Agonistic 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 activation proteins (FAPs), methods for making these molecules, and methods for using them. [Background technology]

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

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

[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 either induce cell death (e.g., DR5-TRAIL, Fas-FasL) or promote survival and inflammation (e.g., TNF-TNFR). The lymphotoxin beta receptor (LTBR) is a member of this second category of TNFR superfamily and is expressed by a variety of cells, including stromal cells, myeloid cells, and tumor cells of epithelial origin in the tumor microenvironment. Ligand-receptor interactions within the TNFR superfamily can be monovalent or multivalent. For example, OX40 and its ligand (OX40L) form a monotypic ligand-receptor pair, whereas LTBR, ​​LTα, LTβ, and LIGHT exhibit multivalent interactions, forming a complex network of interconnected pathways. Activation of LTBR by lymphotoxin α1β1 (LTα1β1) or LIGHT ligand binding induces receptor oligomerization, signaling through canonical and non-canonical NFκB pathways and upregulating 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 fail to develop lymph nodes or Peyer's patches (Fuetterer et al., Immunity 1998,9(1),59-70,doi:10.1016 / s1074-7613(00)80588-9). Furthermore, it is 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 are detected histologically, consisting of T cells, activated dendritic cells (DCs), B cells, and HEVs.Clinical evidence has shown that the presence of these ectopic lymphoid organs, also known as tertiary lymphoid tissue-like structures (TLS), and the presence of HEVs correlates with improved prognosis in several tumor indications (Dieu-Nosjean et al., Immunol. Rev. 2016, 271(1), 260-275, doi:10.1111 / imr.12405). TLS and HEVs are thought to form in response to the same molecular cues 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 antitumor immune responses.

[0005] Indeed, preclinical evidence supports the hypothesis that LTBR activation increases immune infiltration, enhances responses to checkpoint inhibitors, and induces the formation of TLS. LTBR activation by 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). Consequently, 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 was 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 an LTBR agonist humanized antibody (hCBE11) in patients with advanced solid tumors (ClinicalTrials.gov, NCT00105170). The trial was halted and subsequently terminated before patient enrollment was complete, suggesting the potential for significant safety issues associated with broad LTBR agonism in humans.

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

[0008] The present invention relates to novel antibodies that specifically bind to the human lymphotoxin beta receptor (LTBR), particularly agonistic hu LTBR antibodies. These antibodies can bind to human LTBR and cynomolgus monkey LTBR with less than a two-fold difference in affinity. Some of the novel agonistic hu LTBR antibodies can also bind to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR. The present invention also relates to multispecific antibodies comprising these novel agonistic antibodies.

[0009] Accordingly, provided herein are agonistic lymphotoxin beta receptor (LTBR) antibodies that specifically bind to human LTBR and cynomolgus monkey LTBR, ​​wherein the antibodies comprise: (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; H(iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 CDR-H1 having the amino acid sequence of SEQ ID NO: 43, CDR-H2 having the amino acid sequence of SEQ ID NO: 44, and CDR-H3 having the amino acid sequence of SEQ ID NO: 45; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L LTBR); or (iii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 75, CDR-H2 having the amino acid sequence of SEQ ID NO: 76, and CDR-H3 having the amino acid sequence of SEQ ID NO: 77; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L LTBR); or (iv) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 83, CDR-H2 having the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 having the amino acid sequence of SEQ ID NO: 85; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO: 88. L LTBR); or (v) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 35, CDR-H2 having the amino acid sequence of SEQ ID NO: 36, and CDR-H3 having the amino acid sequence of SEQ ID NO: 37; H a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 38, CDR-L2 having the amino acid sequence of SEQ ID NO: 39, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40;L LTBR), or (vi) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 51, CDR-H2 having the amino acid sequence of SEQ ID NO: 52, and CDR-H3 having the amino acid sequence of SEQ ID NO: 53; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 54, CDR-L2 having the amino acid sequence of SEQ ID NO: 55, and CDR-L3 having the amino acid sequence of SEQ ID NO: 56. L LTBR), or (vii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 59, CDR-H2 having the amino acid sequence of SEQ ID NO: 60, and CDR-H3 having the amino acid sequence of SEQ ID NO: 61; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 62, CDR-L2 having the amino acid sequence of SEQ ID NO: 63, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64. L LTBR); or (viii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 67, CDR-H2 having the amino acid sequence of SEQ ID NO: 68, and CDR-H3 having the amino acid sequence of SEQ ID NO: 69 H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 70, CDR-L2 having the amino acid sequence of SEQ ID NO: 71, and CDR-L3 having the amino acid sequence of SEQ ID NO: 72. L LTBR) Includes:

[0010] In one aspect, an agonist LTBR antibody as described herein is provided, wherein the agonist antibody has the following characteristics: (a) binds to human LTBR and cynomolgus monkey LTBR with less than a two-fold difference in affinity; or (b) EC<4 nM as measured by ELISA 50 binds to the human LTBR extracellular domain at 1000 kJ / s and has an EC of less than 5 nM as measured by ELISA 50 binds to the cynomolgus monkey LTBR extracellular domain; or (c) human LTBR activation requires cross-linking for its agonistic activity; or (d) induces upregulation of ICAM in human umbilical vein endothelial cells or cancer-associated fibroblasts, requiring cross-linking for its agonist activity; or (e) inhibiting the interaction between human LTBR and its human ligands, lymphotoxin α1β2 and LIGHT The present invention has at least one of the following:

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

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

[0013] In one aspect, an agonist LTBR antibody as described herein above is provided, wherein the agonist LTBR antibody further specifically binds to murine LTBR. In one aspect, the agonist LTBR antibody has an EC50 of less than 1 nM as measured by ELISA. 50 It binds to the mouse LTBR extracellular domain.

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

[0015] In one embodiment, an agonist LTBR antibody that further specifically binds to mouse LTBR is A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 33 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 81 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 82 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 89 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 90 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 97 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 98 L LTBR) Includes:

[0016] In one particular embodiment, the agonist LTBR antibodies described herein bind to the epitope region of SEQ ID NO: 351 on human LTBR. In one embodiment, such agonist LTBR antibodies comprise a heavy chain variable region (VH1) 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. H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 In one embodiment, the heavy chain variable region (VLTBR) comprises the amino acid sequence of SEQ ID NO: 33. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L LTBR).

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

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

[0019] In one aspect, an agonistic LTBR antibody is provided, which is a bispecific antibody capable of specifically binding to lymphotoxin beta receptor (LTBR) and fibroblast activation protein (FAP), and thus combines an antigen-binding domain that specifically binds to FAP with at least one antigen-binding domain that can agonistically bind to LTBR, ​​particularly hu LTBR, ​​wherein LTBR-mediated activation is provided by cross-linking via binding to FAP expressed on tumor stromal cells. In contrast to conventional non-targeting agonistic LTBR antibodies, targeting a tumor-associated target (TAA) such as FAP can restrict LTBR agonism to only the tumor microenvironment (tumor endothelium and cancer-associated fibroblasts), thereby reducing potential side effects.

[0020] Fibroblast activation 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 has very limited expression in other normal tissues. The widespread presence of FAP in various cancer indications allows its use as a targeting moiety for drugs that accumulate within the tumor stroma.

[0021] Thus, in one embodiment, a bispecific agonist LTBR antibody comprising: (a) a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP); (b) a second 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, the Fc domain containing one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor and / or the effector function. Bispecific agonist LTBR antibodies are provided, comprising:

[0022] The bispecific antibody has an Fc domain composed of a first subunit and a second subunit containing one or more amino acid substitutions that reduce the antibody's binding affinity to the Fc receptor and / or effector function. This releases Fc receptor-mediated cross-linking, and tumor-specific activation is achieved by cross-linking via the binding of antigen-binding domains that specifically bind 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 comprising: (a) a first Fab fragment that specifically binds to fibroblast activation protein (FAP); (b) a second Fab fragment that specifically binds to the lymphotoxin beta receptor (LTBR), and (c) an Fc domain composed of a first subunit and a second subunit and containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor; Bispecific agonist LTBR antibodies are provided, comprising:

[0024] In one aspect, a bispecific agonist LTBR antibody is provided, which comprises a third antigen-binding domain that specifically binds to LTBR, ​​i.e., (a) a first antigen-binding domain that specifically binds to fibroblast activation 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 and comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor.

[0025] In a specific 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 antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR) and the third antigen-binding domain 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 aspect, the bispecific agonist LTBR antibody disclosed herein comprises a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP), (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: 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; H(iv) a light chain variable region (V FAP) comprising a light chain complementarity determining region (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; L FAP), or (ii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 19, CDR-H2 having the amino acid sequence of SEQ ID NO: 20, and CDR-H3 having the amino acid sequence of SEQ ID NO: 21; H and a light chain variable region (VFAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 22, CDR-L2 having the amino acid sequence of SEQ ID NO: 23, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24. L FAP), or (iii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 11, CDR-H2 having the amino acid sequence of SEQ ID NO: 12, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13; H and a light chain variable region (V FAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 15, and CDR-L3 having the amino acid sequence of SEQ ID NO: 16. L FAP) The antibody comprises a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP), comprising:

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

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

[0029] In another aspect, the bispecific agonist LTBR antibodies disclosed herein comprise a second antigen-binding domain that specifically binds to LTBR, (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; H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 CDR-H1 having the amino acid sequence of SEQ ID NO: 43, CDR-H2 having the amino acid sequence of SEQ ID NO: 44, and CDR-H3 having the amino acid sequence of SEQ ID NO: 45; HLTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L LTBR); or (iii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 75, CDR-H2 having the amino acid sequence of SEQ ID NO: 76, and CDR-H3 having the amino acid sequence of SEQ ID NO: 77; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L LTBR); or (iv) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 83, CDR-H2 having the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 having the amino acid sequence of SEQ ID NO: 85; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO: 88. L LTBR); or (v) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 35, CDR-H2 having the amino acid sequence of SEQ ID NO: 36, and CDR-H3 having the amino acid sequence of SEQ ID NO: 37; H a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 38, CDR-L2 having the amino acid sequence of SEQ ID NO: 39, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40; L LTBR), or (vi) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 51, CDR-H2 having the amino acid sequence of SEQ ID NO: 52, and CDR-H3 having the amino acid sequence of SEQ ID NO: 53; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 54, CDR-L2 having the amino acid sequence of SEQ ID NO: 55, and CDR-L3 having the amino acid sequence of SEQ ID NO: 56. LLTBR), or (vii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 59, CDR-H2 having the amino acid sequence of SEQ ID NO: 60, and CDR-H3 having the amino acid sequence of SEQ ID NO: 61; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 62, CDR-L2 having the amino acid sequence of SEQ ID NO: 63, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64. L LTBR); or (viii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 67, CDR-H2 having the amino acid sequence of SEQ ID NO: 68, and CDR-H3 having the amino acid sequence of SEQ ID NO: 69 H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 70, CDR-L2 having the amino acid sequence of SEQ ID NO: 71, and CDR-L3 having the amino acid sequence of SEQ ID NO: 72. L LTBR) and a second antigen-binding domain that specifically binds to LTBR, ​​comprising:

[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; H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 CDR-H1 having the amino acid sequence of SEQ ID NO: 43, CDR-H2 having the amino acid sequence of SEQ ID NO: 44, and CDR-H3 having the amino acid sequence of SEQ ID NO: 45; HLTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L LTBR); or (iii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 75, CDR-H2 having the amino acid sequence of SEQ ID NO: 76, and CDR-H3 having the amino acid sequence of SEQ ID NO: 77; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L LTBR); or (iv) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 83, CDR-H2 having the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 having the amino acid sequence of SEQ ID NO: 85; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO: 88. L LTBR); or (v) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 35, CDR-H2 having the amino acid sequence of SEQ ID NO: 36, and CDR-H3 having the amino acid sequence of SEQ ID NO: 37; H a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 38, CDR-L2 having the amino acid sequence of SEQ ID NO: 39, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40; L LTBR), or (vi) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 51, CDR-H2 having the amino acid sequence of SEQ ID NO: 52, and CDR-H3 having the amino acid sequence of SEQ ID NO: 53; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 54, CDR-L2 having the amino acid sequence of SEQ ID NO: 55, and CDR-L3 having the amino acid sequence of SEQ ID NO: 56. LLTBR), or (vii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 59, CDR-H2 having the amino acid sequence of SEQ ID NO: 60, and CDR-H3 having the amino acid sequence of SEQ ID NO: 61; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 62, CDR-L2 having the amino acid sequence of SEQ ID NO: 63, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64. L LTBR); or (viii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 67, CDR-H2 having the amino acid sequence of SEQ ID NO: 68, and CDR-H3 having the amino acid sequence of SEQ ID NO: 69 H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 70, CDR-L2 having the amino acid sequence of SEQ ID NO: 71, and CDR-L3 having the amino acid sequence of SEQ ID NO: 72. L LTBR) Includes:

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

[0032] In one aspect, the bispecific agonist LTBR antibodies disclosed herein comprise a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR, (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 33 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L LTBR), or (ii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L LTBR), or (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 81 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 82 L LTBR), or (iv) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 89 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 90 L LTBR), or (v) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 97 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 98 L LTBR), or (vi) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L LTBR), or (vii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 57 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 58 L LTBR), or (viii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 65 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 66 L LTBR), (ix) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 73 HLTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 74 L LTBR) and optionally a third antigen-binding domain) that specifically binds to LTBR, ​​comprising:

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

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

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

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

[0037] Thus, in one particular embodiment, the bispecific agonist LTBR antibody described herein comprises: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 9; HFAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10 L (b) a first antigen-binding domain that specifically binds to FAP, comprising the amino acid sequence of SEQ ID NO: 33; and (c) a heavy chain variable region (V H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L In another aspect, the bispecific agonist LTBR antibody described herein comprises a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR, ​​comprising: (a) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 9; H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10 L (b) a first antigen-binding domain that specifically binds to FAP, comprising the amino acid sequence of SEQ ID NO: 41; and (c) a heavy chain variable region (V H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L In yet another aspect, the bispecific antigen-binding molecule described herein comprises a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR, ​​comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 9. H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10 L (b) a first antigen-binding domain that specifically binds to FAP, comprising the amino acid sequence of SEQ ID NO: 49; and (c) a heavy chain variable region (V H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L LTBR), and a second antigen-binding domain (and optionally a third antigen-binding domain) that specifically binds to LTBR.

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

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

[0040] In a further embodiment, the bispecific agonist LTBR antibody as defined herein above, (a) a first Fab fragment that specifically binds to a FAP; (b) a second Fab fragment that specifically binds to LTBR, ​​and (c) an Fc domain composed of a first subunit and a second subunit, the Fc domain containing one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor and / or the effector function. 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 aspect, (a) a first Fab fragment that specifically binds to a FAP; (b) a second Fab fragment and a third Fab fragment that specifically bind to LTBR, ​​and (c) an Fc domain composed of a first subunit and a second subunit, the Fc domain containing one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor and / or the effector function. a first Fab fragment that specifically binds to FAP fused at its N-terminus to the C-terminus of one of the Fc domain subunits, and a second Fab fragment and a third Fab fragment that specifically bind to LTBR each fused at their C-terminus to the N-terminus of one of the Fc domain subunits.

[0042] Thus, bispecific agonist LTBR antibodies are provided that provide bivalent binding to LTBR and monovalent binding to FAP.

[0043] According to another aspect of the present invention, there are provided one or more isolated polynucleotides encoding the agonist LTBR antibody or bispecific agonist LTBR antibody described hereinabove. 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 embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell. In another aspect, there is provided a method for producing the agonist LTBR antibody or bispecific agonist LTBR antibody described hereinabove, comprising culturing such a host cell under conditions suitable for 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 hereinabove and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises an additional therapeutic agent.

[0045] The present invention also encompasses an agonist LTBR antibody or a bispecific agonist LTBR antibody as described herein above, or a pharmaceutical composition comprising a bispecific agonist LTBR antibody for use as a medicament.

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

[0047] In a particular aspect, there is provided an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein, or a pharmaceutical composition of the invention, for use in the treatment of cancer. In another particular aspect, the present invention provides an agonist LTBR antibody or bispecific agonist LTBR antibody as 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, radiation, and / or other agent for use in cancer immunotherapy. In one aspect, the agonist LTBR antibody or bispecific agonist LTBR antibody described herein is for use in the treatment of cancer, wherein 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 aspect, there is provided an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein, or a pharmaceutical composition of the invention, for use in upregulating or extending cytotoxic T cell activity.

[0048] In a further aspect, the present invention provides a method of inhibiting tumor cell growth in an individual, comprising administering to the individual an effective amount of an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein above, or a pharmaceutical composition of the invention, thereby inhibiting tumor cell growth. In another aspect, the present invention provides a method of treating or delaying cancer in an individual, comprising administering to the individual an effective amount of an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein above, or a pharmaceutical composition of the invention.

[0049] Also provided is the use of a bispecific antigen-binding molecule as described herein for the manufacture of a medicament for the treatment of a disease, particularly for the manufacture of a medicament for the treatment of cancer, in an individual in need thereof, as well as a method for treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising an agonist LTBR antibody or a bispecific agonist LTBR antibody of the present invention in a pharmaceutically acceptable form. In a particular embodiment, the disease is cancer. In any of the above embodiments, the individual is a mammal, particularly a human. [Brief explanation of the drawings]

[0050] [Figure 1A-G] Figures 1A-1G show schematic diagrams of recombinant soluble proteins (receptor, ligand, and tool proteins) used as antigens for phage display and immunization to generate anti-LTBR antibodies. Figure 1A shows a schematic diagram of the so-called biotinylated Fc depleter (Fc depleter kh NC avi biotinylated, P1AA0981). This is composed of a knob-into-hole (kh) Fc domain, which is 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 the monomeric huLTBR ectodomain ECD (S28-M227) as Fc-fused avi biotinylated (P1AE2835). Figure 1E shows a schematic diagram of monomeric muLTBR ectodomain ECD (S28-L223) Fc-fused avi biotinylated (P1AE4410). Figure 1F shows a schematic diagram of monomeric cynoLTBR ectodomain ECD (S28-M227) Fc-fused avi biotinylated (P1AE4411). Figure 1G shows a schematic diagram of monomeric N-terminal human LTBR (ECD full-length) hu IgG1 Fc-fused kih HRYF avi (P1AE1217). [Figure 2A-F]Figures 2A-2F show schematic diagrams of additional antigens used in screening anti-LTBR and bispecific FAP-LTBR antibodies and clone characterization. Figure 2A shows a schematic diagram of a dimeric hu LTBR-Fc fusion biotinylated (P1AE2401). Figure 2B shows a schematic diagram of an N- and C-terminal extension of human LTBR ECD (S28-M227)-Fc fusion wt kih HRYF-Avi-His biotinylated (P1AE7979). Figure 2C shows a schematic diagram of a monomeric N-terminal cyno LTbR (full-length ECD) hu IgG1 Fc fusion kih HRYF avi biotinylated (P1AE2656). Figure 2D shows a schematic diagram of a dimeric mouse LTBR-Fc disulfide-linked homodimer biotinylated (P1AE2655). Figure 2E shows a schematic diagram of human FAP-Avi-His biotinylated (P1AA5347), and Figure 2F shows a schematic diagram of mouse FAP-Avi-His biotinylated (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, which determined specific binding to human LTBR (P1AE2835) and mouse LTBR (P1AE4410). This antibody was derived from phage display. Because the antigen used for phage display was an Fc-tagged fusion protein, a human Fc fragment (P1AA0981) was used to confirm specific binding to LTBR and rule out Fc binding. The Y-axis represents OD450-900. [Figure 4] 4 shows the inhibition of LTBR-lymphotoxin α1β2 interaction by anti-LTBR IgG antibodies. Selected anti-LTBR IgGs were titrated in 3-fold dilutions starting at 100 nM (15 μg / ml). [Figure 5] Figure 5 shows the inhibition of LTBR-LIGHT interaction by anti-LTBR IgG antibodies. Selected anti-LTBR IgGs were titrated in 3-fold dilutions starting at 100 nM (15 μg / ml). [Figure 6]Figure 6 shows the results of various anti-LTBR IgGs in the absence or presence of a bridging antibody (linker) as determined in 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 addition of luciferase detection solution. [Figure 7] Figure 7 shows the results of assessment of cell surface LTBR binding of anti-LTBR IgG (FACS on recombinant human LTBR CHO cells) plotted as median fluorescence intensity versus concentration. Anti-LTBR IgG was used in a titration with 3-fold dilution steps starting at 20 μg / ml. [Figure 8] FIG. 8 shows an epitope binning heat map based on the binding values ​​(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 with crossed V domains 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 with crossed CH1 / Ck domains 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 with crossed CH1 / Ck domains 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 the 2+1 anti-LTBR / anti-FAP bispecific antibody as a human IgG1 PG LALA crossMab with crossed V domains in both anti-LTBR Fab arms and charged CH1 / Ck domains of the anti-Fab arms fused to the C-terminus of the Fc domain. [Figure 10]Figure 10 shows inhibition of LTBR-lymphotoxin α1β2 interaction by anti-FAP / anti-LTBR bispecific antibodies. Selected anti-FAP / anti-LTBR bispecific antibodies were titrated in 3-fold dilutions starting at 100 nM (15 μg / ml). [Figure 11] Figure 11 shows inhibition of LTBR-LIGHT interaction by anti-FAP / anti-LTBR bispecific antibodies. Selected anti-FAP / anti-LTBR bispecific antibodies were titrated in 3-fold dilutions starting at 100 nM (15 μg / ml). [Figure 12A-C] Figures 12A and 12B show schematic diagrams of murine surrogate bispecific FAP-LTBR antibodies. Figure 12A shows a schematic diagram of a 1+1 anti-LTBR / anti-FAP bispecific antibody as a murine IgG1 DA PG crossMab with crossed V domains in the anti-Fab arms and charged CH1 / Ck domains in the anti-muLTBR Fab arms. Figure 12B shows a schematic diagram of a 2+1 anti-LTBR / anti-FAP bispecific antibody as a murine IgG1 DA PG crossMab with charged CH1 / Ck domains in both anti-LTBR Fabs and crossed CH1 / Ck domains of the anti-FAP Fab fragments fused to the C-terminus of the Fc domain. Figure 12C shows a schematic diagram of a 2+1 anti-LTBR / non-targeting (DP47) bispecific antibody as a murine IgG1 DA PG crossMab with charged CH1 / Ck domains in both anti-LTBR Fabs and crossed CH1 / Ck domains of the non-targeting (DP47) Fab fragment fused to the C-terminus of the Fc domain. [Figures 13A-D] Figures 13A-13D show that anti-LTBR agonist antibodies upregulate ICAM on endothelial cells in a crosslinking-dependent manner. Median fluorescence intensity of ICAM on human umbilical vein endothelial cells (HUVECs) treated with anti-human LTBR agonist antibodies in the presence (Figures 13A and 13C) or absence (Figures 13B and 13D) of an Fc crosslinking antibody (anti-Fc crosslinker) is shown. Data are normalized to the activity of the anti-LTBR antibody CBE11 (P1AE1873). [Figure 14A-D]Figures 14A-14D show that anti-LTBR agonist antibodies upregulate ICAM on cancer-associated fibroblasts (CAFs) in a crosslinking-dependent manner. Median fluorescence intensity of ICAM on immortalized CAFs treated with anti-human LTBR agonist antibodies in the presence (Figures 14A and 14C) or absence (Figures 14B and 14D) of an anti-Fc crosslinker is shown. Data are normalized to the activity of the anti-LTBR antibody CBE11 (P1AE1873). [Figure 15A-C] Figures 15A-15F show binding of FAP-LTBR bispecific antigen-binding molecules to human (Figures 15A and 15D), cynomolgus monkey (Figures 15B and 15E), and mouse LTBR (Figures 15C and 15F) on CHO-K1 cells engineered to overexpress human, cynomolgus monkey, or mouse LTBR. Median fluorescence intensity values ​​of anti-human Fc secondary antibodies detecting bound FAP-LTBR bispecific molecules on cells overexpressing human LTBR (Figures 15A and 15D), cynomolgus monkey LTBR (Figures 15B and 15E), or mouse LTBR (Figures 15C and 15F) are shown. Data are normalized to baseline. [Figure 15D-F] Figures 15A-15F show binding of FAP-LTBR bispecific antigen-binding molecules to human (Figures 15A and 15D), cynomolgus monkey (Figures 15B and 15E), and mouse LTBR (Figures 15C and 15F) on CHO-K1 cells engineered to overexpress human, cynomolgus monkey, or mouse LTBR. Median fluorescence intensity values ​​of anti-human Fc secondary antibodies detecting bound FAP-LTBR bispecific molecules on cells overexpressing human LTBR (Figures 15A and 15D), cynomolgus monkey LTBR (Figures 15B and 15E), or mouse LTBR (Figures 15C and 15F) are shown. Data are normalized to baseline. [Figures 16A-D]Figures 16A-16D show that the FAP-LTBR bispecific antigen binding molecule up-regulates ICAM on cancer-associated fibroblasts in a FAP-dependent manner. Median fluorescence intensity of ICAM on immortalized CAFs endogenously expressing LTBR and FAP (Figures 16A and 16C, hTERT CAFs) or LTBR alone (Figures 16B and 16D, hTERT CAF_delFAP) is shown. Data are normalized to ICAM expression in untreated controls. [Figures 17A-D] Figures 17A-17D show that the FAP-LTBR bispecific molecule upregulates ICAM on endothelial cells in a FAP-dependent manner. Median fluorescence intensity of ICAM on CD31+ HUVECs cocultured with NIH-3T3 cells overexpressing FAP (Figures 17A and 17C) or not expressing FAP (Figures 17B and 17D) is shown. Data are normalized to ICAM expression in untreated controls, and EC50 values ​​are shown in the legend. [Figures 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 from HUVECs cocultured with NIH-3T3 cells overexpressing FAP (Figures 18A, 18C, and 18E) or non-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 by 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 from HUVECs cocultured with NIH-3T3 cells overexpressing FAP (Figures 18A, 18C, and 18E) or non-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 by Bio-plex. [Figure 19]Figure 19 shows increased T cell adhesion on endothelium stimulated with the FAP-LTBR bispecific antigen-binding molecule. The area of ​​labeled T cells adhered to HUVEC cultures stimulated with the FAP-LTBR bispecific antibody (2 nM) in the presence (black bars) or absence (white bars) of FAP is shown. TNFα (0.5 ng / mL) is used as a positive control. [Figure 20A-B] Figures 20A and 20B demonstrate that FAP-LTBR bispecific antibody surrogate molecules upregulate adhesion molecules on mouse fibroblasts in a FAP-dependent manner in vitro. Median fluorescence intensity of VCAM on NIH-3T3 cells (Figure 20A) or NIH-3T3 FAP-overexpressing cells (Figure 20B) treated with FAP-LTBR antibody surrogate molecules or anti-mouse LTBR agonist antibody (5G11) is shown. Data are normalized to baseline VCAM expression in untreated controls. [Figure 21A-B] Figures 21A and 21B show that the FAP-LTBR bispecific antibody surrogate molecule upregulates adhesion molecules on endothelial cells in a FAP-dependent manner in vitro. Median fluorescence intensity of ICAM on CD31+ HUVECs cocultured with NIH-3T3 cells (Figure 21A) or FAP-overexpressing NIH-3T3 cells (Figure 21B) is shown. [Figure 22] Figure 22 shows the study design for an in vivo mouse study to evaluate the safety, efficacy, and pharmacodynamic profile of a FAP-LTBR bispecific antibody alone or in combination with an anti-PD-L1 antibody in the subcutaneous MC38-hu CEA tumor model. The timeline describes treatment and sacrifice time points, and the table details the treatment arms, doses, and schedule for the study. [Figure 23A-B]Figure 23A shows that the FAP-LTBR surrogate molecule inhibits tumor growth and improves response to aPD-L1 treatment in the MC38-huCEA subcutaneous tumor model as a monotherapy. Tumor growth curves for mice treated with vehicle, P1AF4664, P1AF4674, aPD-L1, P1AF4664 + aPD-L1, and P1AF4674 + aPD-L1 are plotted in Figure 23A. Comparison of tumor volumes on day 12 of treatment showed statistically significantly larger volumes for 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 followed by Holm-Sidak's multiple comparison test (*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 the FAP-LTBR surrogate molecule was able to induce endothelial activation in the 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 ICAM fluorescence intensity (Figures 24A and 24B) was quantified. The data show that treatment with P1AF4664 and P1AF4674 increased ICAM expression on tumor endothelial cells throughout the treatment period. Statistical analysis was performed using one-way ANOVA followed by Holm-Sidak's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001). [Figure 25]Figure 25 shows the study design for an in vivo mouse study to evaluate the safety, efficacy, and pharmacodynamic profile of a FAP-LTBR bispecific antibody alone or in combination with an anti-PD-L1 antibody in the subcutaneous KPC4662-huCEA tumor model. The timeline describes treatment and sacrifice time points, and the table details the treatment arms, doses, and schedule for this KPC4662-huCEA trial protocol. [Figure 26A-C] Figures 26A-26C demonstrate that the FAP-LTBR surrogate molecule induces high endothelial venules in the KPC4662-huCEA subcutaneous tumor model. A significant increase in Meca79-positive vessels, as a percentage of total vessels, was noted in the treatment group compared with the vehicle group. Statistical analysis was performed using one-way ANOVA followed by Holm-Sidak's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001) (Figure 26A). Representative immunofluorescence staining for CD31 (total vessels) and Meca79 (HEVs) in P1AF4664-treated samples 6 days after treatment initiation is shown in Figures 26B and 26C, respectively. [Figure 27] Figure 27 shows the study design for an in vivo mouse study to evaluate the safety, efficacy, and pharmacodynamic profile of LTBR bispecific antigen-binding molecules alone or in combination with an anti-PD-L1 antibody in an orthotopic EMT6 tumor model. The timeline lists treatment and sacrifice time points, and the table details the treatment groups, doses, and schedule for this EMT6 study protocol. Vehicle and the mouse surrogate LTBR bispecific antigen-binding molecules P1AG5459 and P1AG5461 (non-targeting control) were injected at all time points listed above (d0, d2, d5, d7, etc.) unless otherwise specified. PDL1 was only injected at the time points indicated by the empty arrows unless otherwise specified. [Figure 28A-B]Figures 28A and 28B show that the murine surrogate FAP-LTBR bispecific antibody (P1AG5459) inhibits tumor growth and improves response to aPD-L1 treatment in the EMT6 orthotopic tumor model as a monotherapy. Median tumor growth curves for vehicle-, P1AG5459-, P1AG5461-, aPD-L1-, and P1AG5459+aPD-L1-treated mice are plotted in Figure 28A. Comparisons of tumor volumes at 22 days after treatment initiation are plotted in Figure 28B, showing statistically significantly larger volumes for 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's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001). [Figure 29A-C] Figures 29A-29C demonstrate that the murine surrogate LTBR bispecific antibody P1AG5459 was able to induce endothelial activation and HEV differentiation in an EMT6 orthotopic tumor model in a FAP-dependent manner. Flow cytometry analysis of tumor single-cell suspensions was performed 13 days after the initiation 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 % of Meca79+ endothelial cells (Figure 29C) were quantified. The data show that treatment with P1AG5459 alone or in combination with aPD-L1, but not the non-targeting control P1AG5461, increased ICAM and VCAM on tumor endothelial cells and increased the frequency of Meca79+ endothelial cells (Figure 29C). The effects on endothelial activation and HEV differentiation were more pronounced in responder mice (□) than in non-responder mice (●). [Figure 30A] Figure 30A shows that treatment with P1AG5459 alone or in combination with aPD-L1, but not P1AG5461, increases the amount of Meca79+ endothelial cells as quantified by immunofluorescence imaging 13 days after initiation of treatment. Example images show the localization of major vascular structures (indicated by CD31 staining) and Meca79+ endothelium. [Fig. 30B-D]Figures 30B, 30C, and 30D show quantification of infiltration of CD8+ T cells (Figure 30B), CD4+ T cells (Figure 30C), and B220+ B cells (Figure 30D) into EMT6 orthotopic tumors, as measured by immunofluorescence imaging 13 days after treatment initiation. P1AG5459, alone or in combination with aPD-L1, and aPD-L1 alone to some extent, increased the abundance of T and B cells in the tumors, but P1AG5459 did not. With P1AG5459 monotherapy, a clear correlation can be observed in responder mice (□) compared to non-responder mice (●). [Figure 31A-C] Figures 31A-31C demonstrate that the murine surrogate FAP-LTBR bispecific antibody (P1AG5459) induces chemokine upregulation in the EMT6 orthotopic tumor model. Concentrations of the chemokines CXCL13 (Figure 31A), CCL5 (Figure 31B), and CCL10 (Figure 31C) were measured in tumor lysates collected 13 days after treatment initiation. P1AG5459-treated tumors tend to have higher amounts of CXCL13, CCL5, and CXCL10. Responding tumors (□) tend to show higher concentrations of chemokines. [Figure 32A] Figures 32A-32C show that tumor growth inhibition mediated by the murine surrogate FAP-LTBR bispecific antibody (P1AG5459) in the EMT6 orthotopic tumor model is mediated by CD8, CD4 T cells, and B cells. Median tumor growth curves are plotted in Figure 32A. Comparisons of tumor volumes at day 15 are plotted in Figure 32B, demonstrating no difference in tumor volume between vehicle- and P1AG5459-treated mice in the absence of CD8 T cells. Comparisons of tumor volumes at day 20 are plotted in Figure 32C, demonstrating no difference in tumor volume between vehicle- and P1AG5459-treated mice in the absence of CD4 T cells or CD20+-expressing B cells. [Fig. 32B-C]Figures 32A-32C show that tumor growth inhibition mediated by the murine surrogate FAP-LTBR bispecific antibody (P1AG5459) in the EMT6 orthotopic tumor model is mediated by CD8, CD4 T cells, and B cells. Median tumor growth curves are plotted in Figure 32A. Comparisons of tumor volumes at day 15 are plotted in Figure 32B, demonstrating no difference in tumor volume between vehicle- and P1AG5459-treated mice in the absence of CD8 T cells. Comparisons of tumor volumes at day 20 are plotted in Figure 32C, demonstrating no difference in tumor volume between vehicle- and P1AG5459-treated mice in the absence of CD4 T cells or CD20+-expressing B cells. [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 borders 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 infiltration 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-bearing mice treated with P1AG5459. Ki67 staining of the whole section allowed identification of the tumor margin, marked by a dashed line. B220 (B cell) staining of the whole section highlighted B cell aggregates, which are magnified in subsequent images. The magnified images characterize the cellular composition of the TLS in more detail, including pNAD / Meca79+ high endothelial venules, CD11c+ myeloid cells, B220+ Ki67+ proliferating cells representing germinal centers, and CD8 and CD4 T cells, some of which co-express the activation marker PD1 and the stemness 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. Median fluorescence intensity of ICAM on CD31+ HUVECs cocultured with NIH-3T3 cells overexpressing FAP (Figure 35A) or not expressing FAP (Figure 35B) is shown. Data are normalized to ICAM expression in untreated controls. [Figure 36A-C] Figures 36A-36C compare FAP-LTBR bispecific antigen-binding molecules containing different LTBR antigen-binding domains in their ability 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. Median fluorescence intensity values ​​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) are 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 a human IgG1 PG LALA with two N-terminal Fab arms specific for LTBR (CBE11) and the 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 a human IgG1 PG LALA crossMab with crossed VL / VH domains in the anti-LTBR Fab arm and charged CH1 / Ck domains in the anti-Fab arm. Figure 37D shows a schematic diagram of a 2+1 anti-LTBR (BHA10) / anti-FAP (4B9) bispecific antibody (P1AH5885) as a human IgG1 PG LALA crossMab with crossed V domains in both anti-LTBR Fab arms and charged CH1 / Ck domains of the anti-Fab arms fused to the C-terminus of the Fc domain (Fc knob chain). Figure 37E shows a schematic diagram of a 2+1 anti-LTBR (P1AE9459) / anti-FAP (212) bispecific antibody (P1AH5886) as a human IgG1 PG LALA crossMab with crossed V domains in the anti-LTBR Fab arms and charged CH1 / Ck domains of the anti-Fab arms fused to the C-terminus of the Fc domain (Fc knob chain). [Figure 38A]Figures 38A-38D show deuteration difference maps between bound human LTBR compared to 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. Bars above the sequences indicate peptides. Boxes indicate suggested epitopes for each antibody / ligand. Below, proposed epitopes are mapped to S28 to M227 of the alphafold model AF-P36941-F1-model_v2 (black areas). The proposed epitopes on human LTBR of P1AE9459 (Figure 38A), P1AE1873 (Figure 38B); P1AH0119 (Figure 38C) and P1AE1235 (Figure 38D, ligand) are shown. [Figure 38B] Figures 38A-38D show deuteration difference maps between bound human LTBR compared to 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. Bars above the sequences indicate peptides. Boxes indicate suggested epitopes for each antibody / ligand. Below, proposed epitopes are mapped to S28 to M227 of the alphafold model AF-P36941-F1-model_v2 (black areas). The proposed epitopes on human LTBR of P1AE9459 (Figure 38A), P1AE1873 (Figure 38B); P1AH0119 (Figure 38C) and P1AE1235 (Figure 38D, ligand) are shown. [Figure 38C]Figures 38A-38D show deuteration difference maps between bound human LTBR compared to 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. Bars above the sequences indicate peptides. Boxes indicate suggested epitopes for each antibody / ligand. Below, proposed epitopes are mapped to S28 to M227 of the alphafold model AF-P36941-F1-model_v2 (black areas). The proposed epitopes on human LTBR of P1AE9459 (Figure 38A), P1AE1873 (Figure 38B); P1AH0119 (Figure 38C) and P1AE1235 (Figure 38D, ligand) are shown. [Figure 38D] Figures 38A-38D show deuteration difference maps between bound human LTBR compared to 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. Bars above the sequences indicate peptides. Boxes indicate suggested epitopes for each antibody / ligand. Below, proposed epitopes are mapped to S28 to M227 of the alphafold model AF-P36941-F1-model_v2 (black areas). The proposed epitopes on human LTBR of P1AE9459 (Figure 38A), P1AE1873 (Figure 38B); P1AH0119 (Figure 38C) and P1AE1235 (Figure 38D, ligand) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0051] definition Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa.

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

[0053] As used herein, the term "antigen-binding domain capable of specifically binding to a target cell antigen" or "moiety capable of specifically binding to a target cell antigen" refers to a polypeptide molecule that specifically binds to an antigen. In one aspect, the antigen-binding domain can activate signal transduction via that target cell antigen. In certain aspects, the antigen-binding domain can direct the entity to which it is bound (e.g., an LTBR agonist antibody) to a target site, such as a specific type of tumor cell or tumor stroma bearing an antigenic determinant. Antigen-binding domains capable of specifically binding to a target cell antigen include antibodies and fragments thereof as further defined herein. Furthermore, antigen-binding domains capable of specifically binding to a target cell antigen include scaffold antigen-binding proteins as further defined herein, such as binding domains based on designed repeat proteins or designed repeat domains (see, e.g., WO 2002 / 020565). In particular, an antigen-binding domain capable of specifically binding to a target cell antigen is an antigen-binding domain capable of specifically binding to fibroblast activation protein (FAP). In the context of an antibody or a fragment thereof, the term "antigen-binding domain capable of specific binding to a target cell antigen" refers to a portion of a molecule comprising a region that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain capable of specific antigen binding can be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). Specifically, an antigen-binding domain capable of specific antigen binding comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) of an antibody. In another embodiment, the "antigen-binding domain capable of specific binding to a target cell antigen" can 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 population of substantially homogeneous antibodies, i.e., each individual antibody in the population is identical and / or binds to the same epitope, except for possible variant antibodies containing, for example, naturally occurring mutations or mutations that arise during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0055] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds 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 contains two antigen-binding sites, each of which is specific for 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. The bispecific antigen-binding molecules described herein may also form part of a multispecific antibody.

[0056] The term "valent" as used herein indicates that a specific number of binding sites specific for one antigenic determinant are present in an antigen-binding molecule that is specific for one antigenic determinant. Accordingly, the terms "bivalent," "tetravalent," and "hexavalent" indicate that an antigen-binding molecule has two, four, and six binding sites specific for a particular antigenic determinant, respectively. In certain aspects of the present invention, bispecific antigen-binding molecules according to the present invention may be monovalent with respect to a particular antigenic determinant (meaning that they have only one binding site for that antigenic determinant), or may be bivalent or tetravalent with respect to a particular antigenic determinant (meaning that they have two or four binding sites for that antigenic determinant, respectively).

[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 native antibody. "Native antibodies" refer to naturally occurring immunoglobulin molecules with a variety of structures. For example, native IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or 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 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 divided 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] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and resulting in increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).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., E. coli or phage), as described herein.

[0059] Papain digestion of an intact antibody produces 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 light-chain constant domain and the first heavy-chain constant domain (CH1). Therefore, as used herein, the term "Fab fragment" refers to an antibody fragment containing a light-chain fragment containing a light-chain (CL) VL domain and a constant domain, and a heavy-chain VH domain and the first heavy-chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy-chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues in the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment containing 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 "cross-Fab fragments" or "crossover Fab fragments," as defined below.

[0060] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. Two possible chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the present invention. On the one hand, the variable regions of the Fab heavy and light chains have been exchanged, i.e., the crossover Fab molecule comprises a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). This crossover Fab molecule is called CrossFab. (VLVH)On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule contains a peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab molecule is called CrossFab. (CLCH1) It is also called.

[0061] A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, where the antibody domains and linker are arranged in the following order from N- to C-terminus: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1, or (d) VL-CH1-linker-VH-CL; and the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules may be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 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 consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker are arranged in the following order from N- to C-terminus: a) VH-CL-linker-VL-CH1; or b) VL-CH1-linker-VH-CL; together, VH and VL form an antigen-binding site that specifically binds to a given antigen, and the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules may be further stabilized by the creation of an interchain disulfide bond by the insertion of cysteine ​​residues (e.g., at 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 fragment of an antibody heavy chain (V) linked using a short linker peptide of 10 to about 25 amino acids. H ) and light chain (V L ) variable region fusion proteins. The linker is usually rich in glycine for flexibility, as well as rich in serine or threonine for solubility, and V H N-terminus of V L The scFv antibody can be linked to the C-terminus of a full-length antibody, or 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, antibody fragments contain a single polypeptide chain characterized by a VH domain (i.e., capable of assembling with a VL domain) or a VL domain (i.e., capable of assembling with a VH domain into a functional antigen-binding site), thereby conferring the antigen-binding properties of a full-length antibody.

[0064] "Scaffold antigen-binding proteins" are known in the art; for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen-binding domains. See, e.g., 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 invention, the scaffold antigen binding protein is selected from the group consisting of CTLA-4 (e.g., cytochrome P456), lipocalin (anticalin), protein A derived molecules such as the Z-domain of protein A (affibody), the A-domain (avimer / maxibody), serum transferrin (transbody); engineered ankyrin repeat proteins (DARPins), variable domains of antibody light or heavy chains (single domain antibodies, sdAb), variable domains of antibody heavy chains (nanobodies, aVH), V NAR Fragments, fibronectin (adnectin), C-type lectin domain (tetranectin); variable domain of novel antigen receptor beta-lactamase (V NAR fragments), human gamma-crystallin or ubiquitin (affilin molecules); Kunitz-type domains of human protease inhibitors, microbodies, e.g., proteins from the knottin family, peptide aptamers, and fibronectin (adnectins). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is primarily a cytotoxic T lymphocyte-associated antigen. +It is a CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. Loops corresponding to antibody CDRs may be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as ebibodies (e.g., U.S. Patent No. 7,166,697 B1). Ebibodies are approximately the same size as the isolated variable regions of antibodies (e.g., domain antibodies). For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have a rigid beta-sheet secondary structure with many loops at the open end of the conical structure that can be engineered to bind to different target antigens. Anticalins are 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Patent No. 7,250,297 B1, and U.S. Patent Application Publication No. 20070224633. Affibodies are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. Domains consist of three helical bundles of approximately 58 amino acids. Libraries are created by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP 1641818 A1. Avimers are multidomain proteins derived from the A-domain scaffold family. Natural domains of approximately 35 amino acids conform to defined disulfide-bonded structures. Diversity is generated by shuffling the natural variation displayed by the A-domain family.For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind to different target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include transbodies. For further details, see J. Biol. Chem 274, 24066-24073 (1999). Designed ankyrin repeat proteins (DARPins) are derived from ankyrins, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha helices and a beta-turn. Single ankyrin repeats can be engineered to bind different target antigens by randomizing residues in the first alpha helix and beta-turn of each repeat. The binding interface can be increased by increasing the number of modules (affinity maturation method). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US Patent Application Publication No. 20040132028 A1. Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domain was derived from the variable domain of a camel-derived antibody heavy chain (nanobody or V). H Furthermore, the term single domain antibody refers to an antibody that contains an autonomous human heavy chain variable domain (aVH) or a shark-derived VH. NARThese include fragments. Fibronectin is a scaffold that can be engineered to bind to antigens. Adnectins consist of a backbone with the native amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the beta-sandwich can be engineered to enable Adnectins to specifically recognize therapeutic targets of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent Application Publication No. 20080139791, WO 2005056764, and U.S. Patent No. 6,818,418 B1. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop that is inserted into the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins that are 25-50 amino acids long and contain three to four cysteine ​​bridges; examples of microproteins include KalataBI, conotoxins, and knottins. Microproteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

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

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

[0067] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to the site on a polypeptide polymer to which an antigen-binding moiety binds (e.g., a contiguous stretch of amino acids or a conformational structure composed of different regions of non-contiguous amino acids), forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, proteins useful as antigens herein can be proteins in any native form from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When a particular protein is referred to herein, the term encompasses not only the "full-length," unprocessed protein, but also any form of protein resulting from processing within the cell. The term also encompasses naturally occurring protein variants, e.g., splice variants or allelic variants.

[0068] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific 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, such as surface plasmon resonance (SPR) techniques (analysis on 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 extent 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, a molecule that binds to an 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, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 M).

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

[0070] An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody which does not possess those alterations, which alterations result in an improvement in the affinity of the antibody for antigen.

[0071] "Target cell antigen," as used herein, refers to an antigenic determinant displayed on the surface of a target cell, particularly a target cell within a tumor, such as a cancer cell or tumor stromal cell. Thus, the target cell antigen is a tumor-associated antigen. Specifically, a "tumor-associated antigen" or TAA is a fibroblast activation protein (FAP).

[0072] The term "fibroblast activation protein (FAP)," also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any naturally occurring FAP derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed FAPs, as well as any form of FAP resulting from intracellular processing. The term also encompasses naturally occurring variants of FAPs, such as splice variants or allelic variants. In one embodiment, the antigen-binding molecule of the present invention 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 position 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 position 761. SEQ ID NO: 265 shows the amino acid sequence of Avi-His-tagged mouse FAP. Preferably, the anti-FAP binding molecules of the invention bind to the extracellular domain of FAP.

[0073] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of an antigen-binding molecule to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th, W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0074] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and that determine antigen-binding specificity, e.g., the "complementarity-determining regions" (CDRs).

[0075] Generally, an antibody comprises six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen 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, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR nomenclature may also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature.

[0077] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining regions (CDRs). The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences typically appear in the VH (or VL) in the following order: 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 and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

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

[0080] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have been further modified or altered from those of the original antibody to produce the properties according to the invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0081] The term "CH1 domain" refers to a portion of an antibody heavy chain polypeptide spanning approximately EU position 118 to EU position 215 (EU numbering system according to Kabat). In one aspect, 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 of EPKSC (SEQ ID NO: 284) follows to connect the CH1 domain to the hinge region, although in the case of a CH1 domain with a free C-terminus (such as a crossfab fragment), this segment may also consist of the amino acid sequence of EPKSCD (SEQ ID NO: 285) or EPKSCS (SEQ ID NO: 286).

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

[0083] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. Typically, the "CH2 domain" of a human IgG Fc region extends from approximately amino acid residue 231 to approximately amino acid residue 340 (EU numbering system according to Kabat). In one aspect, the CH2 domain has the amino acid sequence APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 290), and is unique in that it is not tightly paired with other domains. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native Fc region. It has been speculated that carbohydrates may provide a surrogate for domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 447 according to the EU numbering system of Kabat for IgG). In one embodiment, the CH3 domain has the amino acid sequence of LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 291).The CH3 region herein may be a native-sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "protuberance" ("knob") in one chain and a corresponding introduced "cavity" ("hole") in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains described herein. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health. The EU numbering system (also called the EU index) is followed, as described in Health, Bethesda, MD, 1991.

[0084] The term "wild-type Fc domain" refers to an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include naturally occurring human IgG1 Fc regions (non-A and A allotypes), naturally occurring human IgG2 Fc regions, naturally occurring human IgG3 Fc regions, and naturally occurring human IgG4 Fc regions, as well as naturally occurring variants thereof. Wild-type Fc regions are set forth in SEQ ID NO: 155 (IgG1, Caucasian allotype), SEQ ID NO: 156 (IgG1, Afro-American allotype), SEQ ID NO: 157 (IgG2), SEQ ID NO: 158 (IgG3), and SEQ ID NO: 159 (IgG4). The term "variant (human) Fc domain" refers to an amino acid sequence that differs from the "wild-type" (human) Fc domain amino acid sequence by at least one "amino acid mutation." In one aspect, the variant Fc region has at least one amino acid mutation, for example, about 1 to about 10 amino acid mutations, compared to a naturally occurring Fc region, and in one aspect, about 1 to about 5 amino acid mutations in a naturally occurring Fc region. In one aspect, 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 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 introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity at the interface of a second polypeptide, so that the protuberance can be positioned within the corresponding cavity, thereby promoting heterodimer formation and preventing homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protuberance and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In a more specific embodiment, the Fc domain subunit containing the knob modification further comprises the amino acid substitution S354C, and the Fc domain subunit containing the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues 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] A "region corresponding to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants having alterations that produce substitutions, additions, or deletions, but that do not substantially reduce the ability of the immunoglobulin to mediate an effector function (e.g., antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantially impairing biological function. Such variants can be selected according to general rules known in the art to have minimal effect on activity (see, e.g., Bowie, JU et al., Science 247:1306-10 (1990)).

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

[0088] Fc receptor binding-dependent effector functions can be mediated by the interaction of the Fc region of an antibody with Fc receptors (FcRs), specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of 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. G. and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are called FcγRs. Fc receptor binding is described, for example, in Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492, Capel, PJ, et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE, et al., Ann. Hematol. 76 (1998) 231-248.

[0089] Cross-linking of receptors (FcγRs) to the Fc region of IgG antibodies triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as control of immune complex clearance and antibody production. In humans, three classes of FcγRs have been characterized: - FcγRI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modification of at least one of the amino acid residues E233-G236, P238, D265, N297, A327, and P329 (numbering according to the Kabat EU index) in the Fc region of IgG reduces binding to FcγRI. IgG2 residues at positions 233-236 substituted into IgG1 and IgG4 reduce binding to FcγRI by 10%. 32-fold reduction in human monocyte responses to antibody-sensitized red blood cells and abolished the human monocyte response to antibody-sensitized red blood cells (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624).

[0090] FcγRII (CD32) binds 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 on many cells involved in killing (e.g., macrophages, monocytes, and neutrophils) and is thought to be able to activate the killing process. FcγRIIB appears to play a role in inhibitory processes and is found on B cells, macrophages, as well as mast cells and eosinophils. On B cells, FcγRIIB appears to function to suppress the production of additional immunoglobulins and isotype switching, for example, to the IgE class. On macrophages, FcγRIIB acts to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B form may serve to suppress activation of these cells via IgE binding to its other receptors. Reduced binding to FcγRIIA is seen, for example, in antibodies comprising an IgG Fc region with mutations in at least one of amino acid residues E233 to G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to the Kabat EU index).

[0091] FcγRIII (CD16) binds IgG with moderate to low affinity and exists as two species. FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. Reduced binding to FcγRIIIA is observed, for example, with antibodies comprising an IgG Fc region with a mutation at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (numbering according to the EU index of Kabat).

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

[0093] The term "ADCC" or "antibody-dependent cellular cytotoxicity" refers to the lysis of target cells by antibodies as reported herein, a function mediated by Fc receptor binding, in the presence of effector cells. The ability of an antibody to trigger the initial steps of mediating ADCC is determined by measuring binding of the antibody to cells expressing Fcγ receptors, such as recombinantly expressing FcγRI and / or FcγRIIA or NK cells (which constitutively express FcγRIIIA). In particular, binding to FcγR on NK cells is measured.

[0094] An "activating Fc receptor" is an Fc receptor that, following engagement by the Fc region of an antibody, initiates signaling events that stimulate a receptor-bearing cell to carry out an effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0095] As used herein, the term "LTBR," unless otherwise specified, refers to any native lymphotoxin beta receptor (LTBR) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed LTBR, ​​as well as any form of LTBR resulting from intracellular processing. The term also encompasses naturally occurring variants of LTBR, ​​such as splice variants or allelic variants. The amino acid sequence of an exemplary human LTBR is set forth in SEQ ID NO: 1 (Uniprot No. P36941), and the amino acid sequence of an exemplary mouse LTBR is set forth in SEQ ID NO: 297 (Uniprot No. B2RRV3). This receptor is expressed on the cell surface in the parenchyma and stroma of most lymphoid organs, but is absent on T and B lymphocytes. LTBR is also referred to as "tumor necrosis factor receptor superfamily member 3 (TNFRSF3)." Signaling through LTBR by the LTα / β heterotrimer (LTα1β2) is important during lymphoid development. LTBR is also known to bind to the ligand LIGHT (TNFSF14). While LTα1β2 is specific for LTBR, ​​LIGHT also binds and activates HVEM (TNFRSF14), a receptor expressed on immune cells and involved in immune cell regulation.

[0096] As used herein, the term "LTBR agonist" includes any moiety that stimulates the interaction of LTBR with its ligand. LTBR as used in this context preferably refers to human LTBR, ​​and thus, an LTBR agonist is preferably an agonist of human LTBR (SEQ ID NO: 1). Typically, this moiety will 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 an antibody that specifically binds to LTBR with sufficient affinity so as to be useful as a diagnostic and / or therapeutic agent targeting LTBR. In one aspect, the extent of binding of the anti-LTBR antibody to an unrelated, non-LTBR protein 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, an antibody that binds to LTBR has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -6 M or less, e.g. 10 -68 ~10 -13 M, e.g. 10 -8 M~10 -10 Dissociation constant (K D )

[0098] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. A suitable, non-immunogenic linker peptide is, for example, (G4S) n , (SG4) n or G4 (SG4) nand a peptide linker, wherein "n" is generally a number from 1 to 10, typically 2 to 4, in particular 2, i.e., 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 including the sequences GSPGSSSSGS (SEQ ID NO:302), (GS) (SEQ ID NO:303), (GS) (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). Peptide linkers of particular interest 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] The term "amino acid," as used herein, refers to the group of naturally occurring carboxy α-amino acids, including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0100] By "fused" or "linked" is meant that the components (e.g., an antibody heavy chain and Fab fragment) are joined by peptide bonds, either directly or via one or more peptide linkers.

[0101] "Percent (%) amino acid sequence identity" to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity for purposes of alignment. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. Those skilled in the art can determine appropriate parameters for performing sequence alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code is on file in the user documentation of the U.S. Copyright Office, Washington DC, 20559, registered under U.S. Copyright Registration No. TXU510087, and described in WO 2001 / 007611. Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or subsequent BLOSUM50 comparison matrices.The FASTA program package was created by W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; W.R. Pearson (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, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring global rather than local alignments. Percent amino acid identity is given in the output alignment header.

[0102] In certain embodiments, amino acid sequence variants of the bispecific antigen-binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the TNF ligand trimer-containing antigen-binding molecules. Amino acid sequence variants of the TNF ligand trimer-containing antigen-binding molecules can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, as long as the final construct possesses the desired characteristics (e.g., antigen binding). Sites of interest for substitutional mutagenesis include HVRs and framework regions (FRs). Conservative substitutions are provided in Table B under the heading "Preferred Substitutions" and are further described below with reference to amino acid side chain classes (1) to (6). Amino acid substitutions can be introduced into the molecule of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF2025500949000002.tif161170

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

[0104] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0105] The term "amino acid sequence variant" includes substantial variants in which amino acid substitutions are present in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing will have modified (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antigen-binding molecule and / or will substantially retain the specific biological properties of the parent antigen-binding molecule. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, variant antigen-binding molecules are those in which one or more CDR residues have been mutated, phage-displayed, and screened for a specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antigen-binding molecule to bind to an antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made to CDRs. A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antigen complex can be used to identify contact points between the antibody and antigen. Such contact and adjacent residues can be targeted or excluded as candidates for substitution. Variants can be screened to determine whether they possess desired properties.

[0106] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is a bispecific antigen-binding molecule of the invention having an N-terminal methionyl residue. Other insertional variants of the molecule include the fusion to the N- or C-terminus of a polypeptide 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 of glycosylation of the antibody. Glycosylation variants of the molecule can be conveniently obtained by modifying the amino acid sequence to create or remove one or more glycosylation sites. When the TNF ligand trimer-containing antigen-binding molecule contains an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides linked by an N-linkage to Asn297 in 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 attached to GlcNAc at the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications may be performed within the TNF family ligand trimer-containing antigen-binding molecule to generate variants with specific improved properties. In one aspect, variants of the bispecific antigen-binding molecule or antibody of the present invention are provided having carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. Such fucosylated variants may have improved ADCC function; see, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) or U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). In another aspect, variants of the bispecific antigen-binding molecule or antibody of the present invention are provided having bisected oligosaccharides, for example, in which a biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. See, e.g., WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Pat. App. Pub. No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.Such antibody variants have improved CDC function and are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0108] In certain embodiments, it may be desirable to create cysteine-engineered variants of the bispecific antigen-binding molecules of the present invention, e.g., "thioMAbs," in which one or more residues of the molecule are replaced with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the molecule. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates. In certain embodiments, any 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. Cysteine-engineered antigen-binding molecules may be created, for example, as described in U.S. Pat. No. 7,521,541.

[0109] The terms "nucleic acid" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically presented from 5' to 3'. As used herein, the term "nucleic acid molecule" encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), particularly messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Additionally, the term nucleic acid molecule encompasses both sense and antisense strands, as well as single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages, or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule, allowing the mRNA to be injected into a subject for in vivo antibody production (see, e.g., Stadler et al., Nature Medicine 2017, doi:10.1038 / nm.4356, published online June 12, 2017, or European Patent No. 2101823).

[0110] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0111] An "isolated nucleic acid encoding a bispecific antigen-binding molecule or antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of a bispecific antigen-binding molecule or antibody, and refers to one or more nucleic acid molecules contained in a single vector or separate vectors, and the one or more nucleic acid molecules 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 a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the nucleotide sequence of the polynucleotide may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. Such modifications of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, and may be either individually interspersed among residues in the reference sequence or interspersed in one or more contiguous groups within the reference sequence. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0113] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide comprising a set of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector comprises, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In a specific embodiment, an expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention, or a fragment thereof.

[0114] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and direct the expression of a specific gene to which it is operably linked in a target cell. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. The expression vector of the invention comprises an expression cassette. The expression vector allows for the stable transcription of large amounts of mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antibody of the invention, or a fragment thereof.

[0115] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to produce the bispecific antigen-binding molecules of the invention. Host cells include cultured cells, e.g., cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, to name just a few, but also include transgenic animals, transgenic plants, or cells contained in cultured plants or animal tissues.

[0116] An "effective amount" of a drug refers to the amount necessary to cause a physiological change in a cell or tissue to which the drug is administered.

[0117] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0118] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0119] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained in the preparation is effective and that does not contain any additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition will be administered.

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

[0121] The term "package insert" is used to refer to instructions customarily included in the commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or precautions regarding the use of that therapeutic product.

[0122] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and achieving remission or improved prognosis. In some embodiments, the molecules of the invention are used to delay the onset of disease or slow the progression of disease.

[0123] The term "cancer", as used herein, includes lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, biliary tract cancer, cancer of the central nervous system, This refers to proliferative disorders such as central nervous system (CNS) neoplasms, spinal axis tumors, brain stem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma (including refractory forms of any of the above cancers, or a combination of one or more of the above cancers).

[0124] The term "chemotherapeutic agent" as used herein refers to a chemical compound useful in the treatment of cancer. In one aspect, the chemotherapeutic agent is an antimetabolite. In one aspect, the antimetabolite is selected from the group consisting of aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine, capecitabine, cytarabine, fluorouracil, floxuridine, and gemcitabine. In a particular aspect, the antimetabolite is capecitabine or gemcitabine. In another aspect, the antimetabolite is fluorouracil. In one aspect, the chemotherapeutic agent is an agent that affects microtubule formation. In one aspect, the agent that affects microtubule formation is selected from the group consisting of paclitaxel, docetaxel, vincristine, vinblastine, vindesine, vinorelbine, taxotere, etoposide, and teniposide. In another embodiment, the chemotherapeutic agent is an alkylating agent such as cyclophosphamide. In one embodiment, the chemotherapeutic agent is a cytotoxic antibiotic such as a topoisomerase II inhibitor. In one embodiment, the topoisomerase II inhibitor is doxorubicin.

[0125] Exemplary Agonist LTBR Antibodies Provided herein are novel antibodies and antibody fragments that specifically bind to lymphotoxin beta receptor (LTBR). Novel antibodies and antibody fragments that specifically bind to an antigen comprising the amino acid sequence of SEQ ID NO: 1 are also provided. Thus, these antibodies specifically bind to human LTBR. These are agonistic hu LTBR antibodies.

[0126] These antibodies can bind to human LTBR and cynomolgus monkey LTBR with an affinity difference of less than 2-fold. Some of the novel agonist hu LTBR antibodies can also bind to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR.

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

[0128] The agonist LTBR antibodies disclosed herein have an EC50 activity of less than 5 nM as measured by ELISA. 50 ligated to the cynomolgus monkey LTBR extracellular domain (ECD of the amino acid sequence of SEQ ID NO: 361) (see Example 1.5). In one aspect, an agonist LTBR antibody has an EC of less than 0.5 nM as measured by ELISA. 50 In one particular embodiment, the agonist LTBR antibody binds to the cynomolgus monkey LTBR ECD with an EC of less than 0.1 nM as measured by ELISA. 50 It binds to the cynomolgus monkey LTBR ECD.

[0129] The agonistic LTBR antibodies described herein require cross-linking for agonistic activity to activate human LTBR, ​​meaning that they can stimulate LTBR only through a cross-linking-dependent mechanism. A "cross-linking-dependent mechanism" can be, for example, an Fc cross-linking-dependent mechanism, in which an antibody must bind to both LTBR and an Fc receptor to stimulate LTBR. Therefore, the antibody must be able to bind to both LTBR and an Fc receptor. If the antibody were cross-linking-independent, it could stimulate LTBR even without Fc receptor binding. This could lead to widespread LTBR activation in humans and associated serious safety issues.

[0130] The agonistic LTBR antibodies described herein also require cross-linking for their agonistic 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 agonistic LTBR antibodies described herein are also capable of inhibiting the interaction between human LTBR and its human ligands, lymphotoxin α1β2 and LIGHT, as shown in ELISA ligand competition experiments described in Example 1.6.

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

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

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

[0135] In one aspect, an agonist LTBR antibody (or antigen binding domain that specifically binds to LTBR) is provided, said antibody or antigen binding domain comprising: A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 33 HLTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 41 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 42 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 57 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 58 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 65 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 66 L LTBR), A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 73 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 74 L LTBR), A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 81 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 82 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 89 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 90 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 97 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 98 L LTBR) Includes:

[0136] In one particular aspect, the agonistic LTBR antibodies described herein are agonistic LTBR antibodies that specifically bind to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR. As used herein, EC 50 Disclosed are agonistic LTBR antibodies that bind to the murine LTBR extracellular domain (ECD of the amino acid sequence of SEQ ID NO: 360) with an EC of less than 1 nM (see Example 1.5). In one embodiment, the agonistic LTBR antibody has an EC of less than 0.15 nM as measured by ELISA. 50 It binds to mouse LTBR ECD at

[0137] In one aspect, an agonist LTBR antibody (or antigen binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR, 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; H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 A heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L LTBR), or A heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77. HLTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L LTBR), or A heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 83, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO: 88. L LTBR) Agonistic LTBR antibodies (or antigen-binding domains) that specifically bind to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR are provided, comprising:

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

[0139] In one specific aspect, the agonistic LTBR antibodies described herein bind to the epitope region of SEQ ID NO: 351 on human LTBR. In one aspect, the agonistic LTBR antibodies described herein bind to an epitope on human LTBR that overlaps with the epitope to which human lymphotoxin α1β2 binds. In one aspect, the agonistic LTBR antibodies described herein bind to a different epitope region on human LTBR than 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 (VH1) 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. H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 In one embodiment, the heavy chain variable region (VLTBR) comprises the amino acid sequence of SEQ ID NO: 33. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L LTBR) (antibody P1AE9459).

[0141] In one aspect, an agonist LTBR antibody (or antigen binding domain that specifically binds to LTBR) derived from immunization in a transgenic rabbit is provided. In one aspect, the antibody (or antigen binding domain) is (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; H(iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 CDR-H1 having the amino acid sequence of SEQ ID NO: 35, CDR-H2 having the amino acid sequence of SEQ ID NO: 36, and CDR-H3 having the amino acid sequence of SEQ ID NO: 37; H a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 38, CDR-L2 having the amino acid sequence of SEQ ID NO: 39, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40; L LTBR), or (iii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 43, CDR-H2 having the amino acid sequence of SEQ ID NO: 44, and CDR-H3 having the amino acid sequence of SEQ ID NO: 45; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L LTBR), or (iv) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 51, CDR-H2 having the amino acid sequence of SEQ ID NO: 52, and CDR-H3 having the amino acid sequence of SEQ ID NO: 53 H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 54, CDR-L2 having the amino acid sequence of SEQ ID NO: 55, and CDR-L3 having the amino acid sequence of SEQ ID NO: 56. L LTBR), or (v) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 59, CDR-H2 having the amino acid sequence of SEQ ID NO: 60, and CDR-H3 having the amino acid sequence of SEQ ID NO: 61; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 62, CDR-L2 having the amino acid sequence of SEQ ID NO: 63, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64.L LTBR), or (vi) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 67, CDR-H2 having the amino acid sequence of SEQ ID NO: 68, and CDR-H3 having the amino acid sequence of SEQ ID NO: 69; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 70, CDR-L2 having the amino acid sequence of SEQ ID NO: 71, and CDR-L3 having 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 comprises 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. H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 a heavy chain variable region (V LTBR) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 43, CDR-H2 having the amino acid sequence of SEQ ID NO: 44, and CDR-H3 having the amino acid sequence of SEQ ID NO: 45. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L In one aspect, the antibody or antigen-binding domain comprises a heavy chain variable region (VLTBR) comprising the amino acid sequence of SEQ ID NO: 33. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L LTBR), or a heavy chain variable region (V HLTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L LTBR).

[0143] In another aspect, a human LTBR antibody or antigen-binding domain derived from immunization of a rat is provided. In particular, the 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 aspect, the antibody or antigen-binding domain comprises a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 75, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L In one aspect, the antibody or antigen-binding domain comprises a heavy chain variable region (VLTBR) comprising the amino acid sequence of SEQ ID NO: 81. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 82 L LTBR).

[0144] In yet another aspect, a human LTBR antibody or antigen-binding domain is provided that is generated from a phage display library. In particular, the 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 aspect, the antibody or antigen-binding domain comprises a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 83, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO: 88. LIn one embodiment, the antibody or antigen-binding domain comprises a heavy chain variable region (VLTBR) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 91, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 92, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 93. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 94, CDR-L2 having the amino acid sequence of SEQ ID NO: 95, and CDR-L3 having the amino acid sequence of SEQ ID NO: 96. L In one aspect, the antibody or antigen-binding domain comprises a heavy chain variable region (VLTBR) comprising the amino acid sequence of SEQ ID NO: 89. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 90 L In one aspect, the antibody or antigen-binding domain comprises a heavy chain variable region (VLTBR) comprising the amino acid sequence of SEQ ID NO: 97. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 98 L 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 comprises the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index). In a particular embodiment, the antibody comprises (i) two heavy chains comprising the amino acid sequence of SEQ ID NO: 227 and two light chains comprising the amino acid sequence of SEQ ID NO: 228, or (ii) two heavy chains comprising the amino acid sequence of SEQ ID NO: 229 and two light chains comprising the amino acid sequence of SEQ ID NO: 230; or (iii) two heavy chains comprising the amino acid sequence of SEQ ID NO: 231 and two light chains comprising the amino acid sequence of SEQ ID NO: 232; or (iv) two heavy chains comprising the amino acid sequence of SEQ ID NO: 233 and two light chains comprising the amino acid sequence of SEQ ID NO: 234; or (v) two heavy chains comprising the amino acid sequence of SEQ ID NO: 235 and two light chains comprising the amino acid sequence of SEQ ID NO: 236; or (vi) two heavy chains comprising the amino acid sequence of SEQ ID NO: 237 and two light chains comprising the amino acid sequence of SEQ ID NO: 238; or (vii) two heavy chains comprising the amino acid sequence of SEQ ID NO: 239 and two light chains comprising the amino acid sequence of SEQ ID NO: 240; or (viii) two heavy chains comprising the amino acid sequence of SEQ ID NO: 241 and two light chains comprising the amino acid sequence of SEQ ID NO: 242; or (ix) two heavy chains comprising the amino acid sequence of SEQ ID NO: 243 and two light chains comprising the amino acid sequence of SEQ ID NO: 244 Includes:

[0146] Bispecific agonist LTBR antibodies Also provided herein are novel bispecific antigen-binding molecules capable of specifically binding to tumor-associated antigens such as lymphotoxin beta receptor (LTBR) and fibroblast activation protein (FAP), which combine an antigen-binding domain that specifically binds to a FAP with at least one antigen-binding domain that can agonistically bind to the LTBR, ​​wherein LTBR-mediated activation is provided by crosslinking via binding to a FAP expressed on tumor stromal cells. The bispecific agonist LTBR antibodies disclosed herein have particularly advantageous properties, such as productivity, stability, binding affinity, biological activity, targeting efficiency, reduced internalization, acceptable pharmacokinetic (PK) properties, reduced toxicity, a wider dosage range that can be administered to patients, and thereby potentially enhanced efficacy.

[0147] Exemplary Bispecific Agonist LTBR Antibodies In one aspect, an agonistic LTBR antibody is provided that is a multispecific antibody, including the agonistic LTBR antibody described hereinabove. In one aspect, an agonistic LTBR antibody is provided that is a bispecific antibody. The bispecific agonistic LTBR antibody preferably comprises an Fc domain of human origin, particularly of the human IgG subclass, more particularly of the human IgG1 subclass. In one aspect, the bispecific agonistic LTBR antibody comprises an Fc domain of the human IgG1 subclass that contains one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor. In one aspect, the bispecific agonistic LTBR antibody comprises an Fc domain of the human IgG1 subclass with amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0148] In one embodiment, the agonist LTBR antibody is a bispecific antibody that specifically binds to LTBR and a tumor-associated antigen (TAA). In particular, the bispecific agonist LTBR antibody is an LTBR agonist that targets FAP. In one embodiment, the bispecific agonist LTBR antibody comprises an Fc region composed of a first subunit and a second subunit that contain mutations that reduce effector function. The use of an Fc region that contains mutations that reduce or eliminate effector function prevents nonspecific agonism due to cross-linking via Fc receptors and prevents ADCC of LTBR-expressing cells. The bispecific agonist LTBR antibodies described herein have the advantage 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] Thus, bispecific agonist LTBR antibodies are characterized by FAP-targeted agonistic binding to LTBR. In the presence of FAP-expressing cells, bispecific antigen-binding molecules can activate cancer-associated fibroblasts (CAFs) (Example 5.2.1), activate endothelial cells (Example 5.2.2), and modulate human endothelium, upregulating adhesion molecules and chemoattractants important in the immune infiltration cascade. The bispecific antigen-binding molecules described herein can induce T cell adhesion (Example 5.2.3).

[0150] In one embodiment, a bispecific agonist LTBR antibody comprising: (a) a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP); (b) a second 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 and containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor; Bispecific agonist LTBR antibodies are provided, comprising:

[0151] The bispecific agonist LTBR antibody has an Fc domain composed of a first subunit and a second subunit containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for the Fc receptor, thereby abrogating Fc receptor-mediated cross-linking and achieving tumor-specific activation by binding-mediated cross-linking of the antigen-binding domain that specifically binds to a FAP via binding to a tumor-associated target.

[0152] In one embodiment, a bispecific agonist LTBR antibody comprising: (a) a first Fab fragment that specifically binds to fibroblast activation protein (FAP); (b) a second Fab fragment that specifically binds to the lymphotoxin beta receptor (LTBR), and (c) an Fc domain composed of a first subunit and a second subunit and containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor; Bispecific agonist LTBR antibodies are provided, comprising:

[0153] In one aspect, the bispecific agonist LTBR antibody disclosed herein comprises a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP), (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: 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; H (iv) a light chain variable region (V FAP) comprising a light chain complementarity determining region (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; L FAP), or (ii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 19, CDR-H2 having the amino acid sequence of SEQ ID NO: 20, and CDR-H3 having the amino acid sequence of SEQ ID NO: 21; H and a light chain variable region (VFAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 22, CDR-L2 having the amino acid sequence of SEQ ID NO: 23, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24. L FAP), or (iii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 11, CDR-H2 having the amino acid sequence of SEQ ID NO: 12, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13; H and a light chain variable region (V FAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 15, and CDR-L3 having the amino acid sequence of SEQ ID NO: 16. L FAP) The antibody comprises a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP), comprising:

[0154] In one embodiment, the first antigen-binding domain that specifically binds to a FAP comprises: (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: 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; H (iv) a light chain variable region (V FAP) comprising a light chain complementarity determining region (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; L FAP), or (ii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 19, CDR-H2 having the amino acid sequence of SEQ ID NO: 20, and CDR-H3 having the amino acid sequence of SEQ ID NO: 21; H and a light chain variable region (VFAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 22, CDR-L2 having the amino acid sequence of SEQ ID NO: 23, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24. L FAP) Includes:

[0155] In one particular aspect, the first antigen-binding domain that specifically binds to a FAP comprises (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: 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. H (iv) a light chain variable region (V FAP) comprising a light chain complementarity determining region (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; L In one embodiment, the first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region (VH1) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 19, CDR-H2 having the amino acid sequence of SEQ ID NO: 20, and CDR-H3 having the amino acid sequence of SEQ ID NO: 21. Hand a light chain variable region (VFAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 22, CDR-L2 having the amino acid sequence of SEQ ID NO: 23, and CDR-L3 having the amino acid sequence of SEQ ID NO: 24. L In another embodiment, the first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region (VH1) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 11, CDR-H2 having the amino acid sequence of SEQ ID NO: 12, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13. H and a light chain variable region (V FAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 15, and CDR-L3 having the amino acid sequence of SEQ ID NO: 16. L FAP).

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

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

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

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

[0160] In a specific embodiment, the second antigen-binding domain that specifically binds to LTBR comprises 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 heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 29. H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 In another embodiment, the second antigen-binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 35, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37. H a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 38, CDR-L2 having the amino acid sequence of SEQ ID NO: 39, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40; L In another embodiment, the second antigen-binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH1) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 43, CDR-H2 having the amino acid sequence of SEQ ID NO: 44, and CDR-H3 having the amino acid sequence of SEQ ID NO: 45. HLTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L In a further aspect, the second antigen-binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 51, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 54, CDR-L2 having the amino acid sequence of SEQ ID NO: 55, and CDR-L3 having the amino acid sequence of SEQ ID NO: 56. L In another embodiment, the second antigen-binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH1) 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 LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 62, CDR-L2 having the amino acid sequence of SEQ ID NO: 63, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64. L In another embodiment, the second antigen-binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH1) 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 LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 70, CDR-L2 having the amino acid sequence of SEQ ID NO: 71, and CDR-L3 having the amino acid sequence of SEQ ID NO: 72. L In yet another embodiment, the second antigen-binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 75, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77. HLTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L In a further aspect, the second antigen-binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 83, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO: 88. L LTBR).

[0161] In another aspect, the second antigen-binding domain that specifically binds to LTBR comprises 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 (VH3) comprising the amino acid sequence of SEQ ID NO: 103. H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 104, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 105, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 106; L In a specific embodiment, the second antigen-binding domain that specifically binds to LTBR may comprise a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 99. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 100 L LTBR) (CBE11).

[0162] In another aspect, the second antigen-binding domain that specifically binds to LTBR comprises 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 (VH1) comprising the amino acid sequence of SEQ ID NO: 345. H(iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 346, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 347, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 348; L In a specific embodiment, the second antigen-binding domain that specifically binds to LTBR may comprise a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 349. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 350 L LTBR) (BHA10).

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

[0023] Bispecific agonist LTBR antibodies are provided that contain a second antigen-binding domain that specifically binds to LTBR, ​​comprising:

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

[0023] Bispecific agonist LTBR antibodies are provided that contain a second antigen-binding domain that specifically binds to LTBR, ​​comprising:

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

[0166] In another embodiment, the second antigen-binding domain that specifically binds LTBR can also specifically bind mouse LTBR; 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; H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 A heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L LTBR), or A heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L LTBR), or A heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 83, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having 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 is also capable of specifically binding to mouse LTBR; A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 33 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 81 HLTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 82 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 89 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 90 L LTBR), or A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 97 H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 98 L LTBR) Includes:

[0168] In one embodiment, the second antigen-binding domain that specifically binds to LTBR is also capable of specifically binding to mouse LTBR and comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 33. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L LTBR).

[0169] In one embodiment, a bispecific agonist LTBR antibody, wherein the bispecific antibody comprises a third antigen-binding domain that specifically binds to LTBR; (a) a first antigen-binding domain that specifically binds to fibroblast activation 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 and containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor; The bispecific agonist LTBR antibody is provided, which means a bispecific antigen-binding molecule comprising:

[0170] In a specific 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 antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR) and the third antigen-binding domain 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 aspect, the bispecific agonist LTBR antibody disclosed herein comprises a second antigen-binding domain that specifically binds to LTBR and a third antigen-binding domain, both of which 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; H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 CDR-H1 having the amino acid sequence of SEQ ID NO: 43, CDR-H2 having the amino acid sequence of SEQ ID NO: 44, and CDR-H3 having the amino acid sequence of SEQ ID NO: 45; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L LTBR); or (iii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 75, CDR-H2 having the amino acid sequence of SEQ ID NO: 76, and CDR-H3 having the amino acid sequence of SEQ ID NO: 77; HLTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L LTBR); or (iv) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 83, CDR-H2 having the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 having the amino acid sequence of SEQ ID NO: 85; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO: 88. L LTBR); or (v) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 35, CDR-H2 having the amino acid sequence of SEQ ID NO: 36, and CDR-H3 having the amino acid sequence of SEQ ID NO: 37; H a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 38, CDR-L2 having the amino acid sequence of SEQ ID NO: 39, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40; L LTBR), or (vi) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 51, CDR-H2 having the amino acid sequence of SEQ ID NO: 52, and CDR-H3 having the amino acid sequence of SEQ ID NO: 53; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 54, CDR-L2 having the amino acid sequence of SEQ ID NO: 55, and CDR-L3 having the amino acid sequence of SEQ ID NO: 56. L LTBR), or (vii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 59, CDR-H2 having the amino acid sequence of SEQ ID NO: 60, and CDR-H3 having the amino acid sequence of SEQ ID NO: 61; H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 62, CDR-L2 having the amino acid sequence of SEQ ID NO: 63, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64. LLTBR); or (viii) a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 67, CDR-H2 having the amino acid sequence of SEQ ID NO: 68, and CDR-H3 having the amino acid sequence of SEQ ID NO: 69 H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 70, CDR-L2 having the amino acid sequence of SEQ ID NO: 71, and CDR-L3 having the amino acid sequence of SEQ ID NO: 72. L LTBR) and a second antigen-binding domain that specifically binds to the LTBR, ​​comprising:

[0172] In one particular aspect, 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 comprise 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. H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 In another embodiment, the second and third antigen-binding domains that specifically bind to LTBR each comprise a heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37. H a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 38, CDR-L2 having the amino acid sequence of SEQ ID NO: 39, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40; LIn another embodiment, the second and third antigen-binding domains that specifically bind to LTBR each comprise a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 43, CDR-H2 having the amino acid sequence of SEQ ID NO: 44, and CDR-H3 having the amino acid sequence of SEQ ID NO: 45. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L In a further aspect, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each comprise a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 51, CDR-H2 having the amino acid sequence of SEQ ID NO: 52, and CDR-H3 having the amino acid sequence of SEQ ID NO: 53. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 54, CDR-L2 having the amino acid sequence of SEQ ID NO: 55, and CDR-L3 having the amino acid sequence of SEQ ID NO: 56. L In another embodiment, the second and third antigen-binding domains that specifically bind to LTBR each comprise a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 59, CDR-H2 having the amino acid sequence of SEQ ID NO: 60, and CDR-H3 having the amino acid sequence of SEQ ID NO: 61. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 62, CDR-L2 having the amino acid sequence of SEQ ID NO: 63, and CDR-L3 having the amino acid sequence of SEQ ID NO: 64. L In another embodiment, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each comprise a heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69. HLTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 70, CDR-L2 having the amino acid sequence of SEQ ID NO: 71, and CDR-L3 having the amino acid sequence of SEQ ID NO: 72. L In yet another embodiment, the second and third antigen-binding domains that specifically bind to LTBR each comprise a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 75, CDR-H2 having the amino acid sequence of SEQ ID NO: 76, and CDR-H3 having the amino acid sequence of SEQ ID NO: 77. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L In a further aspect, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each comprise a heavy chain variable region (V) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 83, CDR-H2 having the amino acid sequence of SEQ ID NO: 84, and CDR-H3 having the amino acid sequence of SEQ ID NO: 85. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO: 88. L LTBR).

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

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

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

[0177] In another aspect, 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: 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 (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 104, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 105, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 106; L In a specific embodiment, the second and third antigen-binding domains that specifically bind to LTBR each comprise a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 99. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 100 L LTBR) (CBE11).

[0178] In another aspect, a bispecific agonist LTBR antibody is provided that specifically binds to LTBR, ​​comprising a second antigen-binding domain and a third antigen-binding domain, 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. H(iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 346, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 347, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 348; L In a specific embodiment, the second and third antigen-binding domains that specifically bind to LTBR each comprise a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 349. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 350 L LTBR) (BHA10).

[0179] In another aspect, a bispecific agonist LTBR antibody that specifically binds human LTBR and can also specifically bind mouse LTBR, ​​comprising a second antigen-binding domain and a third antigen-binding domain, wherein the second antigen-binding domain and the third antigen-binding domain are each: 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; H (iv) a light chain variable region (V LTBR) comprising a light chain complementarity determining region (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 A heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 47, and CDR-L3 having the amino acid sequence of SEQ ID NO: 48. L LTBR), or A heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 75, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77. HLTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 78, CDR-L2 having the amino acid sequence of SEQ ID NO: 79, and CDR-L3 having the amino acid sequence of SEQ ID NO: 80. L LTBR), or A heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 83, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85. H LTBR), and a light chain variable region (V LTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO: 88. L LTBR) The present invention provides a bispecific agonist LTBR antibody comprising a second antigen-binding domain and a third antigen-binding domain that specifically binds to human LTBR and can also specifically bind to mouse LTBR, ​​the second antigen-binding domain comprising:

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

[0181] In one 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) comprising the amino acid sequence of SEQ ID NO: 33. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 34 L LTBR).

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

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

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

[0185] In another aspect, a bispecific agonist LTBR antibody is provided, the antibody comprising: (i) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 9; H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 10 L (ii) a first antigen-binding domain capable of specifically binding to FAP, comprising a FAP; and (iii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 99. H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 100 L The antibody comprises a second antigen-binding domain capable of specifically binding to LTBR, ​​and a third antigen-binding domain capable of specifically binding to LTBR.

[0186] Bispecific, monovalent agonist LTBR antibody (1+1 format) In one aspect, a bispecific agonist LTBR antibody is provided, comprising: a first antigen-binding domain that specifically binds to fibroblast activation 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 and comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor, wherein the bispecific agonist LTBR antibody is (i) a first light chain and a first heavy chain of a full-length antibody that specifically binds to a FAP; and (ii) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to LTBR, ​​in which the variable domains VL and VH are replaced with each other and / or the constant domains CL and CH1 are replaced with each other. Includes:

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

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

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

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

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

[0192] Bispecific agonist LTBR antibody (2+1 format) that is bivalent for binding to LTBR and monovalent for binding to FAP In one aspect, a bispecific agonist LTBR antibody is provided, comprising a first antigen-binding domain that specifically binds to fibroblast activation 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 composed of a first subunit and a second subunit, the Fc domain comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an 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 a FAP, the Fab fragment or crossFab fragment being linked to one of the C-termini of the two heavy chains via a peptide linker; Includes:

[0193] In another aspect, a bispecific antigen-binding molecule is provided, comprising: a first antigen-binding domain that specifically binds to fibroblast activation 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, the Fc domain comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule for an Fc receptor, wherein the bispecific antigen-binding molecule comprises: (i) a first light chain and a first heavy chain of a full-length antibody that specifically binds to a 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 a CrossFab fragment that specifically binds to LTBR is linked to the C-terminus of the second heavy chain via a peptide linker. Includes:

[0194] Thus, a bispecific antigen-binding molecule is provided that has bivalent binding to LTBR and monovalent binding to FAP.

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

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

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

[0198] Mouse alternative agonist LTBR antibody Also provided herein is a murine surrogate molecule. In one aspect, a bispecific agonist LTBR antibody comprising: (a) a first antigen-binding domain that specifically binds to FAP, the first antigen-binding domain comprising a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 17; H FAP) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 18 L a first antigen-binding domain that specifically binds to FAP, comprising FAP; (b) a second antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), the heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 49; H LTBR) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50 L a second antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), including a LTBR; (c) an Fc domain composed of a first subunit and a second subunit and containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor; Bispecific agonist LTBR antibodies are provided, comprising:

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

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

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

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

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

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

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

[0206] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function compared to a non-engineered Fc domain. In certain embodiments, the Fc domain of a bispecific agonist LTBR antibody comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In another embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least two-fold, at least five-fold, or at least ten-fold. In one embodiment, a bispecific agonist LTBR antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity to an Fc receptor compared to a bispecific antibody of the present invention comprising a non-engineered Fc domain. In certain embodiments, the Fc receptor is an Fcγ receptor. In another embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an inhibitory Fc receptor. In a specific embodiment, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding of each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly C1q, is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., maintaining the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain of a bispecific antigen-binding molecule according to the present invention (or a bispecific agonist LTBR antibody comprising said Fc domain) exhibits more than about 70% of the binding affinity to FcRn of a non-engineered Fc domain (or a bispecific agonist LTBR antibody comprising said non-engineered Fc domain).The Fc domain, or a bispecific agonist LTBR antibody comprising the Fc domain, may exhibit greater than about 80%, or even greater than about 90%, of such affinity. In certain embodiments, the Fc domain of the bispecific agonist LTBR antibody is engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector function includes, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0207] Antibodies with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581). Specific antibody variants with improved or diminished binding to FcRs have been described (e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields, R. et al., J. Biol. Chem. 276 (2001) 6591-6604).

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

[0209] In one aspect, the Fc domain is an IgG4 Fc domain. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising the amino acid substitutions L235E, S228P, and P329G. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).

[0210] Antibodies with increased half-lives 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 comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan, A.R., and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0211] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment, such as a BIAcore instrument (GE Healthcare), and Fc receptors that can be obtained, for example, by recombinant expression. Suitable binding assays are described herein. Alternatively, the binding affinity of an Fc domain or a cell-activating bispecific antigen-binding molecule comprising an Fc domain to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor). The effector function of an Fc domain or a bispecific agonist LTBR antibody comprising an Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83, 70597063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 14991502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 13511361 (1987). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0212] The following sections describe preferred embodiments of the bispecific agonist LTBR antibodies described herein, which include Fc domain modifications that reduce Fc receptor binding and / or effector function. In one embodiment, a bispecific agonist LTBR antibody is provided, which includes: (a) a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP), (b) a second (and optionally a 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 includes one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor, particularly an Fcγ receptor. In another aspect, a bispecific agonist LTBR antibody is provided, comprising: (a) a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP), (b) a second (and optionally a 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 comprises one or more amino acid substitutions that reduce effector function. In a specific aspect, the Fc domain is an Fc domain of the human IgG1 subclass having amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0213] Fc domain modifications that promote heterodimerization The bispecific agonist LTBR antibodies described herein contain different antigen-binding sites fused to one or the other of the two subunits of the Fc domain, and therefore the two subunits of the Fc domain may be contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Therefore, to improve the yield and purity of the bispecific agonist LTBR antibodies of the present invention during recombinant production, it would be advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecules of the present invention that promote the association of the desired polypeptides.

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

[0215] In a specific embodiment, the modification is a so-called "knob-into-hole" modification, which comprises 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 composed of 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 comprises the knob and the second subunit of the Fc domain comprises the hole. In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0216] Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity at the interface of a second polypeptide, respectively, so that the protuberance can be positioned within the corresponding cavity, thereby promoting heterodimer formation and preventing homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protuberance is created on the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (eg, alanine or threonine).

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

[0218] In yet a further embodiment, the serine residue at position 354 in the first subunit of the Fc domain is further replaced with a cysteine ​​residue (S354C), and the tyrosine residue at position 349 in the second subunit of the Fc domain is further replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of disulfide bridges 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 the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0219] In an alternative embodiment, the modification that promotes association of the first and second Fc domain subunits comprises a modification that mediates an electrostatic steering effect, e.g., as described in WO 2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favorable.

[0220] The C-terminus of the heavy chain of the bispecific agonist LTBR antibody reported herein can be a complete C-terminus ending at amino acid residue PGK. The C-terminus of the heavy chain can be a truncated 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 truncated C-terminus ending at PG. In certain embodiments, the C-terminus of the heavy chain is a truncated C-terminus ending at P. In one aspect of all embodiments reported herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one aspect of all embodiments reported herein, a bispecific agonist LTBR antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, numbered according to the Kabat EU index).

[0221] Modifications in the Fab domain In one aspect, 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, wherein in one of the Fab fragments, either the variable domains VH and VL are exchanged or the constant domains CH1 and CL are exchanged. The bispecific antibody is prepared according to crossmab technology.

[0222] Multispecific antibodies with domain replacement / swapping in one binding arm (CrossMabVH-VL or CrossMabCH-CL) are described in WO 2009 / 080252 and Schaefer, W. et al., PNAS, 108 (2011) 11187-1191. These multispecific antibodies clearly reduce by-products caused by mismatches between a light chain for one antigen and an incorrect heavy chain for a second antigen (compared to approaches without such domain swapping).

[0223] In one aspect, 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 interchanged 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 interchanged such that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

[0224] In another aspect, a bispecific agonist LTBR antibody is provided, comprising: (a) a first Fab fragment capable of specifically binding to a FAP, wherein the constant domains CL and CH1 are interchanged 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 a FAP.

[0225] In another aspect, there is provided a bispecific agonist LTBR antibody comprising: (a) a first Fab fragment that specifically binds to fibroblast activation protein (FAP); (b) a second Fab fragment and a third Fab fragment that specifically bind to lymphotoxin beta receptor (LTBR); and (c) an Fc domain composed of a first subunit and a second subunit and comprising one or more amino acid substitutions that reduce the binding affinity to an Fc receptor and / or the effector function of the antigen-binding molecule, wherein in the second and third Fab fragments specific for LTBR, ​​the variable domains VL and VH are swapped with each other such that the VH domain is part of a light chain and the VL domain is part of a heavy chain.

[0226] In another aspect, to further improve accurate pairing, a bispecific agonist LTBR antibody comprising (a) a first Fab fragment capable of specifically binding to LTBR, ​​(b) a second Fab fragment capable of specifically binding to FAP, and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association may contain differently charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the crossover or non-crossover CH1 and CL domains. In a specific aspect, the present invention relates to a bispecific antigen-binding molecule in which, in one of the CL domains, the amino acid at position 123 (EU numbering) is replaced with arginine (R), the amino acid at position 124 (EU numbering) is replaced with lysine (K), and in one of the CH1 domains, the amino acid at positions 147 (EU numbering) and / or 213 (EU numbering) is replaced with glutamic acid (E).

[0227] Polynucleotides Additionally provided are isolated polynucleotides encoding the agonist LTBR antibodies or bispecific agonist LTBR antibodies or fragments thereof described herein.

[0228] An isolated polynucleotide encoding an agonist LTBR antibody or a bispecific agonist LTBR antibody can be expressed as a single polynucleotide encoding the entire antigen-binding molecule, or as multiple (e.g., two or more) co-expressed polynucleotides. Polypeptides encoded by co-expressed polynucleotides may associate, for example, via disulfide bonds or other means, to form a functional antigen-binding molecule. For example, the light chain portion of an immunoglobulin can be encoded by a polynucleotide separate from the heavy chain portion of the immunoglobulin. When co-expressed, the heavy chain polypeptide associates with the light chain polypeptide to form...

Claims

1. 1. An agonistic lymphotoxin beta receptor (LTBR) antibody that specifically binds to human LTBR and cynomolgus monkey LTBR, ​​said antibody comprising: (i) a heavy chain complementarity determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 27, a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 28, and a heavy chain variable region (VH3) comprising the amino acid sequence of SEQ ID NO: 29; Ｈ (iv) a light chain variable region (VLTBR) comprising a light chain complementarity determining region (CDR-L1), a light chain complementarity determining region (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 30, a light chain variable region (VVL1), a light chain complementarity determining region (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region (VVL3) comprising the amino acid sequence of SEQ ID NO:

32. Ｌ LTBR); or (ii) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 43, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO:46, CDR-L2 having the amino acid sequence of SEQ ID NO:47, and CDR-L3 having the amino acid sequence of SEQ ID NO:

48. Ｌ LTBR); or (iii) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 75, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO:78, CDR-L2 having the amino acid sequence of SEQ ID NO:79, and CDR-L3 having the amino acid sequence of SEQ ID NO:

80. Ｌ LTBR); or (iv) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 83, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO:

88. Ｌ LTBR); or (v) a heavy chain variable region (V) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 37; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 38, CDR-L2 having the amino acid sequence of SEQ ID NO: 39, and CDR-L3 having the amino acid sequence of SEQ ID NO:

40. Ｌ LTBR), or (vi) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 51, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO:54, CDR-L2 having the amino acid sequence of SEQ ID NO:55, and CDR-L3 having the amino acid sequence of SEQ ID NO:

56. Ｌ LTBR), or (vii) a heavy chain variable region (VH1) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 59, CDR-H2 having the amino acid sequence of SEQ ID NO: 60, and CDR-H3 having the amino acid sequence of SEQ ID NO: 61; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO:62, CDR-L2 having the amino acid sequence of SEQ ID NO:63, and CDR-L3 having the amino acid sequence of SEQ ID NO:

64. Ｌ LTBR); or (viii) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 67, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO:70, CDR-L2 having the amino acid sequence of SEQ ID NO:71, and CDR-L3 having the amino acid sequence of SEQ ID NO:

72. Ｌ L.T.B.R.

2. An agonistic lymphotoxin beta receptor (LTBR) antibody that specifically binds to human LTBR and cynomolgus monkey LTBR, ​​comprising:

2. Agonist antibodies have the following characteristics: (a) binds to human LTBR and cynomolgus monkey LTBR with less than a two-fold difference in affinity; or (b) an EC of less than 4 nM as measured by ELISA ５０ and binds to the human LTBR extracellular domain at 500 ng / s and has an EC of less than 5 nM as measured by ELISA. ５０ or (c) activates human LTBR, ​​requiring cross-linking for its agonist activity; or (d) induces upregulation of ICAM in human umbilical vein endothelial cells or cancer-associated fibroblasts, requiring cross-linking for its agonistic activity; or (e) inhibiting the interaction between human LTBR and its human ligands, lymphotoxin α1β2 and LIGHT The agonist LTBR antibody of claim 1 , having at least one of the following:

3. The agonist antibody (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (iii) a heavy chain variable region (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 89 (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97 (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (vii) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 57 Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65 (V Ｈ LTBR) and a light chain variable region (V Ｌ L.T.B.R. (ix) a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 73 Ｈ LTBR) and a light chain variable region (V Ｌ L.T.B.R. The agonist LTBR antibody of claim 1 or 2, comprising:

4. The agonistic LTBR antibody of any one of claims 1 to 3, wherein the agonistic antibody further binds to mouse LTBR.

5. Agonist antibodies have an EC50 of less than 1 nM as measured by ELISA. ５０ The agonist LTBR antibody of any one of claims 1 to 4, which binds to the mouse LTBR extracellular domain at

6. The agonist antibody (i) a heavy chain complementarity determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 27, a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 28, and a heavy chain variable region (VH3) comprising the amino acid sequence of SEQ ID NO: 29; Ｈ (iv) a light chain variable region (VLTBR) comprising a light chain complementarity determining region (CDR-L1), a light chain complementarity determining region (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 30, a light chain variable region (VVL1), a light chain complementarity determining region (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region (VVL3) comprising the amino acid sequence of SEQ ID NO:

32. Ｌ LTBR), or (ii) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 43, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO:46, CDR-L2 having the amino acid sequence of SEQ ID NO:47, and CDR-L3 having the amino acid sequence of SEQ ID NO:

48. Ｌ LTBR), or (iii) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 75, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO:78, CDR-L2 having the amino acid sequence of SEQ ID NO:79, and CDR-L3 having the amino acid sequence of SEQ ID NO:

80. Ｌ LTBR), or (iv) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 83, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 85; Ｈ and a light chain variable region (VLTBR) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 86, CDR-L2 having the amino acid sequence of SEQ ID NO: 87, and CDR-L3 having the amino acid sequence of SEQ ID NO:

88. Ｌ L.T.B.R. The agonist LTBR antibody of any one of claims 1 to 5, comprising:

7. The agonist antibody (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (iii) a heavy chain variable region (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 89 (V Ｈ LTBR) and a light chain variable region (V Ｌ LTBR), or (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97 (V Ｈ LTBR) and a light chain variable region (V Ｌ L.T.B.R. The agonist LTBR antibody of any one of claims 1 to 6, comprising:

8. The agonist LTBR antibody of any one of claims 1 to 7, wherein the antibody binds to the epitope region of SEQ ID NO: 351 on human LTBR.

9. The antibody comprises a heavy chain variable region (VH1) comprising CDR-H1 having the amino acid sequence of SEQ ID NO:27, CDR-H2 having the amino acid sequence of SEQ ID NO:28, and CDR-H3 having the amino acid sequence of SEQ ID NO:

29. Ｈ (iv) a light chain variable region (VLTBR) comprising a light chain complementarity determining region (CDR-L1), a light chain complementarity determining region (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 30, a light chain variable region (VVL1), a light chain complementarity determining region (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 31, and a light chain variable region (VVL3) comprising the amino acid sequence of SEQ ID NO:

32. Ｌ The agonist LTBR antibody of any one of claims 1 to 8, comprising:

10. The antibody comprises a heavy chain variable region (V Ｈ LTBR) and a light chain variable region (V Ｌ The agonist LTBR antibody of any one of claims 1 to 9, comprising:

11. The agonistic LTBR antibody of any one of claims 1 to 10, wherein the agonistic antibody is a multispecific antibody, in particular a bispecific antibody.

12. The agonistic LTBR antibody of any one of claims 1 to 11, wherein the agonistic antibody comprises an Fc domain of human origin, in particular of the human IgG subclass, more particularly of the human IgG1 subclass.

13. The agonistic LTBR antibody of any one of claims 1 to 12, wherein the agonistic antibody comprises an Fc domain of the human IgG1 subclass that contains one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor.

14. The agonistic LTBR antibody of any one of claims 1 to 13, wherein the agonistic antibody comprises an Fc domain of the human IgG1 subclass having the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

15. The agonistic LTBR antibody of any one of claims 1 to 14, wherein the agonistic antibody is a bispecific antibody that binds to LTBR and a tumor-associated antigen (TAA), in particular fibroblast activation protein (FAP).

16. The agonist antibody (a) a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP); (b) a second antigen-binding domain that specifically binds to LTBR; and (c) an Fc domain of the human IgG1 subclass comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor; The agonist LTBR antibody of any one of claims 1 to 15, which is a bispecific antibody comprising:

17. 17. The agonist LTBR antibody of claim 16, wherein the bispecific antibody activates LTBR upon binding to FAP.

18. The agonist LTBR antibody of claim 16 or 17, wherein the bispecific antibody comprises a third antigen-binding domain that binds to human LTBR.

19. 20. The agonistic LTBR antibody of claim 18, wherein the third antigen-binding domain that binds to human LTBR is identical to the second antigen-binding domain that binds to LTBR.

20. a first antigen-binding domain that specifically binds to FAP, (i) a heavy chain complementarity determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3, a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain variable region (VH3) comprising the amino acid sequence of SEQ ID NO: 5; Ｈ (iv) a light chain variable region (V) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 6, a CDR-L2 having the amino acid sequence of SEQ ID NO: 7, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 8; Ｌ FAP), or (ii) a heavy chain variable region (VH1) comprising the amino acid sequence of SEQ ID NO: 19, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21; Ｈ and a light chain variable region (VFAP) comprising CDR-L1 having the amino acid sequence of SEQ ID NO:22, CDR-L2 having the amino acid sequence of SEQ ID NO:23, and CDR-L3 having the amino acid sequence of SEQ ID NO:

24. Ｌ FAP) The agonist LTBR antibody of any one of claims 16 to 19, comprising:

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

9. Ｈ A light chain variable region (V) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:

10. Ｌ A first antigen-binding domain that specifically binds to FAP or comprises a heavy chain variable region (V) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:

25. Ｈ and a light chain variable region (V Ｌ The agonist LTBR antibody of any one of claims 16 to 20, comprising a nucleotide sequence identical to that of claim 15 (FAP).

22. The first antigen-binding domain that specifically binds to FAP comprises a heavy chain variable region (V Ｈ FAP), and a light chain variable region (V Ｌ The first antigen-binding domain comprises or specifically binds to FAP, and the first antigen-binding domain comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:

25. Ｈ FAP), and a light chain variable region (V Ｌ The agonist LTBR antibody of any one of claims 16 to 21, comprising a nucleotide sequence identical to that of claim 15, further comprising a nucleotide sequence identical to that of claim 16, further comprising a nucleotide sequence identical to that of claim 15, and a nucleotide sequence identical to that of claim 16.

23. The agonist LTBR antibody is (a) a heavy chain variable region (V Ｈ FAP) and a light chain variable region (V Ｌ a first antigen-binding domain that specifically binds to FAP, comprising (b) a heavy chain variable region (V Ｈ LTBR) and a light chain variable region (V Ｌ A second antigen-binding domain that specifically binds to LTBR, ​​comprising The agonist LTBR antibody of any one of claims 1 to 22, which is a bispecific antibody comprising:

24. The agonist LTBR antibody is (a) a first Fab fragment that binds to FAP; (b) a second Fab fragment that binds to human LTBR, ​​and (c) an Fc domain of the human IgG1 subclass comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor; The agonist LTBR antibody of any one of claims 1 to 23, which is a bispecific antibody comprising:

25. 25. The agonist LTBR antibody of claim 24, wherein the second Fab fragment that specifically binds to human LTBR is a crossFab fragment.

26. The agonist LTBR antibody is (a) 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) an Fc domain of the human IgG1 subclass comprising one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to an Fc receptor; a bispecific antibody comprising The agonistic LTBR antibody of any one of claims 1 to 23, wherein a first Fab fragment that binds to FAP is fused at its N-terminus to the C-terminus of one of the Fc domain subunits, and a second Fab fragment and a third Fab fragment that specifically bind to LTBR are each fused at their C-terminus to the N-terminus of one of the Fc domain subunits.

27. 27. One or more isolated polynucleotides encoding the agonist LTBR antibody of any one of claims 1-26.

28. 28. An expression vector comprising one or more isolated polynucleotides of claim 27.

29. 29. A prokaryotic or eukaryotic host cell comprising one or more isolated polynucleotides of claim 27 or an expression vector of claim 28.

30. A method for producing a bispecific antigen-binding molecule, comprising: a) culturing a prokaryotic or eukaryotic host cell according to claim 29 under conditions suitable for expression of an agonistic LTBR antibody; and b) optionally recovering the agonistic LTBR antibody.

31. A pharmaceutical composition comprising an agonist LTBR antibody according to any one of claims 1 to 26 and a pharma- ceutically acceptable excipient.

32. An agonistic LTBR antibody according to any one of claims 1 to 26 for use as a medicament.

33. An agonistic LTBR antibody according to any one of claims 1 to 26 for use in (a) inducing ICAM upregulation on endothelial cells or cancer-associated fibroblasts, or (b) enhancing T cell adhesion.

34. 27. An agonistic LTBR antibody according to any one of claims 1 to 26 for use in the treatment of cancer.

35. 27. The agonistic LTBR antibody of any one of claims 1 to 26, for use in the treatment of cancer, wherein the agonistic LTBR antibody or pharmaceutical composition is for administration in combination with chemotherapeutic agents, radiation and / or other agents for use in cancer immunotherapy.

36. The agonistic LTBR antibody of any one of claims 1 to 26, for use in the treatment of cancer, wherein the agonistic LTBR antibody is for administration in combination with an agent that blocks PD-L1 / PD-1 interaction.

37. 32. Use of an agonist LTBR antibody according to any one of claims 1 to 26, or a pharmaceutical composition according to claim 30, in the manufacture of a medicament for the treatment of cancer.

38. A method of treating an individual having cancer, comprising administering to the individual an effective amount of an agonist LTBR antibody of any one of claims 1 to 26, or a pharmaceutical composition of claim 31.

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