Agonistic LTBR antibodies and bispecific antibodies comprising same

Abstract

The present invention relates to novel antibodies that bind to the lymphotoxin beta receptor (LTBR), and to bispecific antigen-binding molecules comprising these novel LTBR antibodies as well as antigen-binding domains that bind tumor-associated antigens, in particular fibroblast activating protein (FAP), to methods of producing these molecules and to methods of using these molecules.

Description

[0001] This invention application is a divisional application based on an invention patent application with an application date of December 19, 2022, application number 202280083563.4 (international application number PCT / EP2022 / 086540), and name “Agonistic LTBR antibodies and bispecific antibodies containing them”. Technical Field

[0002] The present invention relates to novel antibodies that bind to lymphotoxin beta receptor (LTBR), and to bispecific antigen-binding molecules comprising these novel LTBR antibodies and an antigen-binding domain that binds to tumor-associated antigens, in particular fibroblast activation protein (FAP), to methods of producing these molecules and to methods of using these molecules. Background Art

[0003] In recent years, the landscape of cancer treatment has changed profoundly after the development and approval of cancer immunotherapies such as agents that block immune checkpoints PD-1 / PD-L1 and CTLA-4. These cancer immunotherapies can achieve durable remissions in cancer indications such as melanoma, non-small cell lung cancer, and bladder cancer, and are therefore becoming a new standard of care alone or in combination with other therapies. However, only a portion of patients (<30%) receive lasting benefits from these therapies, and most patients relapse due to primary or acquired resistance mechanisms. In addition, several common cancer indications (e.g., colorectal cancer and pancreatic cancer) are mostly refractory to these immunomodulators. Therefore, there is an urgent medical need for developing therapies to address resistance mechanisms and increase responses to cancer immunotherapies (including checkpoint inhibitors).

[0004] Clinical data show that patients who do not respond or respond poorly to checkpoint inhibitors exhibit a non-T cell inflammatory immune phenotype, characterized by the absence of cytotoxic T cells or their confinement to the tumor stroma. Therefore, novel therapies aimed at increasing immune infiltration will be of great use to improve the response rate to checkpoint inhibitors and expand their clinical benefits.

[0005] The TNFR superfamily consists of 19 ligands and 29 receptors that share certain structural similarities but are involved in many different physiological functions. In general, members of this superfamily can induce cell death (e.g., DR5-TRAIL, Fas-FasL), or they can promote survival and inflammation (e.g., TNF-TNFR). Lymphotoxin beta receptor (LTBR) is a member of the TNFR superfamily belonging to the second class and is expressed by a variety of cells, including stromal cells of the tumor microenvironment, myeloid cells, and tumor cells of epithelial origin. 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, while LTBR, ​​LTα, LTβ, and LIGHT show multivalent interactions that form a complex network of interconnected pathways. Activation of LTBR by lymphotoxin α1β1 (LTα1β1) or LIGHT ligand binding induces receptor oligomerization and signaling via canonical and non-canonical NFκB pathways, leading to upregulation of inflammatory and developmental genes such as adhesion molecules (ICAM and VCAM), chemoattractants (CXCL9, 10, 11), and lymphoid tissue 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 shown by the phenotype of ltbr knockout mice, which fail to develop lymph nodes and Peyer's patches (Fütterer et al., Immunity 1998, 9(1), 59-70, doi: 10.1016 / s1074-7613(00)80588-9). In addition, it is also critical for the development and maintenance of high endothelial venules (HEV) (Browning et al., Immunity 2005, 23(5), 539-550, doi: 10.1016 / j.immuni.2005.10.002). Multicellular aggregates similar to secondary lymphoid organs have been detected histologically in many solid tumors, consisting of T cells and activated dendritic cells (DC), B cells and HEV. Clinical evidence shows that the presence of such ectopic lymphoid organs, also known as tertiary lymphoid structures (TLS), or the presence of HEV is associated with better prognosis in several tumor indications (Dieu-Nosjean et al., Immunol. Rev. 2016, 271(1), 260-275, doi: 10.1111 / imr.12405). TLS and HEV are thought to form in response to the same molecular signals involved in lymph node development, such as activation of the LTBR pathway.Therefore, activation of LTBR has the potential to promote the formation of TLS in the tumor microenvironment and induce antitumor immune responses.

[0006] In fact, preclinical evidence supports the hypothesis that LTBR activation can increase immune infiltration, enhance response to checkpoint inhibitors and induce TLS formation. Activation of LTBR by agonistic antibodies or targeting 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). Therefore, combination therapy when checkpoint inhibitors are combined with LTBR agonists is more effective (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). In addition, evidence of TLS formation and HEV development in response to such agonists has also been reported.

[0007] In addition to the key role of LTBR in the development of TLS and HEV, activation of LTBR has also been found 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 study evaluated the safety and tolerability of a LTBR agonist humanized antibody (hCBE11) in patients with advanced solid tumors (ClinicalTrials.gov, NCT00105170). The study was suspended before completing patient enrollment and was subsequently terminated. This suggests the possibility of serious safety issues associated with widespread LTBR agonism in humans.

[0008] Given the great therapeutic potential of LTBR agonists in improving cancer immunotherapy, there is a need to provide agonistic LTBR antibodies with excellent pharmacological properties, or bispecific antibodies with favorable safety profiles and the ability to activate LTBR only in the tumor microenvironment and not in other LTBR-expressing tissues. Summary of the invention

[0009] The present invention relates to novel antibodies that specifically bind to human lymphotoxin beta receptor (LTBR), in particular agonistic human LTBR antibodies. These antibodies are able to bind to human LTBR and to cynomolgus monkey LTBR with an affinity difference of less than 2-fold. Some novel agonistic human LTBR antibodies are even able to bind to human LTBR, ​​cynomolgus monkey LTBR and mouse LTBR. The present invention also relates to multispecific antibodies comprising these novel agonistic antibodies.

[0010] Thus, provided herein is an agonist lymphotoxin beta receptor (LTBR) antibody that specifically binds to human LTBR and to cynomolgus monkey LTBR, ​​wherein the antibody comprises

[0011] (i) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0012] (ii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0013] (iii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V LLTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0014] (iv) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 88; or

[0015] (v) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40; or

[0016] (vi) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56; or

[0017] (vii) Heavy chain variable region (VH LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64; or

[0018] (viii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:72.

[0019] In one aspect, an agonistic LTBR antibody as previously described herein is provided, wherein the agonistic antibody has at least one of the following properties:

[0020] (a) binds to human LTBR and to cynomolgus monkey LTBR with an affinity that differs by less than 2-fold; or

[0021] (b) With an EC of less than 4 nM as measured by ELISA 50 Binds to the human LTBR extracellular domain with an EC of less than 5 nM as measured by ELISA 50 Binds to the extracellular domain of cynomolgus monkey LTBR; or

[0022] (c) requires cross-linking for its agonistic activity to activate human LTBR; or

[0023] (d) requires cross-linking for its agonistic activity to induce ICAM upregulation in human umbilical vein endothelial cells or cancer-associated fibroblasts; or

[0024] (e) Inhibits the interaction between human LTBR and its human ligands lymphotoxin α1β2 and LIGHT.

[0025] In one aspect, an agonist LTBR antibody is provided, the agonist LTBR antibody comprising (i) a heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:34; or

[0026] (ii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:50; or

[0027] (iii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:82; or

[0028] (iv) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:90; or

[0029] (v) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:98;

[0030] (vi) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:42;

[0031] (vii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:58; or

[0032] (viii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:66; or

[0033] (ix) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:74.

[0034] In one aspect, an agonist LTBR antibody as previously described herein comprises

[0035] (i) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0036] (ii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0037] (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0038] (iv) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (VL LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0039] (v) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98; or

[0040] (vi) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:42; or

[0041] (vii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:58; or

[0042] (viii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 66;

[0043] (ix) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:74.

[0044] In a specific aspect, an agonistic LTBR antibody as described herein above is provided, wherein the agonistic LTBR antibody further specifically binds to murine LTBR. In one aspect, the agonistic LTBR antibody is expressed with an EC of less than 1 nM as measured by ELISA. 50 Binds to the extracellular domain of murine LTBR.

[0045] In one aspect, an agonist LTBR antibody that further specifically binds to murine LTBR comprises

[0046] Heavy chain variable region (V HLTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0047] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0048] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0049] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V LLTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88.

[0050] In one aspect, the agonist LTBR antibody that further specifically binds to murine LTBR comprises a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0051] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0052] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0053] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0054] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98.

[0055] In a specific aspect, an agonist LTBR antibody as described herein binds to the epitope region of SEQ ID NO: 351 on human LTBR. In one aspect, such an agonist LTBR antibody comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (VL LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32. In one aspect, it comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:34.

[0056] The present invention also relates to agonistic LTBR antibodies, which are multispecific antibodies comprising an agonistic LTBR antibody as described herein before. In a specific aspect, an agonistic LTBR antibody as a bispecific antibody is provided. In any aspect described herein before, the agonistic LTBR antibody preferably comprises an Fc domain of human origin, particularly human IgG subclass, more particularly human IgG1 subclass. In one aspect, the agonistic LTBR antibody comprises an Fc domain of human IgG1 subclass, the Fc domain comprising one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to the Fc receptor and / or the effector function. In one aspect, the agonistic LTBR antibody comprises an Fc domain of human IgG1 subclass, the Fc domain having amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

[0057] In one aspect, the agonistic LTBR antibody is a bispecific antibody that specifically binds to LTBR and a tumor associated antigen (TAA).

[0058] In one aspect, an agonistic LTBR antibody is provided, which is a bispecific antibody that can specifically bind 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, ​​especially human LTBR, ​​wherein activation by LTBR is provided by cross-linking with FAP expressed on tumor stromal cells. Compared with conventional non-targeted agonistic LTBR antibodies, targeting tumor-associated antigens (TAA) such as FAP can limit LTBR agonism to the tumor microenvironment (tumor endothelium and cancer-associated fibroblasts), thereby reducing potential side effects.

[0059] Fibroblast activation protein (FAP) is a serine protease that is highly expressed on the cell surface of cancer-associated stromal cells and on fibroblast reticular cells of secondary lymphoid organs, but has very limited expression in normal tissues. FAP is highly prevalent in various cancer indications, making it useful as a targeting moiety for drugs that should accumulate within the tumor stroma.

[0060] Thus, in one aspect, a bispecific agonist LTBR antibody is provided, comprising

[0061] (a) a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP),

[0062] (b) a second antigen binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), and

[0063] (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 of the antibody to an Fc receptor and / or the effector function.

[0064] The bispecific antibody has 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 of the antibody to Fc receptors and / or effector functions. Fc receptor-mediated cross-linking is thereby eliminated, and tumor-specific activation is achieved by cross-linking through binding to an antigen binding domain that specifically binds to FAP by binding to its tumor-associated target. A bispecific antibody is provided that activates LTBR only after binding to FAP. Thus, a bispecific antibody that activates LTBR in the tumor stroma is provided.

[0065] In one aspect, a bispecific agonist LTBR antibody is provided, comprising

[0066] (a) a first Fab fragment that specifically binds to fibroblast activation protein (FAP),

[0067] (b) a second Fab fragment that specifically binds to lymphotoxin beta receptor (LTBR), and (c) an Fc domain composed of the first subunit and the second subunit and comprising one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to the Fc receptor and / or the effector function.

[0068] In one aspect, a bispecific agonist LTBR antibody is provided, wherein the bispecific agonist LTBR antibody comprises a third antigen binding domain that specifically binds to LTBR, ​​i.e., 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 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 of the antigen binding molecule to the Fc receptor and / or the effector function.

[0069] In a specific aspect, the third antigen binding domain that specifically binds to LTBR is the same as the second antigen binding domain that specifically binds to LTBR, ​​which means that the second antigen binding domain and the third antigen binding domain that specifically bind to lymphotoxin beta receptor (LTBR) are the same. In one aspect, the second antigen binding domain and the third antigen binding domain that specifically bind to LTBR are Fab fragments that specifically bind to LTBR. In one aspect, the Fab fragment that specifically binds to LTBR is a crossfab fragment. In another aspect, the first antigen binding domain that specifically binds to FAP is a Fab fragment.

[0070] In one aspect, a bispecific agonist LTBR antibody as disclosed herein comprises a first antigen binding domain that specifically binds to fibroblast activation protein (FAP), the first antigen binding domain comprising (i) a heavy chain variable region (V H FAP), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 3; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 4; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 5; and a light chain variable region (V L FAP), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 6; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 7; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 8; or

[0071] (ii) Heavy chain variable region (V H FAP), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 19; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 20; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 21; and a light chain variable region (VL FAP), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 22; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 23; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 24; or

[0072] (iii) Heavy chain variable region (V H FAP), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 11; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 12; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 13; and a light chain variable region (V L FAP), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 14; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 15; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 16.

[0073] In one aspect, the first antigen binding domain that specifically binds to FAP comprises (i) a heavy chain variable region (V H FAP), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 3; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 4; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 5; and a light chain variable region (V L FAP), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 6; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 7; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 8; or (ii) a heavy chain variable region (V H FAP), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 19; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 20; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 21; and a light chain variable region (V L FAP), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 22; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 23; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 24. In a specific aspect, the first antigen-binding domain that specifically binds to FAP comprises (i) a heavy chain variable region (V HFAP), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 3; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 4; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 5; and a light chain variable region (V L FAP), the light chain variable region comprising: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 6; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 7; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 8.

[0074] In one aspect, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 10; or the first antigen binding domain comprises: a heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 26; or the first antigen binding domain comprises: a heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 18. In one aspect, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10; or the first antigen binding domain comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 26; or the first antigen binding domain comprises: a heavy chain variable region (V HFAP), which comprises the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 18. In a specific aspect, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10. In particular, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10, and the first antigen binding domain is a Fab fragment.

[0075] In another aspect, a bispecific agonist LTBR antibody as disclosed herein comprises a second antigen binding domain that specifically binds to LTBR, ​​the second antigen binding domain comprising

[0076] (i) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0077] (ii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V LLTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0078] (iii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0079] (iv) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 88; or

[0080] (v) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40; or

[0081] (vi) Heavy chain variable region (VH LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56; or

[0082] (vii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64; or

[0083] (viii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:72.

[0084] In one aspect, the second antigen binding domain and the third antigen binding domain that specifically bind to LTBR comprise

[0085] (i) Heavy chain variable region (V HLTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0086] (ii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0087] (iii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0088] (iv) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V LLTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 88; or

[0089] (v) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40; or

[0090] (vi) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56; or

[0091] (vii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64; or

[0092] (viii) Heavy chain variable region (V HLTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:72.

[0093] In one aspect, a bispecific agonist LTBR antibody as disclosed herein is provided, wherein the second antigen binding domain (and optionally the third antigen binding domain) that specifically binds to LTBR each comprises (i) a heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:34; or

[0094] (ii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:50; or

[0095] (iii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:82; or

[0096] (iv) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:90; or

[0097] (v) Heavy chain variable region (V HLTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:98;

[0098] (vi) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:42;

[0099] (vii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:58; or

[0100] (viii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:66; or

[0101] (ix) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:74.

[0102] In one aspect, a bispecific agonist LTBR antibody as disclosed herein comprises a second antigen binding domain (and optionally a third antigen binding domain) that specifically binds to LTBR, ​​the second antigen binding domain (and optionally the third antigen binding domain) comprising

[0103] (i) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0104] (ii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0105] (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0106] (iv) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0107] (v) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98; or

[0108] (vi) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:42; or

[0109] (vii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:58; or

[0110] (viii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 66;

[0111] (ix) Heavy chain variable region (V HLTBR), which comprises the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:74.

[0112] In another aspect, a bispecific agonist LTBR antibody as 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 murine LTBR comprises

[0113] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0114] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0115] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V LLTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0116] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88.

[0117] In one aspect, the bispecific agonist LTBR antibody comprises a second antigen binding domain (and optionally a third antigen binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and murine LTBR, ​​the second antigen binding domain (and optionally the third antigen binding domain) comprising

[0118] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0119] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0120] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0121] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V LLTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0122] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98.

[0123] In another aspect, the second antigen binding domain (and optionally the third antigen binding domain) that specifically binds to LTBR comprises

[0124] (i) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0125] (ii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:42; or

[0126] (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49, and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:50.

[0127] In one specific aspect, the second antigen binding domain (and optionally the third antigen binding domain) that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34. In another specific aspect, the second antigen binding domain (and optionally the third antigen binding domain) that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 42. In yet another specific aspect, the second antigen binding domain (and optionally the third antigen binding domain) that specifically binds to LTBR comprises: a heavy chain variable region (VH LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:50.

[0128] Thus, in one specific aspect, a bispecific agonist LTBR antibody as described herein comprises: (a) a first antigen binding domain that specifically binds to FAP, the first antigen binding domain comprising: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10; and (b) a second antigen binding domain (and optionally a third antigen binding domain) that specifically binds to LTBR, ​​the second antigen binding domain (and optionally the third antigen binding domain) comprising: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34. In another aspect, the bispecific antigen-binding molecule as described herein comprises: (a) a first antigen-binding domain that specifically binds to FAP, the first antigen-binding domain comprising: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10; and (b) a second antigen binding domain (and optionally a third antigen binding domain) that specifically binds to LTBR, ​​the second antigen binding domain (and optionally the third antigen binding domain) comprising: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 42. In another aspect, the bispecific antigen-binding molecule as described herein comprises: (a) a first antigen-binding domain that specifically binds to FAP, the first antigen-binding domain comprising: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V LFAP), which comprises the amino acid sequence of SEQ ID NO: 10; and (b) a second antigen binding domain (and optionally a third antigen binding domain) that specifically binds to LTBR, ​​the second antigen binding domain (and optionally the third antigen binding domain) comprising: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:50.

[0129] In all of these aspects previously described herein, the bispecific agonist LTBR antibody comprises an Fc domain consisting of a first subunit and a second subunit and comprising one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to the Fc receptor and / or effector function. In one aspect, the Fc domain consisting of the first subunit and the second subunit is an IgG Fc domain. In a specific aspect, the Fc domain consisting of the first subunit and the second subunit is an IgG1 Fc domain or an IgG4 Fc domain. In a specific aspect, the Fc domain belongs to the human IgG1 subclass, which has amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

[0130] In another aspect, a bispecific agonist LTBR antibody as defined herein before is provided, wherein according to the knobs into holes approach, the first subunit of the Fc region comprises a knob and the second subunit of the Fc region comprises a hole. In particular, a bispecific antigen binding molecule is provided, wherein (i) the first subunit of the Fc region comprises amino acid substitutions S354C and T366W (numbered according to the Kabat EU index), and the second subunit of the Fc region comprises amino acid substitutions Y349C, T366S and Y407V (numbered according to the Kabat EU index), or (ii) the first subunit of the Fc region comprises amino acid substitutions K392D and K409D (numbered according to the Kabat EU index), and the second subunit of the Fc region comprises amino acid substitutions E356K and D399K (numbered according to the Kabat EU index). More particularly, a bispecific agonist LTBR antibody is provided, wherein the first subunit of the Fc region comprises amino acid substitutions S354C and T366W (numbered according to the Kabat EU index), and the second subunit of the Fc region comprises amino acid substitutions Y349C, T366S and Y407V (numbered according to the Kabat EU index).

[0131] In another aspect, there is provided a bispecific agonist LTBR antibody as defined herein before, comprising

[0132] (a) a first Fab fragment that specifically binds to FAP,

[0133] (b) a second Fab fragment that specifically binds to LTBR, ​​and

[0134] (c) an Fc domain consisting of a first subunit and a second subunit and comprising one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor and / or effector function. In one aspect, 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.

[0135] In another aspect, a bispecific agonist LTBR antibody is provided, comprising

[0136] (a) a first Fab fragment that specifically binds to FAP,

[0137] (b) a second Fab fragment and a third Fab fragment that specifically bind to LTBR, ​​and

[0138] (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 of the antibody to an Fc receptor and / or the effector function, 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 binds to LTBR are each fused at their C-termini to the N-terminus of one of the Fc domain subunits.

[0139] Thus, a bispecific agonist LTBR antibody is provided that provides bivalent binding to LTBR and monovalent binding to FAP.

[0140] According to another aspect of the present invention, there is provided an isolated one or more isolated polynucleotides encoding an agonistic LTBR antibody or a bispecific agonistic LTBR antibody as described herein before. The present invention further provides a vector, in particular an expression vector, comprising an isolated polynucleic acid of the present invention; and provides a host cell comprising an isolated nucleic acid or an expression vector of the present invention. In some aspects, the host cell is a eukaryotic cell, in particular a mammalian cell. On the other hand, there is provided a method for producing an agonistic LTBR antibody or a bispecific agonistic LTBR antibody as described herein before, the method comprising culturing a host cell as described above under conditions suitable for expressing the agonistic LTBR antibody or the bispecific agonistic LTBR antibody, and isolating the agonistic LTBR antibody or the bispecific agonistic LTBR antibody. The present invention also encompasses an agonistic LTBR antibody or a bispecific agonistic LTBR antibody as produced by the method of the present invention.

[0141] The present invention further provides a pharmaceutical composition comprising an agonistic LTBR antibody or a bispecific agonistic LTBR antibody as described herein before and a pharmaceutically acceptable carrier. In one aspect, the pharmaceutical composition comprises an additional therapeutic agent.

[0142] The present invention also encompasses an agonist LTBR antibody or a bispecific agonist LTBR antibody as described herein before, or a pharmaceutical composition comprising the bispecific agonist LTBR antibody, for use as a medicament.

[0143] In one aspect, a bispecific agonistic LTBR antibody or a pharmaceutical composition of the invention as described herein before is provided for use in (a) inducing upregulation of ICAM on endothelial cells or cancer-associated fibroblasts, or (b) enhancing T cell adhesion.

[0144] In one specific aspect, an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein before or a pharmaceutical composition of the invention is provided for use in treating cancer. In another specific aspect, the invention provides an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein before for use in treating 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, an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein is used to treat cancer, wherein the agonist LTBR antibody or bispecific agonist LTBR antibody is used to be administered in combination with an agent that blocks the PD-L1 / PD-1 interaction. In another aspect, an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein before or a pharmaceutical composition of the invention is provided for upregulating or prolonging cytotoxic T cell activity.

[0145] In another aspect, the present invention provides a method of inhibiting tumor cell growth in an individual, the method comprising administering to the individual an effective amount of an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein before, or a pharmaceutical composition of the present invention, to inhibit tumor cell growth. In another aspect, the present invention provides a method of treating or delaying cancer in an individual, the method comprising administering to the individual an effective amount of an agonist LTBR antibody or bispecific agonist LTBR antibody as described herein before, or a pharmaceutical composition of the present invention.

[0146] In addition, provided is the use of a bispecific antigen binding molecule as described above in the manufacture of a medicament for treating a disease in an individual in need thereof, in particular in the manufacture of a medicament for treating cancer, and a method of treating a disease in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition comprising an agonist LTBR antibody or a bispecific agonist LTBR antibody of the invention, the composition being in a pharmaceutical form. In a specific aspect, the disease is cancer. In any of the above aspects, the individual is a mammal, in particular a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] Figures 1A to 1G Schematic representation of recombinant soluble proteins (receptor, ligand and tool protein) used as antigens for phage display and immunization to generate anti-LTBR antibodies is shown. Figure 1A Shown is a schematic diagram of the so-called biotinylated Fc depleting agent (Fc depleting agent kh NC avi biotinylated, P1AA0981), which contains a knob-hole (kh) Fc domain that is used as a pre-clearing agent to avoid binding of clones to the Fc domain. Figure 1BA schematic diagram of human lymphotoxin α1β2 single chain avi his (P1AE1235) is shown. Figure 1C A schematic diagram of murine lymphotoxin α1β2 single chain avi his (P1AE1236) is shown. Figure 1D Schematic representation of monomeric huLTBR ectodomain ECD (S28-M227) as Fc fusion avi biotinylated (P1AE2835) is shown. Figure 1E Schematic representation of monomeric muLTBR ectodomain ECD (S28-L223) Fc fusion avi biotinylation (P1AE4410) is shown. Figure 1F Schematic representation of monomeric cynomolgus LTBR ectodomain ECD (S28-M227) Fc fusion avi biotinylated (P1AE4411) is shown. Figure 1G Schematic representation of the monomeric N-terminal human LTBR (ECD full length) hu IgG1 Fc-fused kih HRYF avi (P1AE1217) is shown.

[0148] Figures 2A to 2F Schematic representation of additional antigens used for screening and clonal characterization of anti-LTBR antibodies and bispecific FAP-LTBR antibodies is shown. Figure 2A Schematic representation of dimeric human LTBR-Fc fusion biotinylation (P1AE2401) is shown. Figure 2B Schematic representation of human LTBR ECD (S28-M227) N-terminal and C-terminal extensions-Fc fusion wt kih HRYF-Avi-His biotinylated (P1AE7979) is shown. Figure 2C Schematic representation of monomeric N-terminal cynomolgus LTbR (ECD full length) hu IgG1 Fc fusion kihHRYF avi biotinylated (P1AE2656) is shown. Figure 2D A schematic diagram of the disulfide-linked biotinylated homodimer of dimeric murine LTBR-Fc (P1AE2655) is shown. Figure 2E Schematic representation of human FAP-Avi-His biotinylation (P1AA5347) is shown. Figure 2F Schematic representation of murine FAP-Avi-His biotinylation (P1AD9907) is shown.

[0149] Figure 3The Fab clone FAP1 tbr.P218.076 (P1AE5929 after IgG conversion) was identified as a cross-reactive antibody in a sandwich ELISA, where specific binding to human LTBR (P1AE2835) and murine LTBR (P1AE4410) was determined. The antibody was derived from phage display. Specific binding to LTBR was confirmed using a human Fc fragment (P1AA0981), and Fc binding was excluded because the antigen used in the phage display was already an Fc-tagged fusion protein. The Y axis represents OD 450-900 .

[0150] Figure 4 Inhibition of LTBR-lymphotoxin α1β2 interaction by anti-LTBR IgG antibodies is shown. The selected anti-LTBR IgG has been titrated starting from 100 nM (15 μg / ml) in 3-fold dilutions.

[0151] Figure 5 Inhibition of LTBR-LIGHT interaction by anti-LTBR IgG antibodies is shown. The selected anti-LTBR IgG has been titrated starting from 100 nM (15 μg / ml) in 3-fold dilutions.

[0152] Figure 6 Results are shown for various anti-LTBR IgGs in the absence or presence of a cross-linking antibody (linker), as determined in the HeLa NFκB luc reporter assay described in Example 1.7. The concentration of anti-LTBR IgG was plotted against the units of emitted light (RLU) measured after incubation and addition of luciferase detection solution.

[0153] Figure 7 Results of the assessment of cell surface LTBR binding of anti-LTBR IgG (FACS on recombinant human LTBR CHO cells) are shown, with median fluorescence intensity plotted against concentration. Anti-LTBR IgG has been used in titrations in 3-fold dilution steps starting from 20 μg / ml.

[0154] Figure 8 A heat map of epitope identification based on the corresponding percentage binding values ​​of eight selected anti-LTBR IgGs is shown.

[0155] Figures 9A to 9E Schematic representations of anti-LTBR antibodies and bispecific FAP-LTBR antibodies are shown. Fig. 9A A schematic diagram of the monospecific anti-LTBR human IgG1 PG LALA antibody is shown. Fig. 9BSchematic representation of a 1+1 anti-LTBR / anti-FAP bispecific antibody that is a human IgG1 PG LALA crossMab with crossed V domains in the anti-LTBR Fab arm and charges in the CH1 / Ck domains of the anti-FAP Fab arm is shown. Fig. 9C Shown is a schematic diagram of a 1+1 anti-LTBR / anti-FAP bispecific antibody that is a human IgG1 PG LALA crossMab with crossed CH1 / Ck domains in the anti-FAP Fab arms. Fig.9D Shown is a schematic diagram of a 2+1 anti-LTBR / anti-FAP bispecific antibody that is 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. Fig.9E Schematic representation of a 2+1 anti-LTBR / anti-FAP bispecific antibody that is a human IgG1 PG LALA crossMab with crossed V domains in both anti-LTBR Fab arms and charges in the CH1 / Ck domains of the anti-FAP Fab fragment fused to the C-terminus of the Fc domain is shown.

[0156] Fig.10 Inhibition of LTBR-lymphotoxin α1β2 interaction by anti-FAP / anti-LTBR bispecific antibodies is shown. The selected anti-FAP / anti-LTBR bispecific antibodies have been titrated starting from 100 nM (15 μg / ml) in 3-fold dilutions.

[0157] Fig.11 Inhibition of LTBR-LIGHT interaction by anti-FAP / anti-LTBR bispecific antibodies is shown. The selected anti-FAP / anti-LTBR bispecific antibodies have been titrated in 3-fold dilutions starting from 100 nM (15 μg / ml).

[0158] Fig. 12A and 12B A schematic diagram of the murine surrogate bispecific FAP-LTBR antibody is shown. Fig. 12A Shown is a schematic of a 1+1 anti-LTBR / anti-FAP bispecific antibody that is a murine IgG1 DA PG crossMab with crossed V domains in the anti-FAP Fab arm and charges in the CH1 / Ck domains of the anti-mu LTBR Fab arm. Fig. 12BSchematic representation of a 2+1 anti-LTBR / anti-FAP bispecific antibody that is a murine IgG1 DA PG crossMab with charges in the CH1 / Ck domains of the two anti-LTBR Fab arms and the crossover CH1 / Ck domains of the anti-FAP Fab fragment fused to the C-terminus of the Fc domain is shown. Fig. 12C Schematic representation of a 2+1 anti-LTBR / non-targeting (DP47) bispecific antibody that is a murine IgG1DA PG crossmab with charges in the CH1 / Ck domains of two anti-LTBR Fabs and the crossover CH1 / Ck domains of the non-targeting (DP47) Fab fragment fused to the C-terminus of the Fc domain is shown.

[0159] Figures 13A to 13D Anti-LTBR agonist antibodies upregulate ICAM on endothelial cells in a cross-linking-dependent manner. Fig.13A and Fig. 13C ) or does not exist ( Fig. 13B and Fig.13D Median fluorescence intensity of ICAM on human umbilical vein endothelial cells (HUVEC) treated with anti-human LTBR agonist antibody in the absence of Fc cross-linking antibody (anti-Fc cross-linker). Data were normalized to the activity of anti-LTBR antibody CBE11 (P1AE1873).

[0160] Figures 14A to 14D Anti-LTBR agonist antibodies upregulate ICAM on cancer-associated fibroblasts (CAFs) in a cross-link-dependent manner. Fig.14A and Fig. 14C ) or does not exist ( Fig. 14B and Fig.14D ) Median fluorescence intensity of ICAM on immortalized CAFs treated with anti-human LTBR agonist antibody in the presence of anti-Fc crosslinker. Data were normalized to the activity of the anti-LTBR antibody CBE11 (P1AE1873).

[0161] Figures 15A to 15F The FAP-LTBR bispecific antigen binding molecules are shown to bind to human ( Fig.15A and Fig.15D )、Cynomolgus monkey( Fig. 15B and Fig.15E ) and mouse LTBR ( Fig. 15C and Fig.15F ) binding. Shown is the detection of overexpressed human LTBR ( Fig.15A and Fig.15D )、Cynomolgus monkey LTBR( Fig. 15B and Fig.15E) or mouse LTBR ( Fig. 15C and Fig.15F ) The median fluorescence intensity of the anti-human Fc secondary antibody of the FAP-LTBR bispecific molecule bound to the cells. The data were normalized to the baseline.

[0162] Figures 16A to 16D The FAP-LTBR bispecific antigen binding molecule is shown to upregulate ICAM on cancer-associated fibroblasts in a FAP-dependent manner. Fig.16A and Fig. 16C , hTERT CAF) or only express LTBR ( Fig. 16B and Fig.16D , median ICAM fluorescence intensity on immortalized CAFs of hTERT CAFs_delFAP). Data were normalized to ICAM expression of untreated controls.

[0163] Figures 17A to 17D The FAP-LTBR bispecific molecule is shown to upregulate ICAM on endothelial cells in a FAP-dependent manner. Fig.17A and Fig. 17C ) or NIH-3T3 cells lacking FAP expression ( Fig. 17B and Fig.17D ) Co-cultured CD31 + Median fluorescence intensity of ICAM on HUVEC. Data were normalized to ICAM expression in untreated controls and EC 50 The values ​​are shown in the legend.

[0164] Figures 18A to 18F The results showed that the FAP-LTBR bispecific antigen binding molecules induced endothelial cells to secrete chemoattractants in a FAP-dependent manner. The FAP-overexpressing NIH-3T3 cells treated with the FAP-LTBR bispecific molecules for 48 hours were measured by Bio-plex. Fig.18A , Fig.18C and Fig.18E ) or NIH-3T3 cells lacking FAP expression ( Fig.18B , Fig.18D and Fig.18F ) The concentrations of different chemokines (CXCL9, CXCL10 and CXCL11) in the supernatant of co-cultured HUVECs.

[0165] Fig.19Shown is an increase in T cell adhesion on endothelium stimulated with FAP-LTBR bispecific antigen binding molecules. Shown is the area of ​​labeled T cells attached to HUVEC cultures stimulated with FAP-LTBR bispecific antibodies (2 nM) in the presence (black bars) or absence (white bars) of FAP. TNFα (0.5 ng / mL) was used as a positive control.

[0166] Fig. 20A and 20B Demonstration that the FAP-LTBR bispecific antibody surrogate molecule upregulates adhesion molecules on mouse fibroblasts in vitro in a FAP-dependent manner. Shown are NIH-3T3 cells ( Fig. 20A ) or NIH-3T3 cells overexpressing FAP ( Fig. 20B ) Median fluorescence intensity of VCAM on . Data were normalized to baseline expression of VCAM in untreated controls.

[0167] Fig.21A and 21B The FAP-LTBR bispecific antibody surrogate molecule was shown to upregulate adhesion molecules on endothelial cells in a FAP-dependent manner in vitro. Fig.21A ) or NIH-3T3 cells overexpressing FAP ( Fig.21B ) Co-cultured CD31 + Median fluorescence intensity of ICAM on HUVECs.

[0168] Fig. 22 The study design for an in vivo mouse study evaluating the safety, efficacy, and pharmacodynamic profile of FAP-LTBR bispecific antibodies alone or in combination with anti-PD-L1 antibodies in a subcutaneous MC38-hu CEA tumor model is shown. The timeline describes the treatment and sacrifice time points, and the table describes the details of the treatment groups, dosing, and schedule of this study.

[0169] exist Fig.23A In the present study, the FAP-LTBR replacement molecule was shown to inhibit tumor growth as a monotherapy in the MC38-huCEA subcutaneous tumor model and improve the response to aPD-L1 treatment. Tumor growth curves of mice treated with vehicle, P1AF4664, P1AF4674, aPD-L1, P1AF4664+aPD-L1, and P1AF4674+aPD-L1 are plotted in Fig.23AComparison of tumor volumes at 12 days after the start of treatment showed statistically significant greater 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, Holm-Sidak multiple comparison test (*p<0.05, **p<0.01, ***p<0.001). ( Fig. 23B ). 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 to 23H middle.

[0170] Fig.24A and 24B The FAP-LTBR surrogate molecule was shown to be able to induce endothelial activation in the MC38-huCEA subcutaneous tumor model. Flow cytometric analysis of tumor single-cell suspensions was performed on days 9 and 16 after the start of treatment. + Podoplanin- and endothelial cells were gated, and the median fluorescence intensity of ICAM ( Fig.24A and Fig. 24B ) were quantified. The data showed that treatment with P1AF4664 and P1AF4674 increased ICAM on tumor endothelial cells throughout the treatment process. Statistical analysis was performed using one-way Anova, Holm-Sidak multiple comparison test (*p<0.05, **p<0.01, ***p<0.001).

[0171] Fig.25 The study design for an in vivo mouse study evaluating the safety, efficacy, and pharmacodynamic profile of FAP-LTBR bispecific antibodies alone or in combination with anti-PD-L1 antibodies in a subcutaneous KPC4662-huCEA tumor model is shown. The timeline describes the treatment and sacrifice time points and the table describes the details of the treatment groups, dosing, and schedule of this KPC4662-huCEA study protocol.

[0172] Figures 26A to 26CThe FAP-LTBR surrogate molecule was demonstrated to induce high endothelial venules in the KPC4662-huCEA subcutaneous tumor model. A significant increase in Meca79-positive vessels (fraction of all vessels) was recorded in the treatment group compared to the vehicle group. Statistical analysis was performed using one-way Anova, Holm-Sidak multiple comparison test (*p<0.05, **p<0.01, ***p<0.001) ( Fig.26A ). Fig.26B and Fig.26C Representative immunofluorescence staining of CD31 (all vessels) and Meca79 (HEV) in P1AF4664-treated samples from day 6 after the start of treatment are shown, respectively.

[0173] Fig. 27 Shown is also able to assess the safety, effectiveness and pharmacodynamic characteristics of the LTBR bispecific antigen binding molecules in the in situ EMT6 tumor model alone or in combination with anti-PD-L1 antibodies. The timeline describes the treatment and execution time points, and the table describes the treatment group, administration and time arrangement details of this EMT6 research program. If not otherwise specified, then at all time points (d0, d2, d5, d7, etc.) above, vehicle and mouse surrogate LTBR bispecific antigen binding molecules P1AG5459 and P1AG5461 (non-targeted control) were injected. If not otherwise specified, PDL1 was injected only at the time point indicated by the empty arrow.

[0174] exist Fig.28A and 28B In the present study, a murine surrogate FAP-LTBR bispecific antibody (P1AG5459) was shown to inhibit tumor growth in the EMT6 orthotopic tumor model as a monotherapy and improve the response to aPD-L1 treatment. Tumor growth curves (medians) of mice treated with vehicle, P1AG5459, P1AG5461, aPD-L1, and P1AG5459+aPD-L1 are plotted in Fig.28A Comparison of tumor volumes at 22 days after the start of treatment is plotted in Fig.28B , and showed that there was a statistically significant greater volume for vehicle versus P1AG5459, vehicle versus P1AG5459+aPD-L1, P1AG5459 versus P1AG5461, and P1AG5461 versus P1AG5459+aPD-L1. Statistical analysis was performed using a one-way Anova, Holm-Sidak multiple comparison test (*p<0.05, **p<0.01, ***p<0.001).

[0175] Figures 29A to 29CThe results showed 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 cytometric analysis of tumor single-cell suspensions was performed on day 13 after the start of treatment. + Podoplanin-gated endothelial cells, and ICAM ( Fig.29A ) and VCAM( Fig.29B ) or the percentage of Meca79+ endothelial cells ( Fig.29C ) were quantified. The data showed that treatment with P1AG5459 alone or in combination with aPD-L1, but not in combination with the non-targeting control P1AG5461, increased ICAM and VCAM on tumor endothelial cells and increased the frequency of Meca79+ endothelial cells ( Fig.29C ). The effects on endothelial activation and HEV differentiation were more pronounced in responder mice (□) than in non-responder mice (●).

[0176] Fig. 30A Shows that treatment with P1AG5459 alone or in combination with aPD-L1 increased the amount of Meca79+ endothelial cells quantified by immunofluorescence imaging at day 13 after the start of treatment, but P1AG5461 did not. Example images show the main vasculature (as shown by CD31 staining) and the localization of Meca79+ endothelial cells.

[0177] Fig. 30B , 30C and 30D show CD8 T cells as measured by immunofluorescence imaging on day 13 after the start of treatment ( Fig. 30B )、CD4 T cells( Fig. 30C ) and B220+B cells ( Fig.30D ) Quantification of infiltration into EMT6-positive tumors. P1AG5459 increased the number of T and B cells in tumors when used alone or in combination with aPD-L1, which also increased the number of T and B cells to some extent when used alone, but P1AG5461 did not increase the number of T and B cells. In the case of P1AG5459 monotherapy, a clear correlation could be observed in responder mice (□) compared to non-responder mice (●).

[0178] Figures 31A to 31C We demonstrated that the murine surrogate FAP-LTBR bispecific antibody (P1AG5459) induced upregulation of chemokines in the EMT6 orthotopic tumor model. We measured the chemokine CXCL13 ( Fig.31A )、CCL5( Fig.31B ) and CCL10( Fig.31C ). Tumors treated with P1AG5459 tended to have higher levels of CXCL13, CCL5, and CXCL10. Responder tumors (□) tended to show higher concentrations of chemokines.

[0179] exist Figures 32A to 32C Figure 5 shows 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 and B cells. Tumor growth curves (median) are plotted on Fig.32A The comparison of tumor volume on day 15 is plotted in Fig.32B and shows that in the absence of CD8 T cells, there was no difference in tumor volume when comparing vehicle and P1AG5459 treated mice. Comparison of tumor volume at day 20 is plotted in Fig.32C and showed in the absence of CD4 T cells or expression of CD20 + In the case of B cells, there was no difference in tumor volume when comparing vehicle- and P1AG5459-treated mice.

[0180] Fig.33 Representative immunofluorescence images of orthotopic colorectal cancer tumors treated with vehicle or a murine surrogate FAP-LTBR bispecific antibody (P1AG5459) are shown. Tumors were identified by Ki67 staining of proliferating cells, and tumor boundaries are marked with 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.

[0181] Fig.34 Shown is the development of tertiary lymphoid structures (TLS) in mice bearing orthotopic CRC following treatment with P1AG5459. Ki67 staining of whole sections guides identification of tumor margins, marked by dashed lines. B220 (B cell) staining of whole sections highlights B cell aggregates, which are magnified in subsequent images. Zoomed images characterize in more detail the cellular composition of TLS, which contain pNAD / Meca79 + High endothelial venules, CD11c + Myeloid cells, B220 + Ki67 + Proliferating cells (indicative of germinal centers), CD8 and CD4 T cells, some of which co-express the activation marker PD1 and the stemness marker TCF1.

[0182] Fig.35A and35B The ability of FAP-LTBR bispecific antigen binding molecules containing different LTBR antigen binding domains to upregulate ICAM on endothelial cells in a FAP-dependent manner was compared. Fig.35A ) or NIH-3T3 cells lacking FAP expression ( Fig.35B ) Co-cultured CD31 + Median fluorescence intensity of ICAM on HUVECs. Data were normalized to ICAM expression in untreated controls.

[0183] Figures 36A to 36C The binding of FAP-LTBR bispecific antigen binding molecules containing different LTBR antigen binding domains to human ( Fig.36A )、Cynomolgus monkey( Fig.36B ) and mouse LTBR ( Fig.36C ) ability. Shown is the detection of overexpressed human LTBR ( Fig.36A )、Cynomolgus monkey LTBR( Fig.36B ) or mouse LTBR ( Fig.36C ) The median fluorescence intensity of the anti-human Fc secondary antibody of the FAP-LTBR bispecific molecule bound to the cells. The data were normalized to the baseline.

[0184] Figures 37A to 37E Schematic representation of anti-LTBR IgG antibody and bispecific FAP-LTBR antibody is shown. Fig.37A A schematic diagram of a monospecific anti-LTBR human IgG1 PG LALA antibody (BHA10, P1AH0119) is shown. Fig.37B Shown is a schematic diagram of a bispecific 2+1 anti-LTBR / anti-FAP (4B9) antibody that is a human IgG1 PG LALA with 2 N-terminal Fab arms specific for LTBR (CBE11) and C-terminal anti-FAP VL and VH domains of clone 4B9 (P1AE1079). Fig.37C Shown is 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 charges in the CH1 / Ck domains of the anti-FAP Fab arm. Fig.37DSchematic representation 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 charges in the CH1 / Ck domains of the anti-FAP Fab fragment fused to the C-terminus of the Fc domain (Fc knob chain) is shown. Fig.37E Schematic representation 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 both anti-LTBR Fab arms and charges in the CH1 / Ck domains of the anti-FAP Fab fragment fused to the C-terminus of the Fc domain (Fc knob chain) is shown.

[0185] Figures 38A to 38D Deuteration difference plot of bound human LTBR compared to non-complexed LTBR is shown. Deuteration difference plot shows the primary sequence of human LTBR (P1AH2680) and the deuteration difference between human LTBR plus antibody / ligand and no antibody / ligand LTBR for five different labeling times 15 seconds, 1 minute, 10 minutes, 60 minutes and 300 minutes. The bars above the sequence show the peptides. The boxes indicate the proposed epitopes for the corresponding antibodies / ligands. The proposed epitope is mapped (black area) onto the alphafold model AF-P36941-F1-model_v2 S28 to M227 below. Shown is the deuteration difference plot on human LTBR for P1AE9459 ( Fig.38A )、P1AE1873( Fig.38B ); P1AH0119( Fig.38C ) and P1AE1235( Fig.38D , ligand) of the proposed epitope. DETAILED DESCRIPTION

[0186] definition

[0187] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the field to which the invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa.

[0188] As used herein, the terms "antigen binding molecules" or "antibodies" are used interchangeably and in their broadest sense refer to molecules that specifically bind to antigenic determinants. Examples of antigen binding molecules are antibodies, bispecific antibodies or multispecific antibodies, antibody fragments, and scaffold antigen binding proteins. The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen binding activity.

[0189] As used herein, the term "antigen binding domain capable of specifically binding to a target cell antigen" or "a portion capable of specifically binding to a target cell antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one aspect, the antigen binding domain is capable of activating signal transduction through its target cell antigen. In a specific aspect, the antigen binding domain is capable of directing an entity connected thereto (e.g., an LTBR agonistic antibody) to a target site, such as a specific type of tumor cell or tumor matrix carrying an antigenic determinant. Antigen binding domains capable of specifically binding to a target cell antigen include antibodies and fragments thereof as further defined herein. In addition, 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 sequence proteins or designed repeat sequence domains (see, e.g., WO 2002 / 020565). In particular, the 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). With respect to antibodies or fragments thereof, the term "antigen binding domain capable of specific binding to a target cell antigen" refers to a portion of a molecule that includes a region that specifically binds to a portion or all of an antigen and is complementary thereto. An antigen binding domain capable of specific antigen binding may be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). In particular, 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. On the other hand, an "antigen binding domain capable of specific binding to a target cell antigen" may also be a Fab fragment or a cross-Fab fragment.

[0190] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are usually present in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. Contrary to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.

[0191] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each binding site binding to the same epitope of the same antigen. The term "bispecific" means that the antigen binding molecule can specifically bind to at least two unique antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which has specificity for different antigenic determinants. In certain embodiments, a bispecific antigen binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two unique cells. Bispecific antigen binding molecules as described herein can also form a part of a multispecific antibody.

[0192] The term "valence" used in the present application means that there is a specific number of binding sites with specificity to a unique antigenic determinant in an antigen binding molecule that is specific to a unique antigenic determinant. Therefore, the terms "divalent", "tetravalent" and "hexavalent" respectively represent that there are two binding sites, four binding sites and six binding sites with specificity to a specific antigenic determinant in an antigen binding molecule. In a particular aspect of the present invention, the bispecific antigen binding molecules according to the present invention can be monovalent to a specific antigenic determinant, meaning that they only have one binding site to the antigenic determinant, or can be divalent or tetravalent to a specific antigenic determinant, meaning that they have two binding sites or four binding sites to the antigenic determinant, respectively.

[0193] The terms "full-length antibody" and "complete antibody" are used interchangeably herein to refer to antibodies with structures substantially similar to natural antibody structures. "Native antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, which are composed of two light chains and two heavy chains bonded by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH) (also referred to as variable heavy chain domain or heavy chain variable domain), followed by three constant domains (CH1, CH2 and CH3) (also referred to as heavy chain constant region). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL) (also referred to as variable light chain domain or light chain variable domain), followed by a light chain constant domain (CL) (also referred to as light chain constant region). The heavy chains of antibodies can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0194] "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 certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269 to 315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen binding sites, which can be bivalent or bispecific, see, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516B1). Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0195] Papain digestion of intact antibodies produces two identical antigen-binding fragments called "Fab" fragments, each of which contains the heavy chain variable domain and the light chain variable domain as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term "Fab fragment" refers to an antibody fragment that contains a light chain fragment, which contains a VL domain and a light chain constant domain (CL), as well as the VH domain and the first constant domain (CH1) of the heavy chain. The difference between Fab' fragments and Fab fragments is that Fab' fragments have some residues added to the carboxyl terminus of the heavy chain CH1 domain, which residues include one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains have a free thiol group. Pepsin treatment produces F(ab') 2 A fragment having two antigen binding sites (two Fab fragments) and a part of the Fc region. According to the invention, the term "Fab fragment" also includes a "crossover Fab fragment" or a "crossover Fab fragment" as defined below.

[0196] The term "crossover Fab fragment" or "xFab fragment" or "exchange Fab fragment" refers to a Fab fragment in which the variable regions or constant regions of the heavy and light chains are exchanged. Two different chain compositions of exchange Fab molecules are possible and are included in the bispecific antibodies of the present invention: in one aspect, the variable regions of the Fab heavy and light chains are exchanged, i.e., the exchange Fab molecule comprises a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). This exchange Fab molecule is also called CrossFab (VLVH) On the other hand, when the constant regions of the Fab heavy chain and light chain are exchanged, the exchange Fab molecule comprises a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL), and a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1). This exchange Fab molecule is also called CrossFab. (CLCH1) .

[0197] "Single-chain Fab fragments" or "scFab" are polypeptides consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragments are stabilized via natural disulfide bonds between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by generating interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0198] "Exchange single-chain Fab fragments" or "x-scFab" are polypeptides consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL together form an antigen binding site that specifically binds to an antigen, and wherein the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules can be further stabilized by generating interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0199] A "single-chain variable fragment (scFv)" is a fragment of the heavy chain variable region (V H ) and light chain variable region (V L ) fusion protein, connected to a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, and rich in serine or threonine for solubility, and V H The N-terminus of LThe antibody fragment is a fragment of a single-chain polypeptide, which is characterized by having a VH domain, i.e., being able to be assembled into a functional antigen binding site together with a VL domain; or having the characteristics of a VL domain, i.e., being able to be assembled into a functional antigen binding site together with a VH domain, thereby providing the antigen binding properties of a full-length antibody.

[0200] "Scaffold antigen binding proteins" are known in the art, for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen binding domains, see, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13:695-701 (2008). In one aspect of the invention, the scaffold antigen binding protein is selected from the group consisting of: CTLA-4 (Evibody), lipocalin (Anticalin), protein A derived molecules (such as the Z domain of protein A (affimer)), A domain (Avimer / giant antibody), serum transferrin (transbody); designed ankyrin repeat protein (DARPin), variable domain of antibody light or heavy chain (single domain antibody, sdAb), variable domain of antibody heavy chain (nanobody, aVH), V NAR fragments, fibronectin (AdNectin), C-type lectin domain (tetranectin); variable domain of neoantigen receptor β-lactamase (V NAR fragments), human γ-crystallin or ubiquitin protein (Affilin molecule); Kunitz-type domains of human protease inhibitors, minibodies (such as proteins from the knottin family), peptide aptamers and fibronectin (adnectin). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is mainly expressed in CD4 +CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. The loops corresponding to the antibody CDR can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also called Evibodies (e.g., US7166697B1). Evibody is roughly the same size as the isolated variable region of an antibody (e.g., a domain antibody). For further details, see Journal of Immunological Methods 248 (1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bile, retinoids, and lipids. They have a rigid β-sheet secondary structure with many loops at the open end of the cone structure, which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), US7250297B1 and US20070224633. Affimers are scaffolds of protein A derived from Staphylococcus aureus that can be engineered to bind antigens. The domain consists of a three-helix bundle of about 58 amino acids. Libraries have been formed by randomization of surface residues. For further details, see Protein Eng.Des.Sel.2004,17,455-462 and EP 1641818A1. Avimers are multi-domain proteins derived from the A domain scaffold family. The natural domain of about 35 amino acids adopts a determined disulfide bonded structure. Diversity is formed by the natural variation exhibited by the recombinant 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 by inserting peptide sequences in the surface loops allowed to bind different target antigens. Examples of engineered transferrin scaffolds include trans-isomers. For further details, see J.Biol.Chem 274, 24066-24073 (1999). Designed ankyrin repeat proteins (DARPins) are derived from ankyrin, which is a protein family that mediates the attachment of integral membrane proteins to cell scaffolds. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn.They can be designed to bind different target antigens by randomizing the residues in the first α-helix and β-turn in each repeat sequence. Their 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), PNAS100 (4), 1700-1705 (2003) and J.Mol.Biol.369,1015-1028 (2007) and US20040132028A1. Single-domain antibodies are antibody fragments consisting of a single monomer variable antibody domain. The first single domain is derived from the variable domain of the antibody heavy chain of camelids (nanoantibodies or V. H H fragment). In addition, the term single domain antibody includes autologous human heavy chain variable domain (aVH) or VH derived from shark. NAR Fragment. Fibronectin can be engineered to bind to the scaffold of the antigen. Adnectin consists of the backbone of the natural amino acid sequence of the 10th domain of 15 repeating units of type III human fibronectin (FN3). The three loops at one end of the β-sandwich can be engineered to enable Adnectin to specifically recognize the target therapeutic target. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764 and US6818418B1. Peptide aptamers are combined recognition molecules that consist of a constant scaffold protein, usually thioredoxin (TrxA), which contains a constrained variable peptide loop inserted at the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Minibodies are derived from naturally occurring microproteins containing 3 to 4 cysteine ​​bridges and 25 to 50 amino acids in length, examples of which include KalataBI and conotoxins and knottins. Microproteins have loops that can be engineered to include up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

[0201] An "antibody that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks the binding of a reference molecule to its antigen by 50% or more in a competition assay, and conversely, the reference molecule blocks the binding of an antigen-binding molecule to its antigen by 50% or more in a competition 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 a reference molecule to its antigen by 50% or more in a competition assay, and conversely, the reference molecule does not block the binding of an antigen-binding molecule to its antigen by 50% or more in a competition assay.

[0202] The term "antigen binding domain" or "antigen binding site" refers to a portion of an antibody that contains a region that specifically binds to and is complementary to a portion or all of an antigen. In the case of a larger antigen, an antibody may bind only to a specific portion of the antigen, which is called an epitope. The antigen binding domain can be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0203] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid or a conformational configuration consisting of different regions of non-continuous amino acids), to which an antigen-binding portion binds, thereby forming an antigen-binding portion-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, in free matter in serum and / or in the extracellular matrix (ECM). Unless otherwise indicated, the protein used as an antigen herein can be any natural form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a specific embodiment, the antigen is a human protein. When referring to a specific protein herein, the term encompasses "full length", unprocessed protein, and any form of protein produced by intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

[0204] "Specific binding" refers to binding that is selective for an antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, for example, as measured by SPR, the degree of binding of an antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. In certain embodiments, the molecule that binds to the antigen has a specific binding affinity 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 lower, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 The dissociation constant (Kd) of the

[0205] "Affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Therefore, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0206] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such alterations.

[0207] As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, particularly a target cell in a tumor (such as a cancer cell or a cell of a tumor stroma). Thus, the target cell antigen is a tumor-associated antigen. In particular, a "tumor-associated antigen" or TAA is fibroblast activation protein (FAP).

[0208] The term "fibroblast activation protein (FAP)" is also referred to as prolyl endopeptidase FAP or Seprase (EC3.4.21), and unless otherwise indicated, the term refers to any natural FAP of any vertebrate origin, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed FAP, as well as any form of FAP produced by processing in cells. The term also encompasses naturally occurring variants of FAP, such as splice variants or allelic variants. In one embodiment, the antigen binding molecules 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 amino acid position 761. SEQ ID NO: 265 shows the amino acids of Avi-His-tagged mouse FAP. Preferably, the anti-FAP binding molecules of the present invention bind to the extracellular domain of FAP.

[0209] The term "variable region" or "variable domain" refers to the domain of the antibody heavy chain or light chain that participates in the binding of an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FR) and three hypervariable regions (HVR). See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0210] As used herein, the term "hypervariable region" or "HVR" refers to individual regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, such as the "complementarity determining region" ("CDR").

[0211] Typically, antibodies contain six CDRs; three in VH (CDR-H1, CDR-H2, CDR-H3), and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

[0212] (a) hypervariable loops occurring at the following amino acid residues: 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0213] (b) CDRs occurring at the following 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

[0214] (c) Antigenic contact points occur at the following amino acid residues: 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2), and 93 to 101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)).

[0215] Unless otherwise indicated, CDRs are determined according to the method described in Kabat et al., supra. One skilled in the art will appreciate that CDR names may also be determined according to the method described in Chothia, supra, the method described in McCallum, supra, or any other scientifically accepted naming system.

[0216] "Framework" or "FR" refers to the variable domain residues excluding the complementarity determining regions (CDRs). The FR of a variable domain is usually composed of the following four FR domains: FR1, FR2, FR3, and FR4. Therefore, CDR and FR sequences usually appear in the following sequence in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0217] 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.

[0218] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as 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.

[0219] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. An antibody in "humanized form," such as a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are antibodies in which the constant region has been additionally modified or altered relative to the original antibody to produce the properties according to the present invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0220] The term "CH1 domain" refers to an antibody heavy chain polypeptide portion extending roughly from EU 118 to EU 215 (according to the EU numbering system of Kabat). In one aspect, the CH1 domain has an amino acid sequence of ASTKGPSVFP LAPSSKSTSGGTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPSNTKVDKKV (SEQ ID NO: 283). Typically, the CH1 domain is then connected to the hinge region by a segment with an amino acid sequence of EPKSC (SEQ ID NO: 284), however, in the case of a CH1 domain with a free C-terminal end (such as in a crossfab fragment), the segment may also include an amino acid sequence of EPKSCD (SEQ ID NO: 285) or EPKSCS (SEQ ID NO: 286).

[0221] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, for example from about position 216 to about position 230 according to the EU numbering system of Kabat, or from about position 226 to about position 230 according to the EU numbering system of Kabat. The hinge region of other IgG subclasses can be determined by comparing the hinge region cysteine ​​residues with the IgG1 subclass sequence. The hinge region is generally a dimer molecule composed of two polypeptides with the same amino acid sequence. The hinge region generally contains up to 25 amino acid residues and is flexible, allowing the associated target binding site to move independently. The hinge region can be subdivided into three domains: an upper hinge region, a middle hinge region, and a lower hinge region (see, for example, 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 S or P. In one aspect, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO: 288), wherein X is S or P. In one aspect, the hinge region has the amino acid sequence CPXCP (SEQ ID NO: 289), wherein X is S or P.

[0222] The term "Fc domain" or "Fc region" herein is used to define the antibody heavy chain C-terminal region containing at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. The "CH2 domain" in the human IgG Fc region generally extends from about the 231st amino acid residue to about the 340th amino acid residue. (According to the EU numbering system of Kabat). In one aspect, the CH2 domain has an amino acid sequence of APELLGGPSVFLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDWLNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 290). The uniqueness of the CH2 domain is that it is not tightly paired with another domain. In contrast, two N-connected branched carbohydrate chains are between the two CH2 domains of the complete native Fc region. It is speculated that carbohydrates may provide alternatives for domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22 (1985) 161-206. In one embodiment, a carbohydrate chain is connected to the CH2 domain. The CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain. A "CH3 domain" comprises a stretch of residues from the C-terminus to the CH2 domain in the Fc region (i.e., from an amino acid residue at about position 341 to an amino acid residue at about position 447 according to the EU numbering system (according to Kabat of IgG). In one aspect, the CH3 domain has an amino acid sequence of GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (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 "protrusion" ("knob") in one of its chains and a corresponding introduced "cavity" ("hole") in the other of its chains; see U.S. Pat. No. 5,821,333, which is expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present.Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0223] The term "wild-type Fc domain" refers to an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Wild-type human Fc domains include native human IgG1 Fc regions (non-A and A allotypes), native human IgG2 Fc regions, native human IgG3 Fc regions, and native human IgG4 Fc regions, as well as naturally occurring variants thereof. Wild-type Fc regions are represented in SEQ ID NO: 155 (IgG1, Caucasian allotype), SEQ ID NO: 156 (IgG1, African American allotype), SEQ ID NO: 157 (IgG2), SEQ ID NO: 158 (IgG3), and SEQ ID NO: 159 (IgG4). The term "variant (human) Fc domain" refers to an amino acid sequence that differs from the amino acid sequence of a "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 compared to the native Fc region, such as from about one to about ten amino acid mutations in the native Fc region, and in one aspect, from about one to about five amino acid mutations. In one aspect, the (variant) Fc region has at least about 95% homology to the wild-type Fc region.

[0224] The "knob-into-hole" technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be positioned in the cavity to promote the formation of heterodimers and hinder the formation of homodimers. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity having the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The projections and cavities can be prepared by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis. In a specific embodiment, the knob is modified to include the amino acid substitution T366W in one of the two subunits of the Fc domain, while the hole is modified to include the amino acid substitutions T366S, L368A and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit comprising the knob-modified Fc domain additionally includes the amino acid substitution S354C, while the subunit comprising the hole-modified Fc domain additionally includes the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bond between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0225] "Regions equivalent to the Fc region of an immunoglobulin" are intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants with modifications that produce substitutions, additions or deletions but do not substantially reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular toxicity). 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 losing biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, JU et al., Science 247: 1306-10 (1990)).

[0226] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0227] Fc receptor binding-dependent effector functions can be mediated by the interaction of the Fc region of an antibody with an Fc receptor (FcR), which is a specific cell surface receptor on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens by engulfing immune complexes, and to lyse red blood cells and other various cell targets (e.g., tumor cells) coated with the corresponding antibodies by antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: the Fc receptors of 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.

[0228] Cross-linking of the Fc region receptors of IgG antibodies (FcγRs) triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, release of inflammatory mediators, and regulation of immune complex clearance and antibody production. Three classes of FcγRs have been identified in humans, including:

[0229] -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 (numbered according to the EU index of Kabat) in the Fc region of IgG reduces binding to FcγRI. Substitution of IgG2 residues at positions 233-236 by IgG1 and IgG4 reduces binding to FcγRI by 10 3 times, and abolished the response of human monocytes to antibody-sensitized erythrocytes (Armour, KL et al., Eur. J. Immunol. 29 (1999) 2613-2624).

[0230] -FcγRII (CD32) binds complexed IgG with moderate to low affinity and is widely expressed. This receptor can be divided into two subtypes, namely FcγRIIA and FcγRIIB. FcγRIIA is present in many cells involved in killing (such as macrophages, monocytes, neutrophils) and seems to be able to activate the killing process. FcγRIIB seems to play a role in the inhibition process and is present in B cells, macrophages, mast cells and eosinophils. On B cells, it seems to play a role in inhibiting further production of immunoglobulins and isotype conversion to, for example, the IgE class. On macrophages, FcγRIIB serves to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B type may help inhibit the activation of these cells through the binding of IgE to its individual receptors. For example, antibodies comprising an IgG Fc region comprising mutations in at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbering according to the EU index of Kabat) were found to have reduced binding to FcγRIIA.

[0231] -FcγRIII (CD16) binds IgG with medium to low affinity, and includes two types. FcγRIIIA is present on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. The expression level of FcγRIIIB on neutrophils is high. It is found that for example antibodies (including IgG Fc regions with mutations of at least one amino acid residue E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 (according to the EU index numbering of Kabat)) have reduced binding to FcγRIIIA.

[0232] Shields, RL et al. (J. Biol. Chem. 276 (2001) 6591-6604) describe the location of the binding site on human IgG1 for Fc receptors, the mutation sites mentioned above, and methods for measuring binding to FcγRI and FcγRIIA.

[0233] The term "ADCC" or "antibody-dependent cellular toxicity" is a function mediated by Fc receptor binding, and refers to the lysis of target cells by the antibodies reported herein in the presence of effector cells. The ability of antibodies to induce the initial steps of mediating ADCC is studied by measuring the binding of antibodies to cells expressing Fcγ receptors, such as cells recombinantly expressing FcγRI and / or FcγRIIA or NK cells (expressing FcγRIIIA in nature). In particular, the binding to FcγR on NK cells is measured.

[0234] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc region of an antibody, causes a signaling event that stimulates cells bearing the receptor to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0235] Unless otherwise indicated, the term "LTBR" as used herein refers to any natural 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 produced by processing in cells. 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 shown in SEQ ID NO: 1 (UniProt Accession No. P36941), and the amino acid sequence of an exemplary mouse LTBR is shown in SEQ ID NO: 297 (UniProt Accession No. B2RRV3). The receptor is expressed on the cell surface in the substance and stroma of most lymphoid organs, but is not present on T lymphocytes and B lymphocytes. LTBR may also be referred to as "tumor necrosis factor receptor superfamily member 3 (TNFRSF3)". Signaling by LTBR of the LTα / β heterotrimer (LTα1β2) is important during lymphoid development. LTBR is also known to bind to the ligand LIGHT (TNFSF14). LTα1β2 is specific for LTBR, ​​while LIGHT also binds to and activates HVEM (TNFRSF14), a receptor expressed on immune cells and involved in the regulation of immune cells.

[0236] As used herein, the term "LTBR agonist" includes any moiety that agonizes the interaction of LTBR with its ligand. As used in this context, LTBR preferably refers to human LTBR, ​​and thus the LTBR agonist is preferably an agonist of human LTBR (SEQ ID NO: 1). Typically, the moiety will be an agonistic LTBR antibody or antibody fragment.

[0237] 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 to be useful as a diagnostic and / or therapeutic agent in targeting LTBR. In one aspect, the extent of binding of an anti-LTBR antibody to an unrelated, non-LTBR protein is less than about 10% of the extent of 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 a binding 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 smaller, e.g. 10 -68 M to 10 -13 M, for example 10 -8 M to 10 -10The dissociation constant (K D ).

[0238] The term "peptide linker" refers to a peptide comprising one or more amino acids (usually about 2 to 20 amino acids). Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, (G 4 S) n , (SG 4 ) n or G 4 (SG 4 ) n A peptide linker wherein "n" is typically a number between 1 and 10, typically between 2 and 4, and particularly 2, i.e. a peptide 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 following sequences: GSPGSSSSGS (SEQ ID NO: 302), (G4S) 3 (SEQ ID NO:303), (G4S) 4 (SEQ ID NO: 304), GSGSGSGS (SEQ ID NO: 305), GSGSGNGS (SEQ ID NO: 306), GGSGSGSG (SEQ ID NO: 307), GGSGSG (SEQ ID NO: 308), GGSG (SEQ ID NO: 309), GGSGNGSG (SEQ ID NO: 310), GGNGSGSG (SEQ ID NO: 311), and GGNGSG (SEQ ID NO: 312). Specific target peptide linkers are (G4S) (SEQ ID NO: 298), (G 4 S) 2 Or GGGGSGGGGS (SEQ ID NO:299), (G4S) 3 (SEQ ID NO:303) and (G4S) 4 (SEQ ID NO:304).

[0239] The term "amino acid" as used in this application refers to the group of naturally occurring carboxy α-amino acids including: alanine (three letter code: ala, single 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).

[0240] By "fused" or "linked" is meant that the components (eg, the heavy chains of an antibody and a Fab fragment) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0241] "Percentage (%) of amino acid sequence identity" relative to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing spaces (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as components of sequence identity for the purpose of alignment. Alignment for determining percentage of amino acid sequence identity can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate percent identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed with user documentation in the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087, and is described in WO 2001 / 007611. Unless otherwise indicated, for purposes herein, amino acid sequence identity percentage values ​​are generated using the BLOSUM50 comparison matrix using the ggsearch program of the FASTA package version 36.3.8c or higher. The FASTA package was created by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36 and is publicly 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 a 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) to ensure that a global rather than a local alignment is performed. The amino acid identity percentage is given in the output alignment header.

[0242] In certain embodiments, the amino acid sequence variants of the bispecific antigen binding molecules provided herein are contemplated. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antigen binding molecules containing TNF ligand trimers. The amino acid sequence variants of the antigen binding molecules containing TNF ligand trimers can be prepared by introducing appropriate modifications into the nucleotide sequence of the coding molecule or by peptide synthesis. Such modifications include, for example, the deletion of residues in the antibody amino acid sequence, and / or insertion and / or substitution. Any combination of deletion, insertion and substitution can be performed to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding. The site of interest for substitution mutagenesis includes HVR and framework (FR). Conservative substitutions are provided under the header "preferred substitutions" in Table B, and are further described below with reference to amino acid side chain categories (1) to (6). Amino acid substitutions can be introduced into the target molecule, and the product is screened for desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0243] Table A

[0244]

[0245]

[0246] Amino acids can be grouped according to common side chain properties:

[0247] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0248] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0249] (3) Acidic: Asp, Glu;

[0250] (4) Basic: His, Lys, Arg;

[0251] (5) Residues that affect chain orientation: Gly, Pro;

[0252] (6) Aromatic: Trp, Tyr, Phe.

[0253] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0254] The term "amino acid sequence variant" includes substantial variants, wherein there is an amino acid substitution in one or more hypervariable region residues of a parent antigen binding molecule (e.g., humanized or human antibody). Typically, relative to the parent antigen binding molecule, one or more resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antigen binding molecule. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. In short, one or more CDR residues are mutated and variant antigen binding molecules are displayed on phage and screened for specific biological activity (e.g., binding affinity). In certain embodiments, substitution, insertion or deletion may occur in one or more HVRs, as long as such changes do not substantially reduce the antigen binding ability of the antigen binding molecule. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in CDR. The method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antigen binding molecule complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution can be targeted or eliminated. Variants can be screened to determine whether they have the desired properties.

[0255] The amino acid sequence insertion includes amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, and intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include bispecific antigen binding molecules of the present invention with an N-terminal methionyl residue. Other insertion variants of the molecule include fusions to the N-terminal or C-terminal end of a polypeptide that increases the serum half-life of the bispecific antigen binding molecule.

[0256] In certain aspects, the bispecific antigen binding molecules provided herein are changed to increase or reduce the degree of antibody glycosylation. One or more glycosylation sites can be produced or removed by changing the amino acid sequence, so as to conveniently obtain the glycosylation variant of the molecule. When the antigen binding molecules containing the TNF ligand trimer include the Fc region, the carbohydrate attached thereto can be changed. The natural antibodies produced by mammalian cells generally include the biantennary oligosaccharides of the branches, which are generally connected to the Asn297 of the CH2 domain of the Fc region by N-bonding. See, for example, Wright et al. TIBTECH15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of the GlcNAc in the "trunk" of the biantennary oligosaccharide structure. In certain embodiments, the oligosaccharides in the antigen binding molecules containing the TNF family ligand trimer can be modified to produce variants with some improved properties. In one aspect, a variant of a bispecific antigen binding molecule or antibody of the present invention is provided, which has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. Such fucosylated variants may have improved ADCC function, see, for example, U.S. Patent Publication No. US2003 / 0157108 (Presta, L.) or US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). On the other hand, the bispecific antigen binding molecule or antibody of the present invention is provided with a variant of a bisected oligosaccharide, for example, wherein the biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, see, for example, WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 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 may have improved CDC function and are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0257] In some aspects, it is desirable to produce a cysteine ​​engineered variant of the bispecific antigen binding molecule of the present invention, such as a "thioMAb", in which one or more residues of the molecule are replaced by cysteine ​​residues. In particular aspects, the substituted residue is present in an accessible site of the molecule. By replacing those residues with cysteine, the reactive thiol group is thus located at an accessible site of the antibody, and can be used to conjugate the antibody with other moieties, such as a drug moiety or a linker-drug moiety, to produce an immunoconjugate. In some aspects, 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) in the heavy chain Fc region. Cysteine ​​engineered antigen binding molecules can be formed as described in, for example, U.S. Patent No. 7,521,541.

[0258] The term "nucleic acid" or "polynucleotide" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by base sequences, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is generally expressed as from 5' to 3'. In this article, the term nucleic acid molecule encompasses synthetic forms of deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes sense strands and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for direct expression in vitro and / or in vivo (e.g., in a host or patient) of the antibodies of the present invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA carrier and / or the expression of the encoding molecule, so that mRNA may be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online on June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1).

[0259] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0260] "Isolated nucleic acid encoding a bispecific antigen-binding molecule or antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of a bispecific antigen-binding molecule or antibody, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0261] With respect to a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include 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 a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with additional nucleotides, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, or may be interspersed individually among the residues of the reference sequence, or may be interspersed in one or more consecutive groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the invention can be routinely determined using known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2).

[0262] The term "expression cassette" refers to a polynucleotide generated by recombination or synthesis, which has a series of specific nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes, in addition to other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0263] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene into a target cell operably associated therewith and to direct the expression of the gene. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows transcription of a large amount of stable mRNA. Once the expression vector is inside the target cell, a ribonucleic acid molecule or protein encoded by the gene is produced by cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0264] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived from the primary transformed cells, regardless of the number of passages. Progeny may not be completely consistent with the nucleic acid content of the parent cell, but may contain mutations. This article includes mutant progeny with the same function or biological activity as screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to generate the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, for example, 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, as well as cells included in transgenic animals, transgenic plants or cultured plants or animal tissues.

[0265] An "effective amount" of an agent is that amount required to produce a physiological change in the cell or tissue to which it is administered.

[0266] 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 preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents an adverse effect of a disease.

[0267] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0268] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in effective form permitting the biological activity of the active ingredient contained therein, and that contains no additional components that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0269] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0270] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0271] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the molecules of the invention are used to delay the development of disease or to slow the progression of disease.

[0272] As used herein, the term "cancer" refers to a proliferative disease such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, bile duct cancer, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, neurilemmoma, 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.

[0273] As used herein, the term "chemotherapeutic agent" refers to a compound that can be used to treat 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 specific aspect, the antimetabolite is capecitabine or gemcitabine. On the other hand, 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. On the other hand, the chemotherapeutic agent is an alkylating agent such as cyclophosphamide. In one aspect, the chemotherapeutic agent is a cytotoxic antibiotic, such as a topoisomerase II inhibitor. In one aspect, the topoisomerase II inhibitor is doxorubicin.

[0274] Exemplary Agonist LTBR Antibodies

[0275] Provided herein are novel antibodies and antibody fragments that specifically bind to lymphotoxin beta receptor (LTBR). Provided are novel antibodies and antibody fragments that specifically bind to an antigen comprising the amino acid sequence of SEQ ID NO: 1. Thus, these antibodies specifically bind to human LTBR. They are agonistic human LTBR antibodies.

[0276] These antibodies can bind to human LTBR and to cynomolgus monkey LTBR with an affinity difference of less than 2-fold. Some of the novel agonistic human LTBR antibodies can even bind to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR.

[0277] Disclosed herein are agonistic LTBR antibodies that have an EC less than 4 nM as measured by ELISA. 50 (See Example 1.5) binds to the human LTBR extracellular domain (ECD of the amino acid sequence of SEQ ID NO: 359). In one aspect, the agonistic LTBR antibody has an EC of less than 1 nM as measured by ELISA. 50 In one specific aspect, the agonist LTBR antibody binds to the human LTBR ECD at an EC of less than 0.15 nM as measured by ELISA. 50 Binds to human LTBR ECD.

[0278] Agonistic LTBR antibodies as disclosed herein have an EC of less than 5 nM as measured by ELISA. 50(See Example 1.5) binds to the extracellular domain of cynomolgus monkey LTBR (ECD of the amino acid sequence of SEQ ID NO: 361). In one aspect, the agonist LTBR antibody has an EC of less than 0.5 nM as measured by ELISA. 50 In one specific aspect, the agonist LTBR antibody binds to the cynomolgus monkey LTBR ECD at an EC of less than 0.1 nM as measured by ELISA. 50 Binds to the cynomolgus monkey LTBR ECD.

[0279] Agonistic LTBR antibodies as described herein require cross-linking for their agonistic activity to activate human LTBR, ​​which means that they can only stimulate LTBR through a cross-linking-dependent mechanism. A "cross-linking-dependent mechanism" can be, for example, an Fc cross-linking-dependent mechanism, in which the antibody must bind to both LTBR and Fc receptors in order to stimulate LTBR. Thus, the antibody must be able to bind to both LTBR and Fc receptors. If the antibody is not cross-link-dependent, it can stimulate LTBR in the absence of binding to Fc receptors. This may lead to widespread LTBR activation in humans and the serious safety issues associated with it.

[0280] Agonistic LTBR antibodies as described herein also need to be cross-linked for agonistic activity to induce ICAM upregulation in human umbilical vein endothelial cells or cancer-associated fibroblasts, as shown in Example 5.2 herein.

[0281] Agonistic LTBR antibodies as described herein are also able to inhibit the interaction between human LTBR and its human ligands lymphotoxin α1β2 and LIGHT. This has been shown in ligand competition experiments by ELISA as described in Example 1.6.

[0282] It has been shown that agonistic LTBR antibodies as described herein compete with human LIGHT, the natural ligand of LTBR comprising the amino acid sequence of SEQ ID NO: 358, for binding to human LTBR.

[0283] In one aspect, provided herein is an agonistic LTBR antibody (or an antigen binding domain that specifically binds to LTBR), wherein the antibody or antigen binding domain comprises

[0284] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V LLTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0285] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40; or

[0286] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0287] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56; or

[0288] Heavy chain variable region (V HLTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64; or

[0289] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 72; or

[0290] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0291] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V LLTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88.

[0292] In one aspect, an agonistic LTBR antibody (or an antigen binding domain that specifically binds to LTBR) is provided, wherein the antibody comprises

[0293] Heavy chain variable region (V H LTBR), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO: 34; or

[0294] Heavy chain variable region (V H LTBR), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO:42; or

[0295] Heavy chain variable region (V H LTBR), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO: 50; or

[0296] Heavy chain variable region (V H LTBR), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO:58; or

[0297] Heavy chain variable region (V H LTBR), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V LLTBR), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO: 66;

[0298] Heavy chain variable region (V H LTBR), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO:74;

[0299] Heavy chain variable region (V H LTBR), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO: 82; or

[0300] Heavy chain variable region (V H LTBR), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO:90; or

[0301] Heavy chain variable region (V H LTBR), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO:98.

[0302] In one aspect, an agonistic LTBR antibody (or an antigen binding domain that specifically binds to LTBR) is provided, wherein the antibody or antigen binding domain comprises

[0303] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0304] Heavy chain variable region (V HLTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:42; or

[0305] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0306] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:58; or

[0307] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 66;

[0308] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:74;

[0309] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0310] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0311] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98.

[0312] In a specific aspect, an agonistic LTBR antibody as described herein is an agonistic LTBR antibody that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and murine LTBR. Disclosed herein are agonistic LTBR antibodies that bind to human LTBR with an EC less than 1 nM as measured by ELISA. 50 (See Example 1.5) binds to the murine LTBR extracellular domain (ECD of the amino acid sequence of SEQ ID NO: 360). In one aspect, the agonist LTBR antibody has an EC of less than 0.15 nM as measured by ELISA. 50 Binds to the ECD of murine LTBR.

[0313] In one aspect, an agonist LTBR antibody (or antigen binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and mouse LTBR is provided, the agonist LTBR antibody comprising

[0314] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0315] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0316] Heavy chain variable region (V HLTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0317] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88.

[0318] In one aspect, an agonist LTBR antibody (or antigen binding domain) that specifically binds to human LTBR, ​​cynomolgus monkey LTBR, ​​and murine LTBR comprises

[0319] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0320] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0321] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0322] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0323] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98.

[0324] In a specific aspect, an agonist LTBR antibody as described herein binds to an epitope region of SEQ ID NO: 351 on human LTBR. In one aspect, an agonist LTBR antibody as described herein binds to an epitope that overlaps with an epitope on human LTBR bound by human lymphotoxin α1β2. In one aspect, an agonist LTBR antibody as described herein binds to a different epitope region on human LTBR than reference antibodies BHA10 and CBE11. This has been demonstrated by hydrogen / deuterium exchange (HDX) mass spectrometry, as described in Example 6.4.

[0325] In one aspect, such agonist LTBR antibodies comprise: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32. In one aspect, it comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:34 (antibody P1AE9459).

[0326] In one aspect, an agonistic LTBR antibody (or an 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) comprises

[0327] (i) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0328] (ii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40; or

[0329] (iii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0330] (iv) Heavy chain variable region (V HLTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56; or

[0331] (v) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64; or

[0332] (vi) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:72.

[0333] In one aspect, the agonistic 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 aspect, the antibody or antigen binding domain comprises: a heavy chain variable region (V HLTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or (ii) a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 48. In one aspect, the antibody or antigen binding domain comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:50.

[0334] In another aspect, a human LTBR antibody or antigen binding domain derived from rat immunization 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 (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V LLTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80. In one aspect, the antibody or antigen binding domain comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:82.

[0335] In another aspect, a human LTBR antibody or antigen binding domain generated from a phage display library 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 (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 88. In one aspect, the antibody or antigen binding domain comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 91; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 93; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 94; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 95; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 96. In one aspect, the antibody or antigen binding domain comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V LLTBR), which comprises the amino acid sequence of SEQ ID NO: 90. In another aspect, the antibody or antigen binding domain comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98.

[0336] In one aspect, the antibody that specifically binds to LTBR is a full-length antibody, in particular of the human IgG1 subclass. In a particular aspect, it comprises the amino acid substitutions L234A, L235A and P329G (numbered according to the Kabat EU index). In a particular aspect, 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

[0337] (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

[0338] (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

[0339] (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

[0340] (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

[0341] (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

[0342] (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

[0343] (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

[0344] (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.

[0345] Bispecific agonistic LTBR antibodies

[0346] Also provided herein are novel bispecific antigen binding molecules capable of specifically binding to lymphotoxin beta receptor (LTBR) and tumor-associated antigens such as fibroblast activation protein (FAP), the bispecific antigen binding molecules combining an antigen binding domain that specifically binds to FAP with at least one antigen binding domain that can agonistically bind to LTBR, ​​wherein activation by LTBR is provided by cross-linking binding to FAP expressed on tumor stromal cells. The bispecific agonistic LTBR antibodies disclosed herein have particularly advantageous properties such as manufacturability, stability, binding affinity, biological activity, targeting efficiency, reduced internalization, acceptable pharmacokinetic (PK) properties, reduced toxicity, an extended dosage range that can be given to patients, and thereby potentially enhanced efficacy.

[0347] Exemplary Bispecific Agonist LTBR Antibodies

[0348] In one aspect, an agonistic LTBR antibody is provided, which is a multispecific antibody comprising an agonistic LTBR antibody as previously described herein. In one aspect, an agonistic LTBR antibody as a bispecific antibody is provided. The bispecific agonistic LTBR antibody preferably comprises an Fc domain of human origin, particularly human IgG subclass, more particularly human IgG1 subclass. In one aspect, the bispecific agonistic LTBR antibody comprises an Fc domain of human IgG1 subclass, the Fc domain comprising one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to the Fc receptor and / or the effector function. In one aspect, the bispecific agonistic LTBR antibody comprises an Fc domain of human IgG1 subclass, the Fc domain having amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

[0349] In one aspect, an agonistic LTBR antibody is a bispecific antibody that specifically binds to LTBR and a tumor-associated antigen (TAA). In particular, a bispecific agonistic LTBR antibody is a LTBR agonist targeted to FAP. In one aspect, a bispecific agonistic LTBR antibody comprises an Fc region composed of a first subunit and a second subunit, the Fc region comprising a mutation that reduces effector function. The use of an Fc region comprising a mutation that reduces or eliminates effector function will prevent nonspecific agonism by cross-linking via Fc receptors and prevent ADCC of cells expressing LTBR. The bispecific agonistic LTBR antibodies described herein have advantages over conventional antibodies because they selectively induce an immune response at target cells, which are typically in the tumor stroma (i.e., adjacent to the tumor).

[0350] Thus, the bispecific agonist LTBR antibodies are characterized by agonistic binding to the targeting FAP of LTBR. In the presence of cells expressing FAP, the bispecific antigen binding molecules are capable of activating cancer associated fibroblasts (CAFs) (Example 5.2.1), activating endothelial cells (Example 5.2.2) and modulating human endothelial cells to upregulate adhesion molecules and chemoattractants (which are essential for the immune infiltration cascade). The bispecific antigen binding molecules described herein are capable of inducing T cell adhesion (Example 5.2.3).

[0351] In one aspect, a bispecific agonist LTBR antibody is provided, comprising

[0352] (a) a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP),

[0353] (b) a second antigen binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), and

[0354] (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 of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0355] The bispecific agonist LTBR antibody has an Fc domain composed of a first subunit and a second subunit, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to the Fc receptor and / or the effector function, thereby eliminating Fc receptor-mediated cross-linking, and achieving tumor-specific activation through cross-linking with the antigen binding domain, which specifically binds to FAP by binding to its tumor-associated target.

[0356] In one aspect, a bispecific agonist LTBR antibody is provided, comprising

[0357] (a) a first Fab fragment that specifically binds to fibroblast activation protein (FAP),

[0358] (b) a second Fab fragment that specifically binds to lymphotoxin beta receptor (LTBR), and (c) an Fc domain composed of the first subunit and the second subunit and comprising one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to the Fc receptor and / or the effector function.

[0359] In one aspect, a bispecific agonist LTBR antibody as disclosed herein comprises a first antigen binding domain that specifically binds to fibroblast activation protein (FAP), the first antigen binding domain comprising (i) a heavy chain variable region (V H FAP), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 3; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 4; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 5; and a light chain variable region (V L FAP), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 6; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 7; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 8; or

[0360] (ii) Heavy chain variable region (V H FAP), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 19; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 20; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 21; and a light chain variable region (V L FAP), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 22; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 23; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 24; or

[0361] (iii) Heavy chain variable region (V HFAP), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 11; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 12; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 13; and a light chain variable region (V L FAP), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 14; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 15; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 16.

[0362] In one aspect, the first antigen binding domain that specifically binds to FAP comprises (i) a heavy chain variable region (V H FAP), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 3; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 4; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 5; and a light chain variable region (V L FAP), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 6; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 7; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 8; or

[0363] (ii) Heavy chain variable region (V H FAP), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 19; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 20; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 21; and a light chain variable region (V L FAP), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:22; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:23; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:24.

[0364] In a specific aspect, the first antigen binding domain that specifically binds to FAP comprises (i) a heavy chain variable region (V HFAP), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 3; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 4; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 5; and a light chain variable region (V L FAP), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 6; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 7; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 8. In one aspect, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 19; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 20; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 21; and a light chain variable region (V L FAP), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 22; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 23; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 24. On the other hand, the first antigen-binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 11; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 12; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 13; and a light chain variable region (V L FAP), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 14; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 15; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 16.

[0365] In one aspect, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises CDR-L1, CDR-L2 and CDR-L3, having the amino acid sequence of SEQ ID NO: 10. On the other hand, it comprises: a heavy chain variable region (VH FAP), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V L FAP), which comprises CDR-L1, CDR-L2 and CDR-L3, having the amino acid sequence of SEQ ID NO: 26. In another aspect, it comprises: a heavy chain variable region (V H FAP), which comprises CDR-H1, CDR-H2 and CDR-H3, having the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises CDR-L1, CDR-L2 and CDR-L3, and has the amino acid sequence of SEQ ID NO: 18.

[0366] In one aspect, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 10; or the first antigen binding domain comprises: a heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 26; or the first antigen binding domain comprises: a heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:18.

[0367] In one aspect, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10; or the first antigen binding domain comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V LFAP), which comprises the amino acid sequence of SEQ ID NO: 26; or the first antigen binding domain comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 18. In a specific aspect, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10. In one aspect, it comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 26. On the other hand, it comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 18. In particular, the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10. In particular, the first antigen-binding domain that specifically binds to FAP is a Fab fragment.

[0368] In one aspect, a bispecific agonist LTBR antibody as disclosed herein comprises a second antigen binding domain that specifically binds to LTBR, ​​the second antigen binding domain comprising

[0369] (i) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V LLTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0370] (ii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0371] (iii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0372] (iv) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 88; or

[0373] (v) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40; or

[0374] (vi) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56; or

[0375] (vii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64; or

[0376] (viii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V LLTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:72.

[0377] In one specific aspect, the second antigen binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32. On the other hand, the second antigen binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40. On the other hand, the second antigen-binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V LLTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 48. On the other hand, the second antigen-binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56. On the other hand, the second antigen-binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64. On the other hand, the second antigen-binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 72. In another aspect, the second antigen binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V HLTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80. On the other hand, the second antigen-binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88.

[0378] In another aspect, the second antigen binding domain that specifically binds to LTBR may comprise: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 101; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 102; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 103; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 104; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 105; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 106. In a specific aspect, the second antigen binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 99; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 100 (CBE11).

[0379] In another aspect, the second antigen binding domain that specifically binds to LTBR may comprise: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 343; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 344; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 345; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 346; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 347; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 348. In a specific aspect, the second antigen binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 349; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 350 (BHA10).

[0380] In one aspect, a bispecific agonist LTBR antibody is provided, the bispecific agonist LTBR antibody comprising a second antigen binding domain that specifically binds to LTBR, ​​the second antigen binding domain comprising (i) a heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:34; or

[0381] (ii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:50; or

[0382] (iii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:82; or

[0383] (iv) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:90; or

[0384] (v) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:98;

[0385] (vi) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:42;

[0386] (vii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:58; or

[0387] (viii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:66; or

[0388] (ix) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:74.

[0389] In one aspect, a bispecific agonist LTBR antibody is provided, the bispecific agonist LTBR antibody comprising a second antigen binding domain that specifically binds to LTBR, ​​the second antigen binding domain comprising (i) a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0390] (ii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0391] (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0392] (iv) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0393] (v) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98; or

[0394] (vi) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:42; or

[0395] (vii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:58; or

[0396] (viii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 66;

[0397] (ix) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:74.

[0398] In one aspect, the second antigen binding domain that specifically binds to LTBR comprises: (i) a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or (ii) a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 42; or (iii) a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50. In a specific aspect, the second antigen binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34. In another specific aspect, the second antigen binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 42. In another specific aspect, the second antigen binding domain that specifically binds to LTBR comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:50.

[0399] In another aspect, the second antigen binding domain that specifically binds to LTBR is also capable of specifically binding to murine LTBR and comprises

[0400] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0401] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0402] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0403] Heavy chain variable region (V HLTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88.

[0404] In one aspect, the second antigen binding domain that specifically binds to LTBR is also capable of specifically binding to murine LTBR and comprises

[0405] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0406] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0407] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0408] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0409] Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98.

[0410] In one aspect, the second antigen binding domain that specifically binds to LTBR is also capable of specifically binding to murine LTBR and comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:34.

[0411] In one aspect, a bispecific agonist LTBR antibody is provided, wherein the bispecific antibody comprises a third antigen binding domain that specifically binds to LTBR, ​​meaning that the bispecific antigen binding molecule comprises

[0412] (a) a first antigen-binding domain that specifically binds to fibroblast activation protein (FAP),

[0413] (b) a second antigen-binding domain and a third antigen-binding domain that specifically bind to lymphotoxin beta receptor (LTBR), and

[0414] (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 of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0415] In a specific aspect, the third antigen binding domain that specifically binds to LTBR is the same as the second antigen binding domain that specifically binds to LTBR, ​​which means that the second antigen binding domain and the third antigen binding domain that specifically bind to lymphotoxin beta receptor (LTBR) are the same. In one aspect, the second antigen binding domain and the third antigen binding domain that specifically bind to LTBR are Fab fragments that specifically bind to LTBR. In one aspect, the Fab fragment that specifically binds to LTBR is a crossfab fragment. In another aspect, the first antigen binding domain that specifically binds to FAP is a Fab fragment.

[0416] In one aspect, the bispecific agonist LTBR antibodies disclosed herein comprise a second antigen binding domain that specifically binds to LTBR and a third antigen binding domain, both of which comprise

[0417] (i) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V LLTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0418] (ii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0419] (iii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0420] (iv) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 88; or

[0421] (v) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40; or

[0422] (vi) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56; or

[0423] (vii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64; or

[0424] (viii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V LLTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:72.

[0425] In a specific aspect, the second antigen binding domain and the third antigen binding domain are identical.

[0426] In one 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 H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32. On the other hand, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each comprise: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40. On the other hand, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each comprise: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V LLTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 48. On the other hand, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each comprise: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56. On the other hand, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each comprise: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64. On the other hand, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each comprise: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V LLTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 72. In another 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 H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80. On the other hand, the second antigen-binding domain and the third antigen-binding domain that specifically bind to LTBR each comprise: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88.

[0427] In one aspect, a bispecific agonist LTBR antibody is provided, comprising a second antigen binding domain that specifically binds to LTBR and a third antigen binding domain, wherein the second antigen binding domain and the third antigen binding domain comprise (i) a heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:34; or

[0428] (ii) Heavy chain variable region (V HLTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:50; or

[0429] (iii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:82; or

[0430] (iv) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:90; or

[0431] (v) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:98;

[0432] (vi) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:42;

[0433] (vii) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:58; or

[0434] (viii) Heavy chain variable region (V HLTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:66; or

[0435] (ix) Heavy chain variable region (V H LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:74.

[0436] In one 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 comprise

[0437] (i) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0438] (ii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0439] (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0440] (iv) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0441] (v) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (VL LTBR), which comprises the amino acid sequence of SEQ ID NO:98; or

[0442] (vi) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:42; or

[0443] (vii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:58; or

[0444] (viii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 66;

[0445] (ix) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:74.

[0446] 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 H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or (ii) a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 42; or (iii) a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50. In a specific aspect, the second antigen binding domain and the third antigen binding domain that specifically bind to LTBR each comprise: a heavy chain variable region (VH LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34. In another specific 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 H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 42. In yet another specific 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 H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:50.

[0447] In one 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 variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 101; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 102; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 103; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 104; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 105; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 106. In a specific 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 H LTBR), which comprises the amino acid sequence of SEQ ID NO: 99; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 100 (CBE11).

[0448] In one 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 variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 343; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 344; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 345; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 346; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 347; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 348. In a specific 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 H LTBR), which comprises the amino acid sequence of SEQ ID NO: 349; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 350 (BHA10).

[0449] In another aspect, a bispecific agonist LTBR antibody is provided comprising a second antigen binding domain that specifically binds to human LTBR and is also capable of specifically binding to murine LTBR and a third antigen binding domain, the second antigen binding domain and the third antigen binding domain each comprising

[0450] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or

[0451] Heavy chain variable region (V HLTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or

[0452] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or

[0453] Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:87; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:88.

[0454] In one aspect, the bispecific agonist LTBR antibody comprises a second antigen binding domain that specifically binds to human LTBR and also specifically binds to murine LTBR and a third antigen binding domain, the second antigen binding domain and the third antigen binding domain each comprising

[0455] (i) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (VL LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0456] (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0457] (vii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0458] (viii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0459] (ix) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98.

[0460] In a specific aspect, the bispecific agonist LTBR antibody comprises a second antigen binding domain that specifically binds to human LTBR and also specifically binds to murine LTBR and a third antigen binding domain, each comprising: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:34.

[0461] Bispecific agonistic LTBR antibodies that bind to FAP and LTBR

[0462] In one aspect, a bispecific agonist LTBR antibody is provided, comprising

[0463] (a) a first antigen-binding domain that specifically binds to FAP, the first antigen-binding domain comprising

[0464] Heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10; or

[0465] Heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 26; or

[0466] Heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 18;

[0467] (b) a second antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), the second antigen-binding domain comprising (i) a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0468] (ii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0469] (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0470] (iv) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0471] (v) Heavy chain variable region (V HLTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98;

[0472] (vi) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:42;

[0473] (vii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:58; or

[0474] (viii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 66; or

[0475] (ix) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:74; and

[0476] (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 of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0477] In another aspect, a bispecific agonist LTBR antibody is provided, comprising

[0478] (a) a first antigen-binding domain that specifically binds to FAP, the first antigen-binding domain comprising

[0479] Heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10; or

[0480] Heavy chain variable region (V HFAP), which comprises the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 26; or

[0481] Heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 18;

[0482] (b) a second antigen-binding domain and a third antigen-binding domain that specifically bind to lymphotoxin beta receptor (LTBR), wherein the second antigen-binding domain and the third antigen-binding domain each comprise (i) a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or

[0483] (ii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or

[0484] (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or

[0485] (iv) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or

[0486] (v) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 97; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98;

[0487] (vi) Heavy chain variable region (V HLTBR), which comprises the amino acid sequence of SEQ ID NO:41; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:42;

[0488] (vii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 57; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:58; or

[0489] (viii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 66; or

[0490] (ix) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 73; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:74; and

[0491] (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 of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0492] In one specific aspect, a bispecific agonist LTBR antibody is provided, the bispecific agonist LTBR antibody comprising: (i) a first antigen binding domain capable of specifically binding to FAP, the first antigen binding domain comprising: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9, and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10, and (ii) a second antigen-binding domain and a third antigen-binding domain that can specifically bind to LTBR, ​​wherein the second antigen-binding domain and the third antigen-binding domain comprise: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33, and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:34.

[0493] In another specific aspect, a bispecific agonist LTBR antibody is provided, the bispecific agonist LTBR antibody comprising: (i) a first antigen binding domain capable of specifically binding to FAP, the first antigen binding domain comprising: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9, and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10, and (ii) a second antigen-binding domain and a third antigen-binding domain that can specifically bind to LTBR, ​​wherein the second antigen-binding domain and the third antigen-binding domain comprise: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 99, and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:100.

[0494] Bispecific monovalent agonistic LTBR antibody (1+1 format)

[0495] 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 of the antigen binding molecule to an Fc receptor and / or effector function, wherein the bispecific antigen binding molecule comprises

[0496] (i) a first light chain and a first heavy chain of a full-length antibody that specifically binds to FAP, and

[0497] (ii) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to LTBR, ​​wherein the variable domains VL and VH are replaced with each other, and / or wherein the constant domains CL and CH1 are replaced with each other.

[0498] 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.

[0499] In another 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 of the antigen binding molecule to the Fc receptor and / or the effector function, 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 FAP, wherein the variable domains VL and VH are replaced with each other, and / or wherein the constant domains CL and CH1 are replaced with each other; and

[0500] (ii) a second light chain and a second heavy chain of a full-length antibody that specifically binds to LTBR.

[0501] 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.

[0502] In one specific aspect, there is provided

[0503] (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

[0504] (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

[0505] (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

[0506] (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

[0507] (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

[0508] (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

[0509] (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

[0510] (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

[0511] (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

[0512] (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

[0513] (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

[0514] (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

[0515] (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

[0516] (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

[0517] (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

[0518] (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.

[0519] In one aspect, a bispecific agonist LTBR antibody is provided, which comprises 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.

[0520] Bispecific agonistic LTBR antibodies that are bivalent for binding to LTBR and monovalent for binding to FAP (2+1 format)

[0521] 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 and comprising 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, wherein the bispecific antigen binding molecule comprises

[0522] (i) two light chains and two heavy chains of a full-length antibody that specifically binds to LTBR, ​​and

[0523] (ii) A Fab fragment or a crossFab fragment that specifically binds to FAP is linked to one of the C-termini of the two heavy chains via a peptide linker.

[0524] 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 and comprising 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, wherein the bispecific antigen-binding molecule comprises

[0525] (i) a first light chain and a first heavy chain of a full-length antibody that specifically binds to FAP, and

[0526] (ii) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to LTBR, ​​wherein the Fab fragment or CrossFab fragment that specifically binds to LTBR is linked to the C-terminus of the second heavy chain via a peptide linker.

[0527] Thus, a bispecific antigen binding molecule is provided, wherein the bispecific antigen binding molecule is bivalent for binding to LTBR and monovalent for binding to FAP.

[0528] In one specific aspect, there is provided:

[0529] (a) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 167 and 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

[0530] (b) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 171 and 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

[0531] (c) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 175 and 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

[0532] (d) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 179 and 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

[0533] (e) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 183 and 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

[0534] (f) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 187 and 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

[0535] (g) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 191 and 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

[0536] (h) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 195 and 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

[0537] (i) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 199 and 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

[0538] (j) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 203 and 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

[0539] (k) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 207 and 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

[0540] (l) A bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO:211 and 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.

[0541] In addition, it provides

[0542] (m) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 326 and 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

[0543] (n) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 333 and 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

[0544] (o) a bispecific agonist LTBR antibody comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 337 and 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.

[0545] In a specific aspect, a bispecific agonist LTBR antibody comprising a bispecific antigen binding molecule is provided, wherein the bispecific antigen binding molecule comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 195 and 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 a light chain comprising the amino acid sequence of SEQ ID NO: 198.

[0546] Mouse surrogate agonistic LTBR antibody

[0547] Murine surrogate molecules are also provided herein. In one aspect, a bispecific agonist LTBR antibody is provided, the bispecific agonist LTBR antibody comprising

[0548] (a) a first antigen-binding domain that specifically binds to FAP, the first antigen-binding domain comprising

[0549] Heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 18;

[0550] (b) a first antigen-binding domain that specifically binds to lymphotoxin beta receptor (LTBR), the first antigen-binding domain comprising: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; and

[0551] (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 of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0552] In a specific aspect, a bispecific agonist LTBR antibody is provided, which comprises 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 (1+1 format).

[0553] In one aspect, a bispecific agonist LTBR antibody is provided, comprising

[0554] (a) a first antigen-binding domain that specifically binds to FAP, the first antigen-binding domain comprising

[0555] Heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 18;

[0556] (b) a second antigen-binding domain and a third antigen-binding domain that specifically bind to lymphotoxin beta receptor (LTBR), each of the second antigen-binding domain and the third antigen-binding domain comprising: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 49; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; and

[0557] (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 of the antigen-binding molecule to an Fc receptor and / or the effector function.

[0558] In one aspect, a bispecific agonist LTBR antibody is provided, which comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:223 and a second heavy chain comprising the amino acid sequence of SEQ ID NO:225, two light chains each comprising the amino acid sequence of SEQ ID NO:224, and a light chain comprising the amino acid sequence of SEQ ID NO:226.

[0559] In a specific aspect, a bispecific agonist LTBR antibody is provided, which comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:318 and 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.

[0560] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0561] The bispecific agonist LTBR antibodies described herein further comprise an Fc domain, which is composed of a first subunit and a second subunit and comprises one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to the Fc receptor and / or the effector function. Thus, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0562] The Fc domain confers favorable pharmacokinetic properties to the bispecific antibodies of the invention, including a long serum half-life and a favorable tissue-blood distribution ratio that contribute to good accumulation in target tissues. However, at the same time it may result in the undesired targeting of the bispecific agonist LTBR antibody to cells expressing Fc receptors, rather than to the preferred antigen-bearing cells. Thus, in a particular aspect, the Fc domain of the bispecific agonist LTBR antibody exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain. More specifically, the Fc domain is an IgG1 Fc domain.

[0563] In one such aspect, the Fc domain (or the bispecific antigen binding molecule of the invention comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity for an Fc receptor compared to a native IgG1 Fc domain (or a bispecific antigen binding molecule of the invention comprising the Fc domain); and / or the Fc domain (or the bispecific antigen binding molecule of the invention comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or a bispecific antigen binding molecule of the invention comprising the Fc domain). In one aspect, the Fc domain (or the bispecific antigen binding molecule of the invention comprising the Fc domain) does not substantially bind to an Fc receptor and / or induce effector function. In a specific aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a specific aspect, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In one aspect, the effector function is one or more of CDC, ADCC, ADCP and cytokine secretion. In a specific aspect, the effector function is ADCC. In one aspect, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the 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 the Fc domain) exhibits greater than about 70%, specifically greater than about 80%, more specifically greater than about 90% of the binding affinity of a native IgG1 Fc domain (or a bispecific agonist LTBR antibody comprising a native IgG1 Fc domain) for FcRn.

[0564] In a specific aspect, 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 a specific aspect, the Fc domain of the bispecific agonist LTBR antibody comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and / or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In another aspect, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In one aspect, the bispecific agonist LTBR antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% binding affinity to an Fc receptor compared to a bispecific antibody of the invention comprising a non-engineered Fc domain. In a specific aspect, the Fc receptor is an Fcγ receptor. In other aspects, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a specific aspect, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In some aspects, the Fc receptor is an activated Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some aspects, the binding affinity to complementary components, especially the specific binding affinity to C1q, is also reduced. In one aspect, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., the binding affinity of the Fc domain to the receptor is maintained, is achieved when the Fc domain (or a bispecific agonist LTBR antibody comprising the Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or a bispecific agonist LTBR antibody comprising the non-engineered form of the Fc domain) to FcRn. The Fc domain or a bispecific agonist LTBR antibody comprising the Fc domain may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain aspects, 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 may include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent 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-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0565] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581). Certain antibody variants with improved or reduced binding to FcR are described. (e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312, and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0566] 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 amino acid substitutions at position P329. In a more specific aspect, the amino acid substitutions are P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises amino acid substitutions at position P329 and comprises additional amino acid substitutions selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a more specific aspect, the Fc domain comprises amino acid mutations L234A, L235A and P329G ("P329G LALA"). The "P329G LALA" combination of amino acid substitutions almost completely eliminates Fcγ receptor binding of human IgG1 Fc domains, as described in PCT patent application number WO 2012 / 130831 A1. The document also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function). Such antibodies are IgG1 with mutations L234A and L235A or with mutations L234A, L235A and P329G (according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, EU index numbering of 1991).

[0567] 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), specifically an IgG4 Fc domain of amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising amino acid substitutions L235E and S228P and P329G. This amino acid substitution reduces the in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).

[0568] Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn), responsible for 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 US 2005 / 0014934. Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those with 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, such as substitutions at Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0569] Binding to Fc receptors can be easily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare), and Fc receptors such as those obtained by recombinant expression. Suitable such binding assays are described herein. Alternatively, cell lines known to express specific Fc receptors (such as human NK cells expressing FcγIIIa receptors) can be used to evaluate the binding affinity of Fc domains or cell-activated bispecific antigen binding molecules comprising Fc domains to Fc receptors. The effector function of bispecific agonist LTBR antibodies comprising Fc domains or Fc domains 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 molecules of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TMNonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0570] The following sections describe preferred aspects of the bispecific agonist LTBR antibodies described herein that comprise Fc domain modifications that reduce Fc receptor binding and / or effector function. In one 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 consisting of a first subunit and a second subunit, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to an Fc receptor, particularly to 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 consisting of a first subunit and a second subunit that are capable of stably associating, wherein the Fc domain com...

Claims

1. An antibody that binds to human agonist lymphotoxin beta receptor (LTBR) and to cynomolgus monkey LTBR, ​​wherein the antibody comprises (i) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or (ii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or (iii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or (iv) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 87; and CDR-L3 comprising the amino acid sequence of SEQ ID NO:88; or (v) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 35; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 36; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 38; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 39; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 40; or (vi) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 51; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 52; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 54; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 55; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 56; or (vii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 59; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 60; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 61; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 64; or (viii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 67; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 68; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 69; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:70; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:71; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

72.

2. The agonistic LTBR antibody of claim 1, wherein the agonistic antibody has at least one of the following properties: (a) binds to human LTBR and to cynomolgus monkey LTBR with an affinity that differs by less than 2-fold; or (b) With an EC of less than 4 nM as measured by ELISA 50 Binds to the human LTBR extracellular domain with an EC of less than 5 nM as measured by ELISA 50 Binds to the extracellular domain of cynomolgus monkey LTBR; or (c) requires cross-linking for its agonistic activity to activate human LTBR; or (d) requires cross-linking for its agonistic activity to induce ICAM upregulation in human umbilical vein endothelial cells or cancer-associated fibroblasts; or (e) Inhibits the interaction between human LTBR and its human ligands lymphotoxin α1β2 and LIGHT.

3. The agonistic LTBR antibody of claim 1 or 2, wherein the agonistic antibody comprises (i) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or (ii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:49; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or (iv) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or (v) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:97; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:98; or (vi) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:41; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:42; or (vii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:57; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:58; or (viii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 65; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 66; (ix) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:73; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:

74.

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

5. The agonistic LTBR antibody of any one of claims 1 to 4, wherein the agonistic antibody has an EC of less than 1 nM as measured by ELISA. 50 Binds to the extracellular domain of murine LTBR.

6. The agonistic LTBR antibody of any one of claims 1 to 5, wherein the agonistic antibody comprises (i) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 32; or (ii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 43; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 44; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 45; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO:46; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:47; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:48; or (iii) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 75; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 76; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 77; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 78; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 79; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 80; or (iv) Heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 83; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 84 or SEQ ID NO: 92; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 85; and a light chain variable region (V L LTBR), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 86; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 87; and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

88.

7. The agonistic LTBR antibody of any one of claims 1 to 6, wherein the agonistic antibody comprises (i) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 34; or (ii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:49; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 50; or (iii) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 81; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO: 82; or (iv) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 89; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:90; or (v) Heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO:97; and the light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:

98.

8. The agonistic 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 agonistic LTBR antibody of any one of claims 1 to 8, wherein the antibody comprises: a heavy chain variable region (V H LTBR), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 27; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 28; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 29; and a light chain variable region (V L LTBR), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO:30; CDR-L2, which comprises the amino acid sequence of SEQ ID NO:31; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

32.

10. The agonistic LTBR antibody of any one of claims 1 to 9, wherein the antibody comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:

34.

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

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

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

16. The agonistic LTBR antibody of any one of claims 1 to 15, wherein the agonistic antibody is a bispecific 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 LTBR, ​​and (c) an Fc domain of human IgG1 subclass, which comprises 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.

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

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

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

20. The agonistic LTBR antibody of any one of claims 16 to 19, wherein the first antigen binding domain that specifically binds to FAP comprises (i) Heavy chain variable region (V H FAP), the heavy chain variable region comprises: a heavy chain complementarity determining region (CDR-H1), which comprises the amino acid sequence of SEQ ID NO: 3; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 4; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 5; and a light chain variable region (V L FAP), the light chain variable region comprises: a light chain complementary determining region CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 6; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 7; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 8; or (ii) Heavy chain variable region (V H FAP), the heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 19; CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 20; and CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 21; and a light chain variable region (V L FAP), the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 22; CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 23; and CDR-L3, which comprises the amino acid sequence of SEQ ID NO:

24.

21. The agonistic LTBR antibody of any one of claims 16 to 20, wherein the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 10; or the first antigen binding domain comprises: a heavy chain variable region (V H FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V L FAP), which comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:

26.

22. The agonist LTBR antibody of any one of claims 16 to 21, wherein the first antigen binding domain that specifically binds to FAP comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10; or the first antigen binding domain comprises: a heavy chain variable region (V H FAP), which comprises the amino acid sequence of SEQ ID NO: 25; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO:

26.

23. The agonistic LTBR antibody of any one of claims 1 to 22, wherein the agonistic LTBR antibody is a bispecific 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 H FAP), which comprises the amino acid sequence of SEQ ID NO: 9; and a light chain variable region (V L FAP), which comprises the amino acid sequence of SEQ ID NO: 10; and (b) a second antigen-binding domain that specifically binds to LTBR, ​​wherein the second antigen-binding domain comprises: a heavy chain variable region (V H LTBR), which comprises the amino acid sequence of SEQ ID NO: 33; and a light chain variable region (V L LTBR), which comprises the amino acid sequence of SEQ ID NO:

34.

24. The agonistic LTBR antibody of any one of claims 1 to 23, wherein the agonistic LTBR antibody is a bispecific antibody comprising (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 human IgG1 subclass, which comprises 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.

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

26. The agonistic LTBR antibody of any one of claims 1 to 23, wherein the agonistic LTBR antibody is a bispecific antibody comprising (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 human IgG1 subclass, which comprises 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, wherein the 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 the second Fab fragment and the third Fab fragment that specifically bind to LTBR are each fused at their C-termini to the N-terminus of one of the Fc domain subunits.

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

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

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

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

31. A pharmaceutical composition comprising the agonistic LTBR antibody of any one of claims 1 to 26 and a pharmaceutically 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 upregulation of ICAM on endothelial cells or cancer-associated fibroblasts, or (b) enhancing T cell adhesion.

34. The agonistic LTBR antibody of any one of claims 1 to 26 for use in treating cancer.

35. The agonist LTBR antibody of any one of claims 1 to 26 for use in treating cancer, wherein the agonist LTBR antibody or pharmaceutical composition is for administration in combination with a chemotherapeutic agent, 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 treating cancer, wherein the agonistic LTBR antibody is for administration in combination with an agent that blocks the PD-L1 / PD-1 interaction.

37. Use of an agonistic 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 treating cancer.

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

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