2 domains in the wild-type antibody heavy chain (e.g., from approximately position 216 to approximately position 230, or from approximately position 226 to approximately position 230, according to the Kabat EU numbering system). Hinge regions of other IgG subclasses can be determined by aligning them with the hinge region cysteine ​​residues of the IgG1 subclass sequence. The hinge region is typically a dimeric molecule consisting of two polypeptides having identical amino acid sequences. The hinge region generally contains up to 25 amino acid residues, is flexible, and allows the associated target binding site to move independently. The hinge region can be subdivided into three domains: upper, middle, and lower hinge domains (see, for example, Roux, et al., J.Immunol. 161 (1998) 4083). In one embodiment, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 287), where X is either S or P. In another embodiment, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO: 288), where X is either S or P. In yet another embodiment, the hinge region has the amino acid sequence CPXCP (SEQ ID NO: 289), where X is either S or P.

[0083] In this specification, the terms “Fc domain” or “Fc region” are used to define the C-terminal region of an antibody heavy chain, including at least a portion of the constant region. This term includes the native sequence Fc region and the variant Fc region. The IgG Fc region includes the IgG CH2 and IgG CH3 domains. Typically, the “CH2 domain” of the human IgG Fc region extends from approximately amino acid residue 231 to approximately amino acid residue 340 (Kabat EU numbering system). In one embodiment, the CH2 domain having the amino acid sequence APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 290) 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 the intact native Fc region. It has been hypothesized that carbohydrates may provide an alternative for domain-domain pairing and help stabilize the CH2 domain. Burton, Mol.Immunol.22(1985)161-206. In one embodiment, a carbohydrate chain is bound to the CH2 domain. The CH2 domain as used herein may be either a natural sequence CH2 domain or a variant CH2 domain. The "CH3 domain" includes a C-terminal stretch of residues relative to the CH2 domain in the Fc region (i.e., from amino acid residue approximately 341 to approximately 447, according to the Kabat EU numbering system for IgG). In one embodiment, the CH3 domain has the amino acid sequence LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 291).The CH3 region as used herein may be either a native sequence CH3 domain or a variant CH3 domain (for example, a CH3 domain in which a “nob” is introduced on one chain and a “hole” corresponding to the other chain is introduced; see U.S. Patent 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 as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the C-terminal lysine (Lys447) of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of It follows the EU numbering system (also known as the EU Index), as described in Health, Bethesda, MD, 1991.

[0084] The term "wild-type Fc domain" refers to an amino acid sequence identical to that of an Fc domain found in nature. Examples of wild-type human Fc domains include the natural human IgG1 Fc region (non-A and A allotypes), the natural human IgG2 Fc region, the natural human IgG3 Fc region, and the natural human IgG4 Fc region, as well as their naturally occurring variants. Wild-type Fc regions are shown in SEQ ID NO: 155 (IgG1, Caucasian allotype), SEQ ID NO: 156 (IgG1, African American allotype), SEQ ID NO: 157 (IgG2), SEQ ID NO: 158 (IgG3), and SEQ ID NO: 159 (IgG4). The term "variant (human) Fc domain" refers to an amino acid sequence that differs from the "wild-type" (human) Fc domain amino acid sequence due to at least one "amino acid mutation." In one embodiment, the variant Fc region has at least one amino acid mutation compared to the natural Fc region, for example, about 1 to about 10 amino acid mutations, and in one embodiment, about 1 to about 5 amino acid mutations in the natural Fc region. In one embodiment, the (variant)Fc region has at least about 95% homology to the wild-type Fc region.

[0085] The “knob-into-hole” technique 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). Typically, this method involves promoting heterodimerization and inhibiting homodimerization by introducing a bump ("knob") at the contact surface of a first polypeptide and a corresponding cavity at the contact surface of a second polypeptide, respectively, so that the bump is located within a corresponding cavity. The bump is constructed by substituting a smaller amino acid side chain (e.g., tyrosine or tryptophan) from the contact surface of the first polypeptide. A complementary cavity of the same or similar size as the bump is created at the interface of the second polypeptide by substituting a larger amino acid side chain (e.g., alanine or threonine) for a smaller amino acid side chain. The bumps and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In specific embodiments, the knob modification includes the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification includes the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In even more specific embodiments, the subunit of the Fc domain containing the knob modification further includes the amino acid substitution S354C, and the subunit of the Fc domain containing the hole modification further includes the amino acid substitution Y349C. The introduction of these two cysteine ​​residues forms a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0086] The “region corresponding to the Fc region of immunoglobulins” is intended to include naturally occurring allelic variants of the Fc region of immunoglobulins, as well as variants that produce substitutions, additions, or deletions but have modifications that do not substantially reduce the effector function of the immunoglobulin (e.g., antibody-dependent cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of immunoglobulins without substantially impairing biological function. Such variants may be selected according to general rules known in the art to have minimal impact on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0087] The term "effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0088] Fc receptor-binding-dependent effector function can be mediated by the interaction between the Fc region of an antibody and the Fc receptor (FcR), a specialized cell surface receptor on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the phagocytosis of antibody-coated pathogens by immune complexes and the lysis of corresponding antibody-coated red blood cells and various other cellular targets (e.g., tumor cells) via antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, J. Gand Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: the Fc receptor for IgG antibodies is called FcγR. Fc receptor binding is described, for example, in Ravetch, J. V. and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492, Capel, P. J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76 (1998) 231-248.

[0089] Cross-linking of the Fc region of IgG antibodies with receptors (FcγRs) induces a variety of effector functions, including phagocytosis, antibody-dependent cell-mediated cytotoxicity, release of inflammatory mediators, and regulation of immune complex clearance and antibody production. In humans, three classes of FcγRs are characterized as follows: -FcγRI(CD64) binds to monomeric IgG with high affinity and is expressed in macrophages, monocytes, neutrophils, and eosinophils. Modification of at least one amino acid residue in the Fc region of IgG, specifically E233-G236, P238, D265, N297, A327, and P329 (numbered according to the Kabat EU index), reduces binding to FcγRI. IgG2 residues at positions 233-236, substituted with IgG1 and IgG4, reduce binding to FcγRI by 10%. 3This reduced the response by a factor of two and eliminated the human monocyte response to antibody-sensitized erythrocytes (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624).

[0090] -FcγRII(CD32) binds to complexed IgG with moderate to low affinity and is widely expressed. This receptor can be divided into two subtypes: FcγRIIA and FcγRIIB. FcγRIIA is found in many cells involved in cell death (e.g., macrophages, monocytes, neutrophils) and is thought to be able to activate the cell death process. FcγRIIB appears to play a role in the inhibitory process and is found in B cells, macrophages, as well as mast cells and eosinophils. On B cells, FcγRIIB appears to function to suppress the production of further immunoglobulins and isotype switching to, for example, the IgE class. On macrophages, FcγRIIB works to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B form may help suppress the activation of these cells via IgE bound to its other receptor. Reduced binding to FcγRIIA is observed, for example, in antibodies containing an IgG Fc region with mutations in at least one amino acid residue E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbered according to the Kabat EU index).

[0091] -FcγRIII(CD16) binds to IgG with moderate to low affinity and exists in two forms. FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Reduced binding to FcγRIIIA is observed, for example, with antibodies containing an IgG Fc region with a mutation in at least 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 (numbered according to Kabat's EU index).

[0092] The mapping of the binding site for the Fc receptor on human IgG1, the aforementioned mutation sites, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604.

[0093] The term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a function mediated by Fc receptor binding, and the lysis of target cells by the antibodies reported herein in the presence of effector cells. The ability of antibodies to induce the initial stages of ADCC is investigated by measuring the binding of antibodies to Fcγ receptor-expressing cells, such as FcγRI and / or FcγRIIA or recombinant NK cells (which essentially express FcγRIIIA). In particular, binding to FcγR on NK cells is measured.

[0094] An "activated Fc receptor" is an Fc receptor that, following involvement by the Fc region of an antibody, triggers a signaling event that stimulates receptor-hosting cells to perform effector functions. Examples of activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activated Fc receptor is human FcγRIIIa (see UniProt accession number P08637, version 141).

[0095] As used herein, the term “LTBR” refers to any native lymphotoxin beta receptor (LTBR) derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses “full-length” unprocessed LTBR, ​​as well as any form of LTBR resulting from intracellular processing. The term also encompasses spontaneous variants of LTBR, ​​such as splice variants or allele variants. The amino acid sequence of an exemplary human LTBR is shown in SEQ ID NO: 1 (Uniprot number P36941), and the amino acid sequence of an exemplary mouse LTBR is shown in SEQ ID NO: 297 (Uniprot number B2RRV3). This receptor is expressed on the cell surface of the parenchyma and stroma of most lymphoid organs, but is absent in T lymphocytes and B lymphocytes. LTBR is also known as “tumor necrosis factor receptor superfamily member 3 (TNFRSF3).” Signal transduction via LTBR by the LTα / β heterotrimer (LTα1β2) is important in the development of the lymphoid system. LTBR is also known to bind to the ligand LIGHT (TNFSF14). While LTα1β2 is specific to LTBR, ​​LIGHT also binds to and activates HVEM (TNFSF14), a receptor expressed on immune cells that is involved in the regulation of immune cells.

[0096] As used herein, the term “LTBR agonist” includes any portion that stimulates the interaction between LTBR and its ligand. In this context, LTBR preferably refers to human LTBR, ​​and therefore, an LTBR agonist is preferably an agonist of human LTBR (SEQ ID NO: 1). Typically, this portion would be an agonist LTBR antibody or antibody fragment.

[0097] The terms “anti-LTBR antibody,” “anti-LTBR,” “LTBR antibody,” and “antibody that specifically binds to LTBR” refer to antibodies that specifically bind to LTBR with sufficient affinity to be useful as diagnostic and / or therapeutic agents targeting LTBR. In one embodiment, the degree of binding of the anti-LTBR antibody to unrelated non-LTBR proteins is less than about 10% of the binding of the antibody to LTBR, ​​as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS). In certain embodiments, the antibody that binds to LTBR has a molecular weight 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, for example, 10 -68 ~10 -13 M, for example 10 -8 M~10 -10 The dissociation constant (K) of M D ) has.

[0098] The term "peptide linker" refers to a peptide containing one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. A suitable non-immunogenic linker peptide is, for example, (G4S) n (SG4) n Or G4 (SG4) nIt is a peptide linker, where "n" is generally a number from 1 to 10, typically 2 to 4, especially 2, that is, the peptide is selected from the group consisting of GGGGS (SEQ ID NO: 298), GGGGSGGGGS (SEQ ID NO: 299), SGGGGSGGGG (SEQ ID NO: 300), and GGGGSGGGGSGGGG (SEQ ID NO: 301), but also includes sequences GSPGSSSSGS (SEQ ID NO: 302), (G4S)3 (SEQ ID NO: 303), (G4S)4 (SEQ ID NO: 304), GSGSGSGS (SEQ ID NO: 305), GSGSGNGS (SEQ ID NO: 306), GGSGSGSG (SEQ ID NO: 307), GGSGSG (SEQ ID NO: 308), GGSG (SEQ ID NO: 309), GGSGNGSG (SEQ ID NO: 310), GGNGSGSG (SEQ ID NO: 311), and GGNGSG (SEQ ID NO: 312). Particularly interesting peptide linkers are (G4S)(SEQ ID NO: 298), (G4S)2 or GGGGSGGGGS(SEQ ID NO: 299), (G4S)3(SEQ ID NO: 303), and (G4S)4(SEQ ID NO: 304).

[0099] As used herein, the term "amino acid" refers to the group of naturally occurring carboxy-α-amino acids, including alanine (three-letter abbreviation: ala, one-letter abbreviation: 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).

[0100] "Fused" or "linked" means that the constituent components (e.g., the antibody heavy chain and Fab fragment) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0101] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or FASTA program packages. Those skilled in the art can determine appropriate parameters for performing sequence alignment, including any algorithm necessary to achieve the maximum alignment relative to the full length of the sequences being compared. Alternatively, the identity percentage value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the U.S. Copyright Office (Washington DC, 20559), registered under U.S. Copyright Registration No. TXU510087, and described in International Publication No. 2001 / 007611. Unless otherwise indicated, for the purposes of this specification, amino acid sequence identity percentage values ​​are generated using the ggsearch program in FASTA package version 36.3.8c, or the subsequent BLOSUM50 comparison matrix.The FASTA program package was created by WRPearson and DJLipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; WRPearson (1996), “Effective protein sequence comparison”, Meth.Enzymol.266:227-258; and Pearson et al. (1997), Genomics 46:24-36, and is available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, you can use the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi to perform a global alignment (not local) and compare sequences using the ggsearch(global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup=2). The amino acid identity percentage is provided in the output alignment header.

[0102] In certain embodiments, amino acid sequence variants of bispecific antigen-binding molecules provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of TNF ligand trimer-containing antigen-binding molecules. Amino acid sequence variants of TNF ligand trimer-containing antigen-binding molecules can be prepared by introducing appropriate modifications to 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 in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to reach the final construct, insofar as the final construct has the desired characteristics (e.g., antigen binding). Sites targeted for substitutional mutagenesis include HVR and framework (FR). Conservative substitutions are given 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 can be introduced into the target molecule, and the resulting product can be screened for desired activity, such as retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. TIFF0007862562000001.tif161170

[0103] Amino acids can be grouped according to their common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0104] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0105] The term “amino acid sequence variant” includes substantial variants in which amino acid substitutions exist in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized antibody or human antibody). Generally, the variant(s) selected for further testing will have modifications (e.g., improvements) (e.g., increased affinity, decreased immunogenicity) of specific biological properties compared to the parent antigen-binding molecule, and / or substantially retain the specific biological properties of the parent antigen-binding molecule. Exemplary substitution variants are affinity-matured antibodies, which can be readily generated, for example, using affinity maturation techniques based on phage display, as described herein. Briefly, they are variant antigen-binding molecules in which one or more CDR residues are mutated, phage-displayed, and screened for specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such modifications 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 to the CDR. 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, residues or target residue groups (e.g., charged residues such as Arg, Asp, His, Lys, Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition to this, the crystal structure of the antigen-antigen binding molecule complex is used to identify contact sites between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or excluded as candidates for substitution. Variants may be screened to determine whether they possess the desired properties.

[0106] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is the bispecific antigen-binding molecule of the present invention having an N-terminal methionyl residue. Other insertion variants of the molecule include the fusion of a polypeptide to the N-terminus or C-terminus that increases the serum half-life of the bispecific antigen-binding molecule.

[0107] In certain embodiments, the bispecific antigen-binding molecules provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Glycosylated variants of the molecule can be conveniently obtained by modifying the amino acid sequence so that one or more glycosylation sites are created or removed. If the TNF ligand trimer-containing antigen-binding molecule contains an Fc region, the carbohydrate that binds to it may be modified. Natural antibodies produced by mammalian cells typically contain branched, bibranched oligosaccharides bound by an N-bond to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc at the "stem" of the bibranched oligosaccharide structure. In some embodiments, the oligosaccharide may be modified within the TNF family ligand trimer-containing antigen-binding molecule to produce variants having specific improved properties. In one embodiment, a variant of the bispecific antigen-binding molecule or antibody of the present invention is provided, having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. Such a fucosylated variant may have improved ADCC function; see, for example, U.S. Patent Application Publication 2003 / 0157108 (Presta, L.) or U.S. Patent Application Publication 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). In another embodiment, a variant of the bispecific antigen-binding molecule or antibody of the present invention is provided, for example, comprising a bifurcated oligosaccharide in which the branched oligosaccharide bound to the Fc region is bifurcated by GlcNAc. Such a variant may have reduced fucosylation and / or improved ADCC function. See, for example, International Publication No. 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6602684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide bound to the Fc domain are also provided.Such antibody variants possess improved CDC function and are described, for example, in International Publication No. 1997 / 30087 (Patel et al.); International Publication No. 1998 / 58964 (Raju, S.); and International Publication No. 1999 / 22764 (Raju, S.).

[0108] In certain embodiments, it may be desirable to create cysteine-modified variants of the bispecific antigen-binding molecule of the present invention, for example, "thioMAb" in which one or more residues of the molecule are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the molecule. By substituting these residues with cysteine, a reactive thiol group is positioned at an accessible site on the antibody, and this reactive thiol group can be used to conjugate the antibody to other parts, such as the drug part or the linker-drug part, to create an immunoconjugate. In certain embodiments, one or more of the following residues can 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. The cysteine-modified antigen-binding molecule may be prepared, for example, as described in U.S. Patent No. 7,521,541.

[0109] The terms “nucleic acid” or “polynucleotide” include any compound and / or substance containing polymers 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. Often, nucleic acid molecules are described by a sequence of bases, thereby representing the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. Here, the term nucleic acid molecule includes, for example, deoxyribonucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially 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 cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone links, or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo in a host or patient, for example. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may or may not be modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that the mRNA can be injected into a target for antibody production in vivo (see, for example, Stadler ert al, Nature Medicine 2017, doi:10.1038 / nm.4356, published online on 12 June 2017, or European Patent No. 2101823).

[0110] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found in cells containing nucleic acid molecules, but these nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0111] "Isolated nucleic acids encoding a bispecific antigen-binding molecule or antibody" means one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of a bispecific antigen-binding molecule or antibody, and such one or more nucleic acid molecules are contained in a single vector or separate vectors, and such one or more nucleic acid molecules are present in one or more locations within a host cell.

[0112] A nucleic acid or polynucleotide having a nucleotide sequence that is, for example, at least 95% "identical" to the reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference nucleotide 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, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. Such modifications to the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or at any position between these terminal positions, and may be individually scattered among the residues in the reference sequence or scattered within the reference sequence in one or more consecutive groups. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the present invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0113] The term "expression cassette" refers to a polynucleotide, generated by recombination or synthesis, comprising a set of specific nucleic acid elements that enable the transcription of a particular nucleic acid in target cells. 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 includes, in particular, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence or fragment thereof encoding the bispecific antigen-binding molecule of the present invention.

[0114] The terms “vector” or “expression vector” are synonymous with “expression construct” and refer to a DNA molecule used to introduce and induce the expression of a specific gene to which it operably binds within a target cell. This term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of the introduced host cell. The expression vector of the present invention comprises an expression cassette. The expression vector enables the transcription of large amounts of stable mRNA. Once the expression vector enters the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation mechanism. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence or fragment thereof encoding the bispecific antibody of the present invention.

[0115] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and also include the offspring of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. The offspring may not have exactly the same nucleic acid content as the parent cells and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the original transformed cells 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. Examples of host cells include cultured cells, e.g., mammalian cultured cells, e.g., to name just a few, 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, but also include cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues.

[0116] The "effective dose" of a drug refers to the amount of that drug required to cause a certain physiological change in the cells or tissues to which it is administered.

[0117] The "therapeutic effective dose" of a drug, such as a pharmaceutical composition, refers to the effective amount in the dosage and duration required to obtain the desired therapeutic or preventive outcome. For example, the therapeutic effective dose of a drug eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0118] The "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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.

[0119] The terms "pharmaceutical composition" or "pharmaceutical preparation" refer to a preparation in which the biological activity of the active ingredient contained therein is effective, and which does not contain any additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is to be administered.

[0120] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient that is non-toxic to the target. pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0121] The term "package insert" is used to refer to the instructions that are typically included in the product packaging of a therapeutic product, and include information on indications, usage, dosage, administration, combination therapies, contraindications and / or precautions regarding the use of the therapeutic product.

[0122] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of an individual being treated, and may be performed for preventive purposes or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and achieving remission or improving prognosis. In some embodiments, the molecules of the present invention are used to delay the onset of a disease or to slow the progression of a disease.

[0123] The term "cancer," as used herein, refers to lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, and central nervous system cancer. System:CNS) refers to proliferative disorders such as neoplasms, axial tumors of the spine, brainstem 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 combinations of one or more of the above cancers).

[0124] The term "chemotherapeutic agent," as used herein, refers to a chemical compound useful for the treatment of cancer. In one embodiment, the chemotherapeutic agent is an antimetabolite. In one embodiment, the antimetabolite is selected from the group consisting of aminopterin, methotrexate, pemetrexed, larcitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine, capecitabine, cytarabine, fluorouracil, floxuridine, and gemcitabine. In a particular embodiment, the antimetabolite is capecitabine or gemcitabine. In another embodiment, the antimetabolite is fluorouracil. In one embodiment, the chemotherapeutic agent is a drug that affects microtubule formation. In one embodiment, the drug 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.

[0125] Exemplary agonist LTBR antibody This specification provides novel antibodies and antibody fragments that specifically bind to the lymphotoxin beta receptor (LTBR). Novel antibodies and antibody fragments that specifically bind to an antigen containing the amino acid sequence of SEQ ID NO: 1 are provided. Therefore, these antibodies specifically bind to human LTBR. These are agonist hu LTBR antibodies.

[0126] These antibodies can bind to human LTBR and cynomolgus monkey LTBR with an affinity difference of less than twofold. Some of the novel agonist hu LTBR antibodies can also bind to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR.

[0127] In this specification, when measured by ELISA, EC 50Disclosed are agonist LTBR antibodies that bind to the human LTBR extracellular domain (ECD of the amino acid sequence of SEQ ID NO: 359) at a concentration of less than 4 nM (see Example 1.5). In one embodiment, the agonist LTBR antibody, when measured by ELISA, has an EC of less than 1 nM. 50 It binds to human LTBR ECD. In a particular embodiment, the agonist LTBR antibody has an EC2 concentration of less than 0.15 nM when measured by ELISA. 50 It binds to human LTBR ECD.

[0128] The agonist LTBR antibodies disclosed herein have an EC of less than 5 nM when measured by ELISA. 50 It binds to the extracellular domain of cynomolgus monkey LTBR (ECD of the amino acid sequence of SEQ ID NO: 361) (see Example 1.5). In one embodiment, the agonist LTBR antibody has an EC of less than 0.5 nM when measured by ELISA. 50 It binds to cynomolgus monkey LTBR ECD. In a particular embodiment, the agonist LTBR antibody has an EC of less than 0.1 nM when measured by ELISA. 50 It binds to cynomolgus macaque LTBR ECD.

[0129] The agonist LTBR antibodies described herein require crosslinking for agonist activity to activate human LTBR, ​​meaning they can only stimulate LTBR via a crosslinking-dependent mechanism. A "crosslinking-dependent mechanism" could be, for example, an Fc crosslinking-dependent mechanism where the antibody must bind to both LTBR and the Fc receptor in order to stimulate LTBR. Therefore, the antibody must be able to bind to both LTBR and the Fc receptor. If the antibody is crosslinking-independent, it can stimulate LTBR without binding to the Fc receptor. This could lead to widespread LTBR activation in the human body and associated serious safety issues.

[0130] The agonist LTBR antibodies described herein also require crosslinking for their agonist activity to induce upregulation of ICAM in human umbilical vein endothelial cells or cancer-associated fibroblasts, as shown in Example 5.2 herein.

[0131] The agonist LTBR antibodies described herein can also inhibit the interaction between human LTBR and its human ligands, lymphotoxin α1β2 and LIGHT. This has been demonstrated in the ligand competition experiment by ELISA described in Example 1.6.

[0132] The agonist LTBR antibodies described herein have been shown to compete for binding to human LIGHT, i.e., the natural ligand of LTBR containing the amino acid sequence of SEQ ID NO: 358, with hu LTBR.

[0133] In one embodiment, an agonist LTBR antibody (or an antigen-binding domain that specifically binds to LTBR) is provided herein, and the antibody or antigen-binding domain is The heavy chain complementarity-determining region (CDR-H1) contains the amino acid sequence of SEQ ID NO: 27, the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 28, and the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 37. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 40. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 61. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 67, CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 69. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 72. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77. HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L LTBR) Includes.

[0134] In one embodiment, an agonist LTBR antibody (or an antigen-binding domain that specifically binds to LTBR) is provided, and the antibody is The heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 33 H LTBR) and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 34 L LTBR), or The heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 41 H LTBR) and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 42 L LTBR), or The heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 49 H LTBR) and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 50 L LTBR), or The heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 57 HLTBR) and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 58 L LTBR), or The heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 65 H LTBR) and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 66 L LTBR), The heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 73 H LTBR) and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 74 L LTBR), The heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 81 H LTBR) and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 82 L LTBR), or The heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 89 H LTBR) and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 90 L LTBR), or The heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 97 H LTBR) and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 98 L LTBR) Includes.

[0135] In one embodiment, an agonist LTBR antibody (or an antigen-binding domain that specifically binds to LTBR) is provided, and the antibody or antigen-binding domain is Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V HLight chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 58 L LTBR), or Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 65 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 66 L LTBR), The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 73 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 74 L LTBR), The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR) Includes.

[0136] In a particular embodiment, the agonist LTBR antibody described herein is an agonist LTBR antibody that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR. In this specification, when measured by ELISA, EC 50 We disclose an agonist LTBR antibody that binds to the mouse LTBR extracellular domain (ECD of the amino acid sequence of SEQ ID NO: 360) at a concentration of less than 1 nM (see Example 1.5). In one embodiment, the agonist LTBR antibody has an EC of less than 0.15 nM when measured by ELISA. 50 It then binds to the mouse LTBR ECD.

[0137] In one embodiment, an agonist LTBR antibody (or antigen-binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR, The heavy chain complementarity-determining region (CDR-H1) contains the amino acid sequence of SEQ ID NO: 27, the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 28, and the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77. HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L LTBR) An agonist LTBR antibody (or antigen-binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR is provided.

[0138] In one embodiment, an agonist LTBR antibody (or antigen-binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98L LTBR) Includes.

[0139] In one specific embodiment, the agonist LTBR antibody described herein binds to the epitope region of SEQ ID NO: 351 on human LTBR. In one embodiment, the agonist LTBR antibody described herein binds to an epitope on human LTBR that overlaps with the epitope to which human lymphotoxin α1β2 binds. In one embodiment, the agonist LTBR antibody described herein binds to an epitope region on human LTBR that is different from that of the reference antibodies BHA10 and CBE11. This has been demonstrated by hydrogen / deuterium exchange (HDX) mass spectrometry, as described in Example 6.4.

[0140] In one embodiment, such an agonist LTBR antibody comprises a heavy chain variable region (V) including CDR-H1 containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L Includes LTBR). In one embodiment, a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L Includes LTBR (antibody P1AE9459).

[0141] In one embodiment, an agonist LTBR antibody (or an antigen-binding domain that specifically binds to LTBR) derived from immunization in transgenic rabbits is provided. In one embodiment, this antibody (or antigen-binding domain) (i) Heavy chain complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 29. HLight chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L LTBR), or (ii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 37 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 40. L LTBR), or (iii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR), or (iv) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56. L LTBR), or (v) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 61 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64.L LTBR), or (vi) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 67, CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 69 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 72. L LTBR) Includes.

[0142] In one embodiment, the agonist LTBR antibody (or antigen-binding domain) derived from transgenic rabbit immunization is a cross-reactive antibody or antigen-binding domain that specifically binds to human, cynomolgus monkey, and mouse LTBR. In one embodiment, the antibody or antigen-binding domain includes a heavy chain complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 29, and a heavy chain variable region (V H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L LTBR), or a heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L It includes LTBR). In one embodiment, the antibody or antigen-binding domain is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or the heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 49 HLight chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L Includes LTBR.

[0143] In another embodiment, a human LTBR antibody or antigen-binding domain derived from rat immunization is provided. In particular, this antibody or antigen-binding domain is a cross-reactive antibody or antigen-binding domain that specifically binds to human, cynomolgus monkey, and mouse LTBR. In one embodiment, the antibody or antigen-binding domain comprises a heavy chain variable region (V) including CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L It includes LTBR). In one embodiment, the antibody or antigen-binding domain is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 81. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L Includes LTBR.

[0144] In yet another embodiment, a human LTBR antibody or antigen-binding domain generated from a phage display library is provided. In particular, this antibody or antigen-binding domain is a cross-reactive antibody or antigen-binding domain that specifically binds to human, cynomolgus monkey, and mouse LTBRs. In one embodiment, the antibody or antigen-binding domain comprises a heavy chain variable region (V) including CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L(LTBR) is included. In one embodiment, the antibody or antigen-binding domain includes a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 91, CDR-H2 containing the amino acid sequence of SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 93. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 94, CDR-L2 containing the amino acid sequence of SEQ ID NO: 95, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 96. L It includes LTBR). In one embodiment, the antibody or antigen-binding domain is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 89. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L It includes LTBR). In one embodiment, the antibody or antigen-binding domain is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 97. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L Includes LTBR.

[0145] In one embodiment, the antibody that specifically binds to LTBR is a full-length antibody, particularly an antibody of the human IgG1 subclass. In a particular embodiment, it includes amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index). In a particular embodiment, the antibody is (i) Two heavy chains containing the amino acid sequence of SEQ ID NO: 227 and two light chains containing the amino acid sequence of SEQ ID NO: 228, or (ii) Two heavy chains containing the amino acid sequence of SEQ ID NO: 229 and two light chains containing the amino acid sequence of SEQ ID NO: 230, or (iii) Two heavy chains containing the amino acid sequence of SEQ ID NO: 231 and two light chains containing the amino acid sequence of SEQ ID NO: 232, or (iv) Two heavy chains containing the amino acid sequence of SEQ ID NO: 233 and two light chains containing the amino acid sequence of SEQ ID NO: 234, or (v) Two heavy chains containing the amino acid sequence of SEQ ID NO: 235 and two light chains containing the amino acid sequence of SEQ ID NO: 236, or (vi) Two heavy chains containing the amino acid sequence of SEQ ID NO: 237 and two light chains containing the amino acid sequence of SEQ ID NO: 238, or (vii) Two heavy chains containing the amino acid sequence of SEQ ID NO: 239 and two light chains containing the amino acid sequence of SEQ ID NO: 240, or (viii) Two heavy chains containing the amino acid sequence of SEQ ID NO: 241 and two light chains containing the amino acid sequence of SEQ ID NO: 242, or (ix) Two heavy chains containing the amino acid sequence of SEQ ID NO: 243 and two light chains containing the amino acid sequence of SEQ ID NO: 244 Includes.

[0146] Bispecific agonist LTBR antibody This specification also provides a novel bispecific antigen-binding molecule capable of specifically binding to tumor-associated antigens such as lymphotoxin beta receptor (LTBR) and fibroblast-activating protein (FAP), comprising an antigen-binding domain that specifically binds to FAP and at least one antigen-binding domain capable of agonist-binding to LTBR, ​​where LTBR-mediated activation is provided by crosslinking via binding to FAP expressed on tumor stromal cells. The bispecific agonist LTBR antibodies disclosed herein have particularly advantageous properties, including productivity, stability, binding affinity, biological activity, targeting efficiency, reduced internal distribution, acceptable pharmacokinetic (PK) properties, reduced toxicity, expanded dose range for patient administration, and thereby possibly enhanced efficacy.

[0147] Exemplary bispecific agonist LTBR antibody In one embodiment, an agonist LTBR antibody is provided that is a multispecific antibody containing the agonist LTBR antibody described herein. In one embodiment, an agonist LTBR antibody is provided that is a bispecific antibody. The bispecific agonist LTBR antibody preferably contains an Fc domain of human origin, particularly a human IgG subclass, more specifically a human IgG1 subclass. In one embodiment, the bispecific agonist LTBR antibody contains an Fc domain of a human IgG1 subclass that includes one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor. In one embodiment, the bispecific agonist LTBR antibody contains an Fc domain of a human IgG1 subclass having amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

[0148] In one embodiment, the agonist LTBR antibody is a bispecific antibody that specifically binds to LTBR and tumor-associated antigens (TAAs). In particular, the bispecific agonist LTBR antibody is an LTBR agonist that targets FAP. In one embodiment, the bispecific agonist LTBR antibody includes an Fc region comprising a first subunit and a second subunit containing a mutation that reduces effector function. The use of an Fc region containing a mutation that reduces or eliminates effector function prevents nonspecific agonism by crosslinking via the Fc receptor and inhibits ADCC in LTBR-expressing cells. The bispecific agonist LTBR antibodies described herein have advantages over conventional antibodies in that they selectively induce an immune response in target cells that are typically located within the tumor stroma, i.e., in the vicinity of the tumor.

[0149] Therefore, the bispecific agonist LTBR antibody is characterized by FAP-targeted agonist binding to LTBR. In the presence of FAP-expressing cells, the bispecific antigen-binding molecule can activate cancer-associated fibroblasts (CAFs) (Example 5.2.1), activate endothelial cells (Example 5.2.2), and modulate human endothelium to upregulate adhesion molecules and chemoattractants important for the immunoinvasion cascade. The bispecific antigen-binding molecule described herein can induce T cell adhesion (Example 5.2.3).

[0150] In one embodiment, a bispecific agonist LTBR antibody, (a) A first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (b) A second antigen-binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. A bispecific agonist LTBR antibody containing the above is provided.

[0151] The bispecific agonist LTBR antibody has an Fc domain comprising a first subunit and a second subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. This inhibits crosslinking via the Fc receptor, and tumor-specific activation is achieved through crosslinking via the binding of the antigen-binding domain, which specifically binds to FAP via binding to tumor-associated targets.

[0152] In one embodiment, a bispecific agonist LTBR antibody, (a) A first Fab fragment that specifically binds to fibroblast-activating protein (FAP), (b) A second Fab fragment that specifically binds to the lymphotoxin beta receptor (LTBR), and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. A bispecific agonist LTBR antibody containing the above is provided.

[0153] In one embodiment, the bispecific agonist LTBR antibody disclosed herein has a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (i) Heavy chain complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 3, CDR-H2 containing the amino acid sequence of SEQ ID NO: 4, and heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 5 H FAP), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 8. L FAP), or (ii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21 H The light chain variable region (V) includes FAP), as well as CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24. L FAP), or (iii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 13 H FAP), and the light chain variable region (V) including CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 16. L FAP) It contains a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP).

[0154] In one embodiment, the first antigen-binding domain that specifically binds to FAP is (i) Heavy chain complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 3, CDR-H2 containing the amino acid sequence of SEQ ID NO: 4, and heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 5 H FAP), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 8. L FAP), or (ii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21 H The light chain variable region (V) includes FAP), as well as CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24. L FAP) Includes.

[0155] In a particular embodiment, the first antigen-binding domain that specifically binds to FAP comprises (i) a heavy chain complementarity-determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 3, a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 4, and a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 5. H FAP), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 8. L The first antigen-binding domain that specifically binds to FAP includes a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21. HThe light chain variable region (V) includes FAP), as well as CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24. L In another embodiment, the first antigen-binding domain that specifically binds to FAP includes a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 11, CDR-H2 containing the amino acid sequence of SEQ ID NO: 12, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 13. H FAP), and the light chain variable region (V) including CDR-L1 containing the amino acid sequence of SEQ ID NO: 14, CDR-L2 containing the amino acid sequence of SEQ ID NO: 15, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 16. L (Includes FAP)

[0156] In one embodiment, the first antigen-binding domain that specifically binds to FAP is a heavy chain variable region (V) containing CDR-H1, CDR-H2, and CDR-H3 of the amino acid sequence of SEQ ID NO: 9. H FAP), and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 10. L It includes FAP). In another embodiment, it includes a light chain variable region (V) comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence of SEQ ID NO: 25. H FAP), and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 26. L It includes FAP). In yet another embodiment, it includes a heavy chain variable region (V) comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequence of SEQ ID NO: 17. H FAP), and the light chain variable region (V) containing CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequence of SEQ ID NO: 18. L (Includes FAP)

[0157] In one embodiment, the first antigen-binding domain that specifically binds to FAP is a heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 9. HFAP), and a light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 10. L The first antigen-binding domain, which contains FAP or specifically binds to FAP, includes a heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 25. H FAP), and a light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 26. L The first antigen-binding domain, which contains FAP or specifically binds to FAP, includes a heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 17. H FAP), and a light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 18. L (Includes FAP)

[0158] In one embodiment, the first antigen-binding domain that specifically binds to FAP is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 9. H FAP), and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10. L The first antigen-binding domain, which contains FAP or specifically binds to FAP, is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 25. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 26 L The first antigen-binding domain, which contains FAP or specifically binds to FAP, is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 17. H FAP), and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 18. L It includes FAP). In a particular embodiment, the first antigen-binding domain that specifically binds to FAP is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 9. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 L It includes FAP). In one embodiment, it includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 25. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 26 LIt includes FAP). In another embodiment, it includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 17. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 18 L It includes FAP. In particular, the first antigen-binding domain that specifically binds to FAP is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 9. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 L It contains FAP. In particular, the first antigen-binding domain that specifically binds to FAP is the Fab fragment.

[0159] In one embodiment, the bispecific agonist LTBR antibody disclosed herein has a second antigen-binding domain that specifically binds to LTBR, (i) Heavy chain complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L LTBR); or (ii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR); or (iii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77 HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L LTBR); or (iv) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L LTBR); or (v) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 37 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 40. L LTBR), or (vi) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56. L LTBR), or (vii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 61 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64. LLTBR); or (viii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 67, CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 69 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 72. L LTBR) It includes a second antigen-binding domain that specifically binds to LTBR.

[0160] In a particular embodiment, the second antigen-binding domain that specifically binds to the LTBR comprises a heavy chain complementarity-determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 28, and a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L The LTBR is included. In another embodiment, the second antigen-binding domain that specifically binds to the LTBR is a heavy chain variable region (V) including CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 37. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 40. L The LTBR is included. In another embodiment, the second antigen-binding domain that specifically binds to the LTBR is a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45. HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L The LTBR includes a second antigen-binding domain that specifically binds to the LTBR, ​​comprising a heavy chain variable region (V) including CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56. L The LTBR is included. In another embodiment, the second antigen-binding domain that specifically binds to the LTBR is a heavy chain variable region (V) including CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 61. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64. L The LTBR is included. In another embodiment, the second antigen-binding domain that specifically binds to the LTBR is a heavy chain variable region (V) including CDR-H1 containing the amino acid sequence of SEQ ID NO: 67, CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 69. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 72. L In yet another embodiment, the second antigen-binding domain that specifically binds to the LTBR includes a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77. HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L The LTBR includes a second antigen-binding domain that specifically binds to the LTBR, ​​comprising a heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L Includes LTBR.

[0161] In another embodiment, the second antigen-binding domain that specifically binds to the LTBR comprises a heavy chain complementarity-determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 101, a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 102, and a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 103. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 104, CDR-L2 containing the amino acid sequence of SEQ ID NO: 105, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 106. L It may include LTBR). In a particular embodiment, the second antigen-binding domain that specifically binds to LTBR is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 99. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 100 L Includes LTBR (CBE11).

[0162] In another embodiment, the second antigen-binding domain that specifically binds to the LTBR comprises a heavy chain complementarity-determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 343, a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 344, and a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 345. HLight chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 346, CDR-L2 containing the amino acid sequence of SEQ ID NO: 347, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 348. L It may include LTBR). In a particular embodiment, the second antigen-binding domain that specifically binds to LTBR is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 349. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 350 L Includes LTBR (BHA10).

[0163] In one embodiment, a bispecific agonist LTBR antibody comprising a second antigen-binding domain that specifically binds to LTBR, (i) Heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 33 H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 34. L LTBR), or (ii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 50. L LTBR), or (iii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 82. L LTBR), or (iv) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 90. L LTBR), or (v) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 98. L LTBR), (vi) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 42. L LTBR), (vii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 58. L LTBR), or (viii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 65 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 66. L LTBR), or (ix) Heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 73 H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 74. L LTBR) A bispecific agonist LTBR antibody is provided, which includes a second antigen-binding domain that specifically binds to LTBR.

[0164] In one embodiment, a bispecific agonist LTBR antibody comprising a second antigen-binding domain that specifically binds to LTBR, (i) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or (iv) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or (v) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR), or (vi) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L LTBR), or (vii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 58 L LTBR), or (viii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO. 65 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 66 L LTBR), (ix) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 73 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 74 L LTBR) A bispecific agonist LTBR antibody is provided, which includes a second antigen-binding domain that specifically binds to LTBR.

[0165] In one embodiment, the second antigen-binding domain that specifically binds to LTBR is (i) a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33.H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L (i) LTBR), or (ii) heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L (LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L It includes LTBR). In a particular embodiment, the second antigen-binding domain that specifically binds to LTBR is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L It includes LTBR). In another particular embodiment, the second antigen-binding domain that specifically binds to LTBR is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 41. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L It includes LTBR). In yet another specific embodiment, the second antigen-binding domain that specifically binds to LTBR is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 49. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L Includes LTBR.

[0166] In another embodiment, the second antigen-binding domain that specifically binds to LTBR can also specifically bind to mouse LTBR. The heavy chain complementarity-determining region (CDR-H1) contains the amino acid sequence of SEQ ID NO: 27, the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 28, and the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L LTBR) Includes.

[0167] In one embodiment, the second antigen-binding domain that specifically binds to LTBR can also specifically bind to mouse LTBR. Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V HLight chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR) Includes.

[0168] In one embodiment, the second antigen-binding domain that specifically binds to LTBR can also specifically bind to mouse LTBR, ​​and the heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33 H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L Includes LTBR.

[0169] In one embodiment, a bispecific agonist LTBR antibody comprises a third antigen-binding domain that specifically binds to LTBR, (a) A first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (b) A second antigen-binding domain and a third antigen-binding domain that specifically bind to the lymphotoxin beta receptor (LTBR), and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor, The bispecific agonist LTBR antibody is provided, which means a bispecific antigen-binding molecule containing a bispecific antigen-binding molecule.

[0170] In one particular embodiment, the third antigen-binding domain that specifically binds to LTBR is identical to the second antigen-binding domain that specifically binds to LTBR, ​​meaning that the second and third antigen-binding domains that specifically bind to the lymphotoxin beta receptor (LTBR) are the same. In one embodiment, the second and third antigen-binding domains that specifically bind to LTBR are Fab fragments that specifically bind to LTBR. In one embodiment, the Fab fragment that specifically binds to LTBR is a crossfab fragment. In a further embodiment, the first antigen-binding domain that specifically binds to FAP is a Fab fragment.

[0171] In one embodiment, the bispecific agonist LTBR antibody disclosed herein comprises a second antigen-binding domain and a third antigen-binding domain that specifically bind to LTBR, ​​both of which (i) Heavy chain complementarity determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L LTBR); or (ii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR); or (iii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77 HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L LTBR); or (iv) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L LTBR); or (v) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 35, CDR-H2 containing the amino acid sequence of SEQ ID NO: 36, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 37 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 40. L LTBR), or (vi) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56. L LTBR), or (vii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 59, CDR-H2 containing the amino acid sequence of SEQ ID NO: 60, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 61 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64. LLTBR); or (viii) Heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 67, CDR-H2 containing the amino acid sequence of SEQ ID NO: 68, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 69 H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 72. L LTBR) It includes a second antigen-binding domain and a third antigen-binding domain that specifically bind to the LTBR.

[0172] In a particular embodiment, the second antigen-binding domain and the third antigen-binding domain are identical.

[0173] In one embodiment, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each include a heavy chain complementarity-determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 28, and a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L The LTBR is included. In another embodiment, the second antigen-binding domain and the third antigen-binding domain that specifically bind to the LTBR each include a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 35 (CDR-H1), the amino acid sequence of SEQ ID NO: 36 (CDR-H2), and the amino acid sequence of SEQ ID NO: 37 (CDR-H3). H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 38, CDR-L2 containing the amino acid sequence of SEQ ID NO: 39, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 40. LThe LTBR is included. In another embodiment, the second and third antigen-binding domains that specifically bind to the LTBR each include a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 43 (CDR-H1), the amino acid sequence of SEQ ID NO: 44 (CDR-H2), and the amino acid sequence of SEQ ID NO: 45 (CDR-H3). H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L The LTBR includes a second antigen-binding domain and a third antigen-binding domain that specifically bind to the LTBR, ​​respectively, comprising a heavy chain variable region (V) containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 55, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 56. L The LTBR is included. In another embodiment, the second and third antigen-binding domains that specifically bind to the LTBR each include a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 59 (CDR-H1), the amino acid sequence of SEQ ID NO: 60 (CDR-H2), and the amino acid sequence of SEQ ID NO: 61 (CDR-H3). H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 62, CDR-L2 containing the amino acid sequence of SEQ ID NO: 63, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 64. L The LTBR is included. In another embodiment, the second antigen-binding domain and the third antigen-binding domain that specifically bind to the LTBR each include a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 67 (CDR-H1), the amino acid sequence of SEQ ID NO: 68 (CDR-H2), and the amino acid sequence of SEQ ID NO: 69 (CDR-H3). HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 70, CDR-L2 containing the amino acid sequence of SEQ ID NO: 71, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 72. L In yet another embodiment, the second antigen-binding domain and the third antigen-binding domain that specifically bind to the LTBR each include a heavy chain variable region (V) comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L The LTBR includes a second antigen-binding domain and a third antigen-binding domain that specifically bind to the LTBR, ​​each comprising a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 83 (CDR-H1), the amino acid sequence of SEQ ID NO: 84 (CDR-H2), and the amino acid sequence of SEQ ID NO: 85 (CDR-H3). H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L Includes LTBR.

[0174] In one embodiment, a bispecific agonist LTBR antibody comprising a second antigen-binding domain and a third antigen-binding domain that specifically bind to LTBR, (i) Heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 33 H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 34. L LTBR), or (ii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 49 (V HLight chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 50. L LTBR), or (iii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 82. L LTBR), or (iv) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 90. L LTBR), or (v) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 98. L LTBR), (vi) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 42. L LTBR), (vii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 58. L LTBR), or (viii) Heavy chain variable region containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 65 (V H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 66. L LTBR), or (ix) Heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 73 H Light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of LTBR) and SEQ ID NO: 74. L LTBR) A bispecific agonist LTBR antibody is provided, comprising a second antigen-binding domain and a third antigen-binding domain that specifically bind to LTBR.

[0175] In one embodiment, a bispecific agonist LTBR antibody comprising a second antigen-binding domain and a third antigen-binding domain that specifically bind to LTBR, (i) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or (iv) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or (v) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR), or (vi) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L LTBR), or (vii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57 (VH Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 58 L LTBR), or (viii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO. 65 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 66 L LTBR), (ix) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 73 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 74 L LTBR) A bispecific agonist LTBR antibody is provided, comprising a second antigen-binding domain and a third antigen-binding domain that specifically bind to LTBR.

[0176] In one embodiment, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each include (i) a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L (i) LTBR), or (ii) heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L (LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L Includes LTBR). In a particular embodiment, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each include a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L Includes LTBR). In another particular embodiment, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each include a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 41. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 LIncludes LTBR). In yet another specific embodiment, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each include a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 49. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L Includes LTBR.

[0177] In another embodiment, a bispecific agonist LTBR antibody is provided, comprising a second antigen-binding domain and a third antigen-binding domain that specifically bind to LTBR, ​​wherein the second and third antigen-binding domains each comprise a heavy chain complementarity-determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 101, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 102, and a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 103. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 104, CDR-L2 containing the amino acid sequence of SEQ ID NO: 105, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 106. L It includes LTBR). In a particular embodiment, the second and third antigen-binding domains that specifically bind to LTBR each include a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 99. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 100 L Includes LTBR (CBE11).

[0178] In another embodiment, a bispecific agonist LTBR antibody is provided, comprising a second antigen-binding domain and a third antigen-binding domain that specifically bind to LTBR, ​​wherein the second antigen-binding domain and the third antigen-binding domain each comprise a heavy chain complementarity-determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 343, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 344, and a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 345. HLight chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 346, CDR-L2 containing the amino acid sequence of SEQ ID NO: 347, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 348. L Includes LTBR). In a particular embodiment, the second and third antigen-binding domains that specifically bind to LTBR each include a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 349. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 350 L Includes LTBR (BHA10).

[0179] In another embodiment, a bispecific agonist LTBR antibody comprising a second antigen-binding domain and a third antigen-binding domain that can specifically bind to human LTBR and also specifically bind to mouse LTBR, ​​wherein the second antigen-binding domain and the third antigen-binding domain are each The heavy chain complementarity-determining region (CDR-H1) contains the amino acid sequence of SEQ ID NO: 27, the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 28, and the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 29. H Light chain variable region (V) containing the LTBR), and the light chain complementarity determining region (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 75, CDR-H2 containing the amino acid sequence of SEQ ID NO: 76, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 77. HLight chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 78, CDR-L2 containing the amino acid sequence of SEQ ID NO: 79, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 80. L LTBR), or The heavy chain variable region (V) includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 83, CDR-H2 containing the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 85. H Light chain variable region (V) including LTBR), and CDR-L1 containing the amino acid sequence of SEQ ID NO: 86, CDR-L2 containing the amino acid sequence of SEQ ID NO: 87, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 88. L LTBR) A bispecific agonist LTBR antibody is provided, comprising a second antigen-binding domain and a third antigen-binding domain, which are capable of specifically binding to human LTBR and also specifically binding to mouse LTBR.

[0180] In one embodiment, the bispecific agonist LTBR antibody comprises a second antigen-binding domain and a third antigen-binding domain that specifically bind to human LTBR and also specifically bind to mouse LTBR, ​​wherein the second antigen-binding domain and the third antigen-binding domain each comprise: (i) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or (vii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or (viii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V HLight chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or (ix) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR) Includes.

[0181] In a particular embodiment, the bispecific agonist LTBR antibody comprises a second antigen-binding domain and a third antigen-binding domain that specifically bind to human LTBR and also specifically bind to mouse LTBR, ​​wherein the second antigen-binding domain and the third antigen-binding domain each comprise a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L Includes LTBR.

[0182] Bispecific agonist LTBR antibody that binds to FAP and LTBR In one embodiment, a bispecific agonist LTBR antibody, (a) A first antigen-binding domain that specifically binds to FAP, Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9 (V H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 L FAP), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 (V H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 26 L FAP), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17 (V H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 18 L A first antigen-binding domain that specifically binds to FAP, including FAP (b) A second antigen-binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), (i) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (VH Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or (iv) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or (v) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR), (vi) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L LTBR), (vii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 58 L LTBR), or (viii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO. 65 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 66 L LTBR), or (ix) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 73 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 74 L LTBR) A second antigen-binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. A bispecific agonist LTBR antibody containing the above is provided.

[0183] In another embodiment, a bispecific agonist LTBR antibody, (a) A first antigen-binding domain that specifically binds to FAP, Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9 (V H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 L FAP), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 (V H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 26 L FAP), or The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17 (V H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 18 L A first antigen-binding domain that specifically binds to FAP, including FAP (b) A second antigen-binding domain and a third antigen-binding domain that specifically bind to the lymphotoxin beta receptor (LTBR), respectively (i) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L LTBR), or (ii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L LTBR), or (iii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 81 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 82 L LTBR), or (iv) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 90 L LTBR), or (v) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 97 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 98 L LTBR), (vi) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 41 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 42 L LTBR), (vii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 57 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 58 L LTBR), or (viii) Heavy chain variable region containing the amino acid sequence of SEQ ID NO. 65 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 66 L LTBR), or (ix) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 73 (V H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 74 L LTBR) This includes a second antigen-binding domain and a third antigen-binding domain that specifically bind to the lymphotoxin beta receptor (LTBR), and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. A bispecific agonist LTBR antibody containing the above is provided.

[0184] In one particular embodiment, a bispecific agonist LTBR antibody is provided, wherein the antibody comprises (i) a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 9. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 L(ii) a first antigen-binding domain that can specifically bind to FAP, and (ii) a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 33. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 34 L It includes an LTBR, ​​a second antigen-binding domain capable of specifically binding to the LTBR, ​​and a third antigen-binding domain.

[0185] In another embodiment, a bispecific agonist LTBR antibody is provided, wherein the antibody comprises (i) a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 9. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 L (ii) a first antigen-binding domain that can specifically bind to FAP, including FAP, and (ii) a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 99. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 100 L It includes an LTBR, ​​a second antigen-binding domain capable of specifically binding to the LTBR, ​​and a third antigen-binding domain.

[0186] Bispecific, monovalent agonist LTBR antibody (1+1 format) In one embodiment, a bispecific agonist LTBR antibody is provided, comprising a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), a second antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), and an Fc domain composed of a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor, wherein the bispecific agonist LTBR antibody is, (i) The first light chain and the first heavy chain of a full-length antibody that specifically binds to FAP, and (ii) A second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to LTBR, ​​wherein the variable domains VL and VH are substituted for each other and / or the constant domains CL and CH1 are substituted for each other. Includes.

[0187] The first antigen-binding domain that specifically binds to FAP is the Fab fragment, and the second antigen-binding domain that specifically binds to LTBR is the crossFab fragment.

[0188] In another embodiment, a bispecific agonist LTBR antibody comprising a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), a second antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), and A bispecific agonist LTBR antibody is provided, comprising a first subunit and a second subunit, and containing an Fc domain having one or more amino acid substitutions that reduce the binding affinity and / or effector function of an antigen-binding molecule to an Fc receptor, wherein the bispecific antigen-binding molecule is (i) A first (modified) light chain and a first (modified) heavy chain of a full-length antibody that specifically binds to FAP, wherein the variable domains VL and VH are substituted for each other and / or the constant domains CL and CH1 are substituted for each other, and (ii) The second light chain and second heavy chain of the full-length antibody that specifically binds to LTBR. Includes.

[0189] The first antigen-binding domain that specifically binds to FAP is the crossFab fragment, and the second antigen-binding domain that specifically binds to LTBR is the Fab fragment.

[0190] In a particular embodiment, the following is provided: (a) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 107, a second heavy chain containing the amino acid sequence of SEQ ID NO: 109, a first light chain containing the amino acid sequence of SEQ ID NO: 108, and a second light chain containing the amino acid sequence of SEQ ID NO: 110, or (b) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 111, a second heavy chain containing the amino acid sequence of SEQ ID NO: 113, a first light chain containing the amino acid sequence of SEQ ID NO: 112, and a second light chain containing the amino acid sequence of SEQ ID NO: 114, or (c) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 115, a second heavy chain containing the amino acid sequence of SEQ ID NO: 117, a first light chain containing the amino acid sequence of SEQ ID NO: 116, and a second light chain containing the amino acid sequence of SEQ ID NO: 118, or (d) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 119, a second heavy chain containing the amino acid sequence of SEQ ID NO: 121, a first light chain containing the amino acid sequence of SEQ ID NO: 120, and a second light chain containing the amino acid sequence of SEQ ID NO: 122, or (e) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 123, a second heavy chain containing the amino acid sequence of SEQ ID NO: 125, a first light chain containing the amino acid sequence of SEQ ID NO: 124, and a second light chain containing the amino acid sequence of SEQ ID NO: 126, or (f) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 127, a second heavy chain containing the amino acid sequence of SEQ ID NO: 129, a first light chain containing the amino acid sequence of SEQ ID NO: 128, and a second light chain containing the amino acid sequence of SEQ ID NO: 130, or (g) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 131, a second heavy chain containing the amino acid sequence of SEQ ID NO: 133, a first light chain containing the amino acid sequence of SEQ ID NO: 132, and a second light chain containing the amino acid sequence of SEQ ID NO: 134, or (h) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 135, a second heavy chain containing the amino acid sequence of SEQ ID NO: 137, a first light chain containing the amino acid sequence of SEQ ID NO: 136, and a second light chain containing the amino acid sequence of SEQ ID NO: 138, or (i) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 139, a second heavy chain containing the amino acid sequence of SEQ ID NO: 141, a first light chain containing the amino acid sequence of SEQ ID NO: 140, and a second light chain containing the amino acid sequence of SEQ ID NO: 142, or (j) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 143, a second heavy chain containing the amino acid sequence of SEQ ID NO: 145, a first light chain containing the amino acid sequence of SEQ ID NO: 144, and a second light chain containing the amino acid sequence of SEQ ID NO: 146, or (k) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 147, a second heavy chain containing the amino acid sequence of SEQ ID NO: 149, a first light chain containing the amino acid sequence of SEQ ID NO: 148, and a second light chain containing the amino acid sequence of SEQ ID NO: 150, or (l) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 151, a second heavy chain containing the amino acid sequence of SEQ ID NO: 153, a first light chain containing the amino acid sequence of SEQ ID NO: 152, and a second light chain containing the amino acid sequence of SEQ ID NO: 154, or (m) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 155, a second heavy chain containing the amino acid sequence of SEQ ID NO: 157, a first light chain containing the amino acid sequence of SEQ ID NO: 156, and a second light chain containing the amino acid sequence of SEQ ID NO: 158, or (n) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 159, a second heavy chain containing the amino acid sequence of SEQ ID NO: 161, a first light chain containing the amino acid sequence of SEQ ID NO: 160, and a second light chain containing the amino acid sequence of SEQ ID NO: 162, or (o) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 163, a second heavy chain containing the amino acid sequence of SEQ ID NO: 165, a first light chain containing the amino acid sequence of SEQ ID NO: 164, and a second light chain containing the amino acid sequence of SEQ ID NO: 166, or (q) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 215, a second heavy chain containing the amino acid sequence of SEQ ID NO: 217, a first light chain containing the amino acid sequence of SEQ ID NO: 216, and a second light chain containing the amino acid sequence of SEQ ID NO: 218.

[0191] In one embodiment, a bispecific agonist LTBR antibody is provided, comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 329, a second heavy chain containing the amino acid sequence of SEQ ID NO: 330, a first light chain containing the amino acid sequence of SEQ ID NO: 331, and a second light chain containing the amino acid sequence of SEQ ID NO: 332.

[0192] A bivalent, bivalent, bivalent agonist antibody (2+1 format) for binding to LTBR and monovalent for binding to FAP. In one embodiment, a bispecific agonist LTBR antibody is provided, comprising a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), a second antigen-binding domain and a third antigen-binding domain that specifically bind to lymphotoxin beta receptor (LTBR), and an Fc domain comprising a first subunit and a second subunit, which includes one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor, wherein the bispecific antigen-binding molecule is (i) Two light chains and two heavy chains of a full-length antibody that specifically binds to LTBR, ​​and (ii) A Fab fragment or crossFab fragment that specifically binds to FAP, wherein the Fab fragment or crossFab fragment is linked to one of the C-terminuses of two heavy chains via a peptide linker. Includes.

[0193] In another embodiment, a bispecific antigen-binding molecule is provided, comprising a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), a second antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), and an Fc domain comprising a first subunit and a second subunit, which includes one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor, wherein the bispecific antigen-binding molecule is, (i) The first light chain and the first heavy chain of a full-length antibody that specifically binds to FAP, and (ii) A second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to LTBR, ​​wherein a Fab fragment or CrossFab fragment that specifically binds to LTBR is linked to the C-terminus of the second heavy chain via a peptide linker. Includes.

[0194] Therefore, a bispecific antigen-binding molecule having a divalent bond to LTBR and a monovalent bond to FAP is provided.

[0195] In one embodiment, the following is provided: (a) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 167, a second heavy chain containing the amino acid sequence of SEQ ID NO: 169, two light chains, each containing the amino acid sequence of SEQ ID NO: 168, and one light chain containing the amino acid sequence of SEQ ID NO: 170, or (b) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 171, a second heavy chain containing the amino acid sequence of SEQ ID NO: 173, two light chains, each containing the amino acid sequence of SEQ ID NO: 172, and one light chain containing the amino acid sequence of SEQ ID NO: 174, or (c) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 175, a second heavy chain containing the amino acid sequence of SEQ ID NO: 177, two light chains, each containing the amino acid sequence of SEQ ID NO: 176, and one light chain containing the amino acid sequence of SEQ ID NO: 178, or (d) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 179, a second heavy chain containing the amino acid sequence of SEQ ID NO: 181, two light chains, each containing the amino acid sequence of SEQ ID NO: 180, and one light chain containing the amino acid sequence of SEQ ID NO: 182, or (e) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 183, a second heavy chain containing the amino acid sequence of SEQ ID NO: 185, two light chains, each containing the amino acid sequence of SEQ ID NO: 184, and one light chain containing the amino acid sequence of SEQ ID NO: 186, or (f) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 187, a second heavy chain containing the amino acid sequence of SEQ ID NO: 189, two light chains, each containing the amino acid sequence of SEQ ID NO: 188, and one light chain containing the amino acid sequence of SEQ ID NO: 190, or (g) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 191, a second heavy chain containing the amino acid sequence of SEQ ID NO: 193, and two light chains, each containing the amino acid sequence of SEQ ID NO: 192 and one light chain containing the amino acid sequence of SEQ ID NO: 194, or (h) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 195, a second heavy chain containing the amino acid sequence of SEQ ID NO: 197, two light chains, each containing the amino acid sequence of SEQ ID NO: 196, and one light chain containing the amino acid sequence of SEQ ID NO: 198, or (i) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 199, a second heavy chain containing the amino acid sequence of SEQ ID NO: 201, two light chains, each containing the amino acid sequence of SEQ ID NO: 200, and one light chain containing the amino acid sequence of SEQ ID NO: 202, or (j) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 203, a second heavy chain containing the amino acid sequence of SEQ ID NO: 205, two light chains, each containing the amino acid sequence of SEQ ID NO: 204, and one light chain containing the amino acid sequence of SEQ ID NO: 206, or (k) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 207, a second heavy chain containing the amino acid sequence of SEQ ID NO: 209, two light chains, each containing the amino acid sequence of SEQ ID NO: 208, and one light chain containing the amino acid sequence of SEQ ID NO: 210, or (l) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 211, a second heavy chain containing the amino acid sequence of SEQ ID NO: 213, and two light chains, each containing the amino acid sequence of SEQ ID NO: 212 and one light chain containing the amino acid sequence of SEQ ID NO: 214.

[0196] Furthermore, the following will be provided: (m) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 326, a second heavy chain containing the amino acid sequence of SEQ ID NO: 328, and two light chains, each containing the amino acid sequence of SEQ ID NO: 327 and one light chain containing the amino acid sequence of SEQ ID NO: 329, or (n) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 333, a second heavy chain containing the amino acid sequence of SEQ ID NO: 334, two light chains, each containing the amino acid sequence of SEQ ID NO: 335, and one light chain containing the amino acid sequence of SEQ ID NO: 336, or (o) A bispecific agonist LTBR antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 337, a second heavy chain containing the amino acid sequence of SEQ ID NO: 338, two light chains, each containing the amino acid sequence of SEQ ID NO: 339, and one light chain containing the amino acid sequence of SEQ ID NO: 340.

[0197] In one particular embodiment, a bispecific agonist LTBR antibody is provided, comprising a bispecific antigen-binding molecule comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 195, a second heavy chain containing the amino acid sequence of SEQ ID NO: 197, and two light chains, each containing the amino acid sequence of SEQ ID NO: 196 and one light chain containing the amino acid sequence of SEQ ID NO: 198.

[0198] Mouse surrogate agonist LTBR antibody This specification also provides mouse surrogate molecules. In one embodiment, a bispecific agonist LTBR antibody, (a) A first antigen-binding domain that specifically binds to FAP, comprising a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 17. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 18 L A first antigen-binding domain that specifically binds to FAP, including FAP (b) A second antigen-binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), comprising a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 49. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L A second antigen-binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), including LTBR, ​​and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. A bispecific agonist LTBR antibody containing the above is provided.

[0199] In one particular embodiment, a bispecific agonist LTBR antibody (1+1 format) is provided, comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 219, a second heavy chain containing the amino acid sequence of SEQ ID NO: 221, a first light chain containing the amino acid sequence of SEQ ID NO: 220, and a second light chain containing the amino acid sequence of SEQ ID NO: 222.

[0200] In one embodiment, a bispecific agonist LTBR antibody, (a) A first antigen-binding domain that specifically binds to FAP, comprising a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 17. H FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 18 L A first antigen-binding domain that specifically binds to FAP, including FAP (b) A second antigen-binding domain and a third antigen-binding domain that specifically bind to the lymphotoxin beta receptor (LTBR), each comprising a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 49. H Light chain variable region (V) containing the amino acid sequence of LTBR) and SEQ ID NO: 50 L A second antigen-binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), including LTBR, ​​and (c) An Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. A bispecific agonist LTBR antibody containing the above is provided.

[0201] In one embodiment, a bispecific agonist LTBR antibody is provided, comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 223, a second heavy chain containing the amino acid sequence of SEQ ID NO: 225, and two light chains, each containing the amino acid sequence of SEQ ID NO: 224 and one light chain containing the amino acid sequence of SEQ ID NO: 226.

[0202] In one embodiment, a bispecific agonist LTBR antibody is provided, comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 318, a second heavy chain containing the amino acid sequence of SEQ ID NO: 320, and two light chains, each containing the amino acid sequence of SEQ ID NO: 319 and one light chain containing the amino acid sequence of SEQ ID NO: 321.

[0203] Fc domain modification that reduces Fc receptor binding and / or effector function. The bispecific agonist LTBR antibodies described herein further include an Fc domain comprising a first subunit and a second subunit, which contains one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor. Thus, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0204] The Fc domain confers desirable pharmacokinetic properties to the bispecific antibody of the present invention, including a long serum half-life and a desirable tissue-to-blood distribution ratio, which contribute to good accumulation in target tissues. However, at the same time, it may result in undesirable targeting of the bispecific agonist LTBR antibody to cells expressing the Fc receptor rather than the preferred antigen-holding cells. Therefore, in certain embodiments, the Fc domain of the bispecific agonist LTBR antibody exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the natural IgG Fc domain, particularly the IgG1 Fc domain or IgG4 Fc domain. More specifically, the Fc domain is the IgG1 Fc domain.

[0205] In one such embodiment, the Fc domain (or the bispecific antigen-binding molecule of the present invention comprising the Fc domain) exhibits a binding affinity to the Fc receptor of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5%, compared to the natural IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention comprising the natural IgG1 Fc domain), and / or exhibits effector function of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5%, compared to the natural IgG1 Fc domain (or the bispecific antigen-binding molecule of the present invention comprising the natural IgG1 Fc domain). In one embodiment, the Fc domain (or the bispecific antigen-binding molecule of the present invention comprising the Fc domain) substantially does not bind to the Fc receptor and / or does not induce effector function. In certain embodiments, 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 activated Fc receptor. In certain embodiments, the Fc receptor is an activated 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 certain embodiments, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human Fc□RIIB. In one embodiment, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In certain embodiments, the effector function is ADCC. In one embodiment, the domain of the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the natural IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the bispecific antigen-binding molecule of the present invention containing said Fc domain) exhibits a binding affinity to FcRn that is greater than about 70%, particularly greater than about 80%, and more specifically greater than about 90%, compared to the natural IgG1 Fc domain (or the bispecific agonist LTBR antibody containing the natural IgG1 Fc domain).

[0206] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to the Fc receptor and / or reduced effector function compared to an unengineered Fc domain. In certain embodiments, the Fc domain of a bispecific agonist LTBR antibody contains one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the 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 mutation reduces the binding affinity of the Fc domain to the Fc receptor. In another embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least half, at least one-fifth, or at least one-tenth. In one embodiment, a bispecific agonist LTBR antibody containing an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more specifically less than 5% of the binding affinity to the Fc receptor compared to the bispecific antibody of the present invention containing an unengineered Fc domain. In certain embodiments, the Fc receptor is the 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 specifically, human Fc□RIIB. In some embodiments, the Fc receptor is an activated Fc receptor. In a particular embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, the binding of each of these receptors is reduced. In some embodiments, the binding affinity to complement components, particularly to C1q, is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., maintenance of the binding affinity of the Fc domain to the receptor, is achieved when the Fc domain of the bispecific antigen-binding molecule according to the present invention (or a bispecific agonist LTBR antibody containing the Fc domain) exhibits more than 70% of the binding affinity of the unmanipulated Fc domain (or a bispecific agonist LTBR antibody containing the unmanipulated Fc domain) to FcRn.An Fc domain, or a bispecific agonist LTBR antibody containing the Fc domain, may exhibit affinity of approximately 80% or even 90%. In certain embodiments, the Fc domain of a bispecific agonist LTBR antibody is manipulated to reduce effector function compared to an unmanipulated Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cell 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 that induces apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0207] Antibodies with reduced effector function include those having 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 variants include those having two or more substitutions at amino acid positions 265, 269, 270, 297, and 327 (including the so-called "DANA" Fc variant with alanine substitutions at residues 265 and 297 (U.S. Patent No. 7,332,581)). Specific antibody variants with improved or reduced binding to FcR have been described (e.g., U.S. Patent No. 6,737,056, International Publication No. 2004 / 056312, and Shields, R. Let al., J. Biol. Chem. 276(2001) 6591-6604).

[0208] In one embodiment, the Fc domain includes amino acid substitutions at positions E233, L234, L235, N297, P331, and P329. In some embodiments, the Fc domain includes 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 embodiment, the Fc domain includes an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain includes an amino acid substitution at position P329 and includes further amino acid substitutions selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In more specific embodiments, the Fc domain includes amino acid mutations L234A, L235A, and P329G ("P329G LALA"). The "P329G LALA" combination of amino acid substitutions almost completely eliminates the binding of the human IgG1 Fc domain to the Fcγ receptor, as described in PCT international publication 2012 / 130831A1. The aforementioned document also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function. Such antibodies are IgG1 with mutations L234A and L235A or IgG1 with mutations L234A, L235A, and P329G (numbering is based on the EU index by Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0209] In one embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing 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 containing 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 38, 84-91 (2010)).

[0210] Antibodies having an increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer, RL et al., J.Immunol. 117 (1976) 587-593, and Kim, JK et al., J.Immunol. 24 (1994) 2429-2434) are described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies contain an Fc region having one or more substitutions within the Fc region that improve the binding of the Fc region to FcRn. Such Fc variants include substitutions in one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitution of 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" Vol. 322 (1988), pp. 738-740; U.S. Patent Nos. 5,648,260; U.S. Patent Nos. 5,624,821; and International Publication No. 94 / 29351.

[0211] Binding to the Fc receptor can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments, such as the BIAcore instrument (GE Healthcare) and Fc receptors that can be obtained, for example, by recombinant expression. Such suitable binding assays are described herein. Alternatively, the binding affinity of an Fc domain or a cell-activating bispecific antigen-binding molecule containing an Fc domain to the Fc receptor may be evaluated using cell lines known to express a particular Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor). The effector function of the Fc domain or a bispecific agonist LTBR antibody containing 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 evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5500362; Hellstrom et al., Proc Natl Acad Sci USA 83, 70597063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 14991502 (1985); U.S. Patent No. 5821337; Bruggemann et al., J Exp Med 166, 13511361 (1987). Alternatively, non-radioactive assay methods may be used (see, for example, 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)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0212] The following sections describe preferred embodiments of the bispecific agonist LTBR antibody described herein, comprising Fc domain modifications that reduce Fc receptor binding and / or effector function. In one embodiment, a bispecific agonist LTBR antibody is provided, comprising (a) a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (b) a second (and optionally third) antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), and (c) an Fc domain composed of a first subunit and a second subunit, wherein the Fc domain comprises one or more amino acid substitutions that reduce the antibody's binding affinity to Fc receptors, particularly Fcγ receptors. In another embodiment, a bispecific agonist LTBR antibody is provided, comprising (a) a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (b) a second (and optionally third) antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, wherein the Fc domain contains one or more amino acid substitutions that reduce effector function. In a particular embodiment, the Fc domain is an Fc domain of a human IgG1 subclass having amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

[0213] Fc domain modification that promotes heterodimerization The bispecific agonist LTBR antibody described herein contains different antigen-binding sites fused to one or the other of two subunits of the Fc domain, and therefore the two subunits of the Fc domain may be contained in two non-identical polypeptide chains. Co-recombination expression of these polypeptides and subsequent dimerization result in several possible combinations of the two polypeptides. Therefore, to improve the yield and purity of the bispecific agonist LTBR antibody of the present invention in recombinant production, it would be advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecule of the present invention that promote the association of the desired polypeptide.

[0214] Accordingly, in certain embodiments, a bispecific agonist LTBR antibody is provided, comprising (a) a first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (b) a second antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), and (c) an Fc domain comprising a first subunit and a second subunit, comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor, wherein the Fc domain comprises a modification that promotes the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is located within the CH3 domain of the Fc domain. Accordingly, in one embodiment, the modification is located within the CH3 domain of the Fc domain.

[0215] In certain embodiments, the modification is a so-called "knob-into-hole" modification, comprising a "knob" modification of one of the two subunits of the Fc domain and a "hole" modification of the other of the two subunits of the Fc domain. Thus, a bispecific agonist LTBR antibody is provided, comprising (a) at least one antigen-binding domain capable of specifically binding to LTBR, ​​(b) at least one antigen-binding domain capable of specifically binding to a target cell antigen, and (c) an Fc domain comprising a first subunit and a second subunit capable of stable association, wherein, according to the knob-into-hole method, the first subunit of the Fc domain contains a knob and the second subunit of the Fc domain contains a hole. In certain embodiments, the first subunit of the Fc domain includes the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain includes the amino acid substitutions Y349C, T366S and Y407V (numbered according to the Kabat EU index).

[0216] The knob-into-hole technique is described, for example, in U.S. Patent Nos. 5,731,168, 7,695,936, Ridgway et al., Prot Eng 9,617-621 (1996), and Carter, J Immunol Meth 248,7-15 (2001). Typically, this method involves promoting heterodimerization and inhibiting homodimerization by introducing a bump ("knob") on the contact surface of a first polypeptide and a corresponding cavity on the contact surface of a second polypeptide, respectively, so that the bump is located within its corresponding cavity. The bump is constructed by substituting a smaller amino acid side chain from the contact surface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the ridge is formed at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0217] 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, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a ridge within the CH3 domain of the first subunit that can be placed in the cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit that can accommodate the ridge within the CH3 domain of the first subunit. The ridge and cavity can be created by changing the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In certain embodiments, the threonine residue at position 366 in the CH3 domain of the first subunit of the Fc domain is replaced with a tryptophan residue (T366W), and the tyrosine residue at position 407 in the CH3 domain of the second subunit of the Fc domain is replaced with a valine residue (Y407V). In one embodiment, the threonine residue at position 366 in the second subunit of the Fc domain is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0218] In a further embodiment, the serine residue at position 354 of the first subunit of the Fc domain is further replaced with a cysteine ​​residue (S354C), and the tyrosine residue at position 349 of the second subunit of the Fc domain is further replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues leads to the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15 (2001)). In a specific embodiment, the first subunit of the Fc domain contains amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains amino acid substitutions Y349C, T366S, and Y407V (numbered according to the Kabat EU index).

[0219] In alternative embodiments, modifications that facilitate the association of the first and second subunits of the Fc domain include modifications that mediate an electrostatic steering effect, such as those described in International Publication No. 2009 / 089004. Generally, this method involves the substitution of one or more amino acid residues with charged amino acid residues at the interface of the two Fc domain subunits such that homodimerization is electrostatically undesirable but heterodimerization is electrostatically desirable.

[0220] The C-terminus of the heavy chain of the bispecific agonist LTBR antibody reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. In certain embodiments, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In certain embodiments, the C-terminus of the heavy chain is a shortened C-terminus ending with P. In one embodiment of all embodiments reported herein, the bispecific antibody containing a heavy chain with the C-terminal CH3 domain specified herein contains a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of all embodiments reported herein, the bispecific agonist LTBR antibody containing a heavy chain with the C-terminal CH3 domain specified herein contains a C-terminal glycine residue (G446, numbered according to the Kabat EU index).

[0221] Modification in the Fab domain In one embodiment, a bispecific agonist LTBR antibody is provided, comprising (a) a first Fab fragment that can specifically bind to FAP, (b) a second Fab fragment that can specifically bind to LTBR, ​​and (c) an Fc domain composed of a first subunit and a second subunit, wherein in one of the Fab fragments, either the variable domain VH and VL are exchanged or the constant domain CH1 and CL are exchanged. The bispecific antibody is prepared according to Crossmab technology.

[0222] Multispecific antibodies with domain substitution / exchange in one binding arm (CrossMabVH-VL or CrossMabCH-CL) are described in International Publication No. 2009 / 080252 and Schaefer, W. et al., PNAS, 108(2011)11187-1191. These multispecific antibodies clearly reduce byproducts resulting from mismatches between the light chain for the first antigen and the wrong heavy chain for the second antigen (compared to methods without such domain exchange).

[0223] In one embodiment, a bispecific agonist LTBR antibody is provided, comprising (a) a first Fab fragment capable of specifically binding to FAP, (b) a second Fab fragment capable of specifically binding to LTBR, ​​and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, wherein in one of the Fab fragments, the variable domains VL and VH are substituted for each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. More specifically, in the second Fab fragment capable of specifically binding to LTBR, ​​the variable domains VL and VH are substituted for each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

[0224] In another embodiment, a bispecific agonist LTBR antibody is provided comprising (a) a first Fab fragment capable of specifically binding to FAP, wherein 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 domain is part of the heavy chain, and (b) a second Fab fragment capable of specifically binding to LTBR. Such a molecule provides monovalent binding to both LTBR and FAP.

[0225] In another embodiment, a bispecific a...

Claims

1. An agonistrin phototoxin beta receptor (LTBR) antibody that binds to human LTBR and cynomolgus monkey LTBR, ​​wherein the antibody is The heavy chain complementarity determination region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, the heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 28, and the heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:

29. Ｈ Light chain variable region (V) including LTBR), a light chain complementarity determining region (CDR-L1) containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

32. Ｌ LTBR) including and Agonist LTBR antibody is an agonist antibody that further binds to mouse LTBR.

2. The agonist LTBR antibody according to claim 1, wherein the antibody binds to the epitope region of Sequence ID No. 351 on a human LTBR.

3. Agonist antibodies Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V Ｈ Light chain variable region (V) containing the LTBR) and the amino acid sequence of SEQ ID NO: 34 Ｌ LTBR) An agonist LTBR antibody according to claim 1 or 2, comprising:

4. The agonist LTBR antibody according to claim 1, wherein the agonist antibody is a multispecific antibody, particularly a bispecific antibody.

5. The agonist LTBR antibody according to claim 1, wherein the agonist antibody comprises a human-derived Fc domain, particularly a human IgG subclass, more specifically a human IgG1 subclass.

6. The agonist LTBR antibody according to claim 5, wherein the agonist antibody comprises an Fc domain of a human IgG1 subclass having one or more amino acid substitutions that reduce the binding affinity and / or effector function of an antigen-binding molecule to an Fc receptor.

7. The agonist LTBR antibody according to claim 6, wherein the agonist antibody comprises an Fc domain of a human IgG1 subclass having amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

8. The agonist LTBR antibody according to claim 4, wherein the agonist antibody is a bispecific antibody that binds to LTBR and tumor-associated antigens (TAAs), particularly fibroblast-activating protein (FAP).

9. Agonist antibodies (a) A first antigen-binding domain that specifically binds to fibroblast-activating protein (FAP), (i) A heavy chain complementarity determination region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 3, a heavy chain variable region (V H FAP) containing the amino acid sequence of SEQ ID NO: 4, a heavy chain variable region (V L FAP) containing the amino acid sequence of SEQ ID NO: 5, a light chain complementarity determination region (CDR-L1) containing the amino acid sequence of SEQ ID NO: 6, a light chain variable region (V L FAP) containing the amino acid sequence of SEQ ID NO: 7, a light chain variable region (V L FAP) containing the amino acid sequence of SEQ ID NO: 8, or (ii) Heavy chain variable region (V H FAP) including CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and light chain variable region (V L FAP) including CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

24. The first antigen-binding domain, (b) A second antigen-binding domain that specifically binds to the LTBR, A second antigen-binding domain comprising a heavy chain complementarity-determining region (CDR-H1) containing the amino acid sequence of SEQ ID NO: 27, a heavy chain variable region (VHLTBR) containing the amino acid sequence of SEQ ID NO: 28, and a light chain variable region (VLLTBR) containing the amino acid sequence of SEQ ID NO: 29, and a light chain complementarity-determining region (CDR-L1) containing the amino acid sequence of SEQ ID NO: 30, a light chain variable region (VLLTBR) containing the amino acid sequence of SEQ ID NO: 31, and a light chain variable region (VLLTBR) containing the amino acid sequence of SEQ ID NO: 32, and (c) Fc domain of a human IgG1 subclass containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. The agonist LTBR antibody according to claim 8, which is a bispecific antibody containing the above.

10. The agonist LTBR antibody according to claim 9, wherein the bispecific antibody comprises a third antigen-binding domain that binds to human LTBR.

11. The agonist LTBR antibody according to claim 10, wherein the third antigen-binding domain that binds to human LTBR is identical to the second antigen-binding domain that binds to LTBR.

12. The first antigen-binding domain that specifically binds to FAP contains a heavy chain variable region (V) that includes an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO:

9. Ｈ FAP), and a light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO:

10. Ｌ A heavy chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO: 25, or a first antigen-binding domain that specifically binds to FAP. Ｈ FAP), and a light chain variable region (V) containing an amino acid sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO:

26. Ｌ The agonist LTBR antibody according to claim 9, comprising FAP.

13. The first antigen-binding domain that specifically binds to FAP comprises a heavy-chain variable region (V Ｈ FAP) comprising the amino acid sequence of SEQ ID NO: 9, and a light-chain variable region (V Ｌ FAP) comprising the amino acid sequence of SEQ ID NO: 10, or the first antigen-binding domain that specifically binds to FAP comprises a heavy-chain variable region (V Ｈ FAP) comprising the amino acid sequence of SEQ ID NO: 25, and a light-chain variable region (V Ｌ FAP) comprising the amino acid sequence of SEQ ID NO: 26, the agonist LTBR antibody according to claim 12.

14. The aforementioned agonist LTBR antibody (a) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9 (V Ｈ FAP) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 10 Ｌ A first antigen-binding domain that specifically binds to FAP, including FAP, and (b) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 (V Ｈ Light chain variable region (V) containing the LTBR) and the amino acid sequence of SEQ ID NO: 34 Ｌ A second antigen-binding domain that specifically binds to LTBR, ​​including LTBR. The agonist LTBR antibody according to claim 13, which is a bispecific antibody containing the above.

15. The aforementioned agonist LTBR antibody (a) First Fab fragment that binds to FAP, (b) A second Fab fragment that binds to human LTBR, ​​and (c) Fc domain of a human IgG1 subclass containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. The agonist LTBR antibody according to claim 9, which is a bispecific antibody containing the above.

16. The agonist LTBR antibody according to claim 15, wherein the second Fab fragment that specifically binds to human LTBR is a crossFab fragment.

17. The aforementioned agonist LTBR antibody (a) First Fab fragment that binds to FAP, (b) A second Fab fragment and a third Fab fragment that bind to human LTBR, ​​and (c) Fc domain of a human IgG1 subclass containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of antigen-binding molecules to the Fc receptor. It is a bispecific antibody that includes, The agonist LTBR antibody according to claim 15, wherein a first Fab fragment that binds to FAP is fused at its N-terminus to one C-terminus of an Fc domain subunit, and a second Fab fragment and a third Fab fragment that specifically bind to LTBR are each fused at their C-terminuses to one N-terminus of an Fc domain subunit.

18. The agonist LTBR antibody according to claim 9, wherein the agonist LTBR antibody is a bispecific antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 199, a second heavy chain containing the amino acid sequence of SEQ ID NO: 201, two light chains each containing the amino acid sequence of SEQ ID NO: 200, and one light chain containing the amino acid sequence of SEQ ID NO:

202.

19. The agonist LTBR antibody according to claim 9, wherein the agonist LTBR antibody is a bispecific antibody comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 195, a second heavy chain containing the amino acid sequence of SEQ ID NO: 197, two light chains each containing the amino acid sequence of SEQ ID NO: 196, and one light chain containing the amino acid sequence of SEQ ID NO:

198.

20. One or more isolated polynucleotides encoding the agonist LTBR antibody described in claim 1.

21. An expression vector comprising one or more isolated polynucleotides as described in claim 20.

22. A prokaryotic or eukaryotic host cell comprising one or more isolated polynucleotides according to claim 20 or an expression vector according to claim 21.

23. A method for producing a bispecific antigen-binding molecule, comprising: a) culturing a prokaryotic or eukaryotic host cell as described in claim 22 under conditions suitable for the expression of an agonist LTBR antibody; and b) selectively recovering an agonist LTBR antibody.

24. A pharmaceutical composition comprising the agonist LTBR antibody described in claim 9 and a pharmaceutically acceptable additive.

25. An agonist LTBR antibody according to claim 9, or a pharmaceutical composition according to claim 24, for use as a pharmaceutical.

26. The agonist LTBR antibody according to claim 9 for use in (a) in inducing ICAM upregulation on endothelial cells or cancer-associated fibroblasts, or (b) in enhancing T cell adhesion.

27. An agonist LTBR antibody according to claim 9, or a pharmaceutical composition according to claim 24, for use in the treatment of cancer.

28. The agonist LTBR antibody according to claim 9, or the pharmaceutical composition according to claim 24, for use in the treatment of cancer, wherein the agonist LTBR antibody or pharmaceutical composition is for administration in combination with chemotherapeutic agents, radiation and / or other agents for use in cancer immunotherapy.

29. An agonist LTBR antibody according to claim 9, or a pharmaceutical composition according to claim 24, for use in the treatment of cancer, wherein the agonist LTBR antibody is for administration in combination with a drug that blocks PD-L1 / PD-1 interaction.

30. Use of the agonist LTBR antibody according to claim 9, or the pharmaceutical composition according to claim 24, in the manufacture of a pharmaceutical for the treatment of cancer.

31. A pharmaceutical for treating an individual having cancer, comprising an effective amount of the agonist LTBR antibody described in claim 9, or the pharmaceutical composition described in claim 24.

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