Multispecific binding molecules containing LTBR and EDB binding domains, and their use

Abstract

Provided herein are anti-LTBR multispecific binding molecules, nucleic acids encoding the anti-LTBR multispecific binding molecules, vectors containing the nucleic acids, host cells containing the vectors, and pharmaceutical compositions containing the anti-LTBR multispecific binding molecules. Also provided are methods for treating cancer in a subject in need thereof, comprising administering the pharmaceutical compositions disclosed herein.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the benefits of U.S. Provisional Application No. 62 / 946,452, filed on 11 December 2019. The entire contents of the aforementioned application are incorporated herein by reference.

[0002] (Field of Invention) This invention relates to an anti-LTBR multispecific binding molecule, a nucleic acid encoding the binding molecule and an expression vector, recombinant cells containing the vector, and a composition containing the binding molecule. Furthermore, methods for producing the binding molecule and methods for using the binding molecule to kill cancer cells are also provided. [Background technology]

[0003] Immunotherapy for cancer has the potential to improve survival rates in cancer patients by promoting an immune response against tumors. While certain patients experience deep and prolonged responses to currently available anti-cancer immunotherapies (e.g., anti-PD-1 / PD-L1 antibodies such as the anti-CTLA4 antibody ipilimumab, pembrolizumab, or nivolumab), the majority of patients do not benefit from such treatments (Ribas et al., "Science," Vol. 359: pp. 1350-1355 (2018)). For example, patients with so-called "low-temperature" or non-inflammatory tumors, characterized by a lack of immune cell infiltration or the absence of inflammatory signatures, benefit less from anti-cancer immunotherapy (Chen and Mellman, "Nature," Vol. 541: pp. 321-330 (2017)). Therefore, there is a need for novel anti-cancer immunotherapies with improved efficacy.

[0004] The lymphphotoxin beta receptor (LTBR / TNFRSF3), a receptor of the TNF superfamily, is one of many possible targets for anti-cancer immunotherapy. LTBR plays a central role in the development and homeostasis of lymph nodes and secondary lymphoid organs by regulating the expression of several homeostatic lymphoid cytokines (e.g., CCL19, CCL21, CXCL13) and adhesion molecules (ICAM-1, VCAM-1, MAdCAM1) via the NF-κB pathway (Dejardin et al., "Immunity", Vol. 17: pp. 525-535 (2002); Schneider et al., "Immunol. Rev.", Vol. 202: pp. 49-66 (2004)). LTBR is activated by two distinct trimer ligands, LIGHT (TNFRF14) and lymphotin α1β2 (LTα1β2). While LTα1β2 is specific to LTBR, ​​LIGHT also binds to and activates HVEM (TNFRSF14), a receptor expressed on immune cells that is involved in the regulation of immune cells (Pasero et al., "Curr. Opin. Pharmacol.", Vol. 12: pp. 478-475 (2012)).

[0005] Of particular interest from the perspective of cancer immunotherapy is the finding that activation of LTBR by its ligand leads to ectopic formation of tertiary lymphoid structures (TLS) (Schrama et al., "Immunity", Vol. 14: pp. 111-121 (2001); Tang et al., "Cell.Mol.Immunol.", Vol. 14: pp. 809-818 (2017)). The presence of TLS in the tumor microenvironment is typically correlated with immune infiltration and is also associated with a better prognosis, suggesting that TLS are involved in the antitumor immune response (Dieu-Nosjean et al., "J. Clin. Oncol.", Vol. 26: pp. 4410-4417 (2008); Weinstein and Storkus, "Adv. Cancer Res.", Vol. 128: pp. 197-233 (2015)). Therefore, activation of LTBR has the potential to promote TLS formation in the tumor microenvironment, induce an antitumor immune response, and improve current cancer immunotherapy.

[0006] The concept of targeting LTBRs for therapeutic purposes to promote a protective antitumor immune response has been established in several preclinical studies.

[0007] Several groups have targeted LTBR using its natural ligand, LIGHT (TNFSF14). LIGHT binds to LTBR and its second receptor, HVEM (TNFSF14), and is expressed on immune cells such as B cells, T cells, NK cells, monocytes, and DCs (Pasero et al., "Curr. Opin. Pharmacol.", Vol. 12: pp. 478-475 (2012)). Therefore, it should be noted that LIGHT-mediated immunobiological effects may depend on either LTBR or HVEM.

[0008] Yu et al. demonstrated that forced expression of the membrane-bound form of LIGHT in mouse tumor cell lines resulted in large-scale infiltration of naive T lymphocytes correlated with upregulation of chemokine products and adhesion molecules, leading to established tumor rejection at both local and distal sites (Yu et al., "Nat.Immunol.", Vol. 5: pp. 141-149 (2004)). Similar findings were made when forced expression of membrane-bound LIGHT was achieved through adenovirus delivery of the LIGHT gene to established tumors (Yu et al., "J.Immunol.", Vol. 179: pp. 1960-1968 (2007)).

[0009] Based on these findings, in an attempt to utilize this mode of action with a more suitable modality for clinical use, Tang et al. generated a homotrimeric single-chain LIGHT variant with improved stability and human-mouse cross-reactivity, referred to as 3xhmLIGHT (Tang et al., "Cancer Cell," Vol. 29: pp. 285-2896 (2016)). When fused to an EGFR-specific tumor-targeting antibody, 3xhmLIGHT induced anti-tumor immunity in mouse and human tumor models by increasing lymphocyte infiltration, thereby overcoming resistance to checkpoint block immunotherapy when combined with an anti-PD-L1 antibody in models with low lymphocyte infiltration. Tang et al. reported on the tolerability of intratumoral injection into mice with tumors. No significant side effects were observed, as no significant changes in body weight or serum cytokines were seen. The authors did not report on tolerability after systemic administration.

[0010] Johansson-Percival et al. developed a fusion construct consisting of mouse LIGHT fused to the C-terminus of a vascular targeting peptide (VTP) (Johansson-Percival et al., "Nat. Immunol.", Vol. 18: pp. 1207-1217 (2017)). In a mouse solid tumor model, the VTP-LIGHT construct homed to tumor blood vessels, promoted vascular normalization, and induced thrombolytic stromal vasoconstriction (TLS). Addition of VTP-LIGHT enhanced the activity of a combination of anti-CTLA4 and anti-PD-1 antibodies, as well as the activity of in vivo antitumor vaccination. Weight loss was observed in treated mice after intravenous administration of VTP-LIGHT.

[0011] Gurney et al. reported in vitro and in vivo studies using a bispecific fusion construct consisting of a heterotrimeric single-chain LTα1β2 moiety fused to a B7-H4-specific tumor-targeting antibody (International Publication No. 2018 / 119118). Importantly, unlike LIGHT used in the various approaches described above, the LTα1β2 fusion construct is a specific agonist of LTBR and does not activate HVEM. Immune cell infiltration, induced cytokine expression, and TLS formation were observed after treatment with the LTα1β2 fusion construct in a mouse tumor model. The antitumor activity of the LTα1β2 antibody fusion combined with an anti-PD-L1 antibody was superior to that of each individual compound. The efficacy model used by Gurney et al. was an artificial model consisting of engineered cell lines overexpressing B7-H4. The activity in models with unengineered B7-H4 expression levels, which are more representative of B7-H4 levels in human tumors, remains unknown. No observations regarding tolerance in mice were reported.

[0012] Michaelson et al. constructed bispecific antibodies targeting TRAIL-R2 and LTBR, ​​suggesting that bispecific antibodies may induce a more enhanced, synergistic, or broader antitumor response than that achieved by treatment with a mixture of the two antibodies (Michaelson et al., "MAbs," Vol. 1: pp. 128-141 (2009)). TRAIL-R2 is a TNF family receptor widely expressed in normal tissues including the colon, lung, liver, and brain (Spierings et al., "J. Histochem. Cytochem.", Vol. 52: pp. 821-831 (2004)), and has also been found to be co-expressed with LTBR on the surface of human epithelial cancer cell lines. The bispecific constructs showed enhanced activity compared to the parent antibody in in vitro and mouse tumor xenograft models. No observations regarding tolerance in mice were reported.

[0013] These studies demonstrate the potential of targeting LTBR for tumor immunotherapy, suggesting that activated LTBR signaling can enhance immune cell infiltration, induce TLS in the tumor environment, and potentially help overcome resistance to checkpoint inhibitor therapy.

[0014] The immune system is tightly regulated to ensure immune-mediated eradication of pathogens without causing tissue damage or autoimmunity. Systemic immunomodulatory therapy brings about a delicate balance that is out of equilibrium, and is commonly observed to cause immune-related adverse events such as interstitial pneumonia, colitis, hepatitis, thyroid dysfunction, skin reactions, and ocular inflammation, which pose challenges to the development of novel immunotherapies, especially in the context of combination therapies where toxicity can be additive or synergistic.

[0015] Due to the widespread expression of LTBR in organisms, agonist LTBR-targeted drugs that can induce TLS and create an activated immune environment have a substantial risk of causing systemic immune-related adverse events. Interestingly, Johansson-Percival et al. reported weight loss in mice after systemic administration of VTP-LIGHT, an LTBR-activating compound (Johansson-Percival et al., "Nat. Immunol.", Vol. 18: pp. 1207-17 (2017)). Therefore, as hypothesized in the prior art, a therapeutic modality that specifically activates LTBR in tumors but not in other tissues is needed to reduce the risk of toxicity and to generate well-tolerated drugs for use in combination therapies (Allen et al., "Oncotarget", Vol. 8: pp. 99207-8 (2017); Tang et al., "Cell Mol. Immunol.", Vol. 14: pp. 809-18 (2017)).

[0016] Several groups have investigated LTBR as a therapeutic target using various LTBR-targeting moieties, but to date, no therapeutic modality that enables specific LTBR activation in tumors has been described.

Summary of the Invention

Means for Solving the Problems

[0017] This specification provides a multispecific binding molecule, such as a bispecific antibody, that can specifically activate the lymphotoxin beta receptor (LTBR) in tumors. The multispecific binding molecule has a first specificity for LTBR and a second specificity for the extra-domain B (EDB) of fibronectin. EDB is a tumor-associated antigen (TAA) of the extracellular matrix. The multispecific binding molecule activates LTBR in tumors expressing EDB, but does not activate LTBR in the absence of EDB, or only slightly activates it, to a degree sufficiently lower than that of its ligands LIGHT and LTα1β2, thereby reducing the risk of undesirable immune-related events. Unlike the LTBR activating molecules described above, efficient LTBR activation in the presence of EDB is achieved by EDB binding via the EDB-specific moiety of the multispecific binding molecule of the present invention, and by LTBR binding via the LTBR-specific moiety of the multispecific binding molecule of the present invention. In the absence of EDB, the multispecific binding molecule does not result in LTBR activation in normal tissue. This represents a significant advantage over molecules described in the prior art, such as those based on natural LTBR ligands, e.g., LIGHT-antibody fusions, as they can activate LTBR independently of TAA and therefore produce far fewer tumors specific to LTBR activation compared to the molecules of the present invention, as shown in the examples herein.

[0018] This specification provides multispecific binding molecules. A multispecific binding molecule may include (i) a first binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), and (ii) a second binding domain that specifically binds to EDB, thereby activating LTBR upon binding to EDB. More specifically, when the multispecific binding molecule binds to both LTBR and EDB simultaneously, it activates LTBR via the respective specific binding domains of these targets. Preferably, this occurs in a tumor environment where cells expressing LTBR and cells expressing EDB are present, resulting in specific activation of LTBR in tumor tissue. In certain embodiments, the multispecific binding molecule activates LTBR in a tumor-specific manner. A multispecific binding molecule may be, for example, a bispecific antibody. In certain embodiments, the multispecific binding molecule includes two antigen-binding domains. In certain embodiments, the multispecific binding molecule includes three antigen-binding domains. The three antigen-binding domains may include, for example, one binding domain that specifically binds to LTBR. The three antigen-binding domains may include, for example, two binding domains that specifically bind to EDB.

[0019] In a particular embodiment, the multispecific binding molecule comprises three antigen-binding domains, and consists of an antibody (e.g., in IgG format) to which an additional binding domain, for example in the form of a single-chain variable domain, is fused, for example, to the N-terminus or C-terminus of the heavy or light chain of the antibody.

[0020] In the multispecific binding molecule of the present invention, which specifically binds to LTBR and TAA present in the extracellular matrix, the TAA present in the extracellular matrix is ​​fibronectin. Preferably, the binding domain that binds to TAA specifically binds to the extracellular domain B (EDB) of fibronectin.

[0021] In certain non-limiting embodiments, the binding domain that specifically binds to LTBR includes a BHA10 antibody or CBE11 antibody or its fragment or derivative, such as a single-chain antibody fragment (scFv), comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and VH and VL comprise any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (ii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (iii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44. For example, VH includes an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence of SEQ ID NO: 44 VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43; VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 43 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43 VL contains an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 44, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; or (v)VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48. For example, VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 48. In contrast, VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 47 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47 VL contains an amino acid sequence having at least 99% identity with respect to the amino acid sequence of SEQ ID NO: 48, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 48; or (vi) Sequence ID 22; or (vii) Sequence ID 23; or (viii) Sequence ID 25.

[0022] In certain non-limiting embodiments, the second binding domain that specifically binds to EDB comprises, for example, a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment comprises any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46. For example, VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 46 VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence having at least 97% identity with respect to the amino acid sequence of SEQ ID NO: 45 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 45; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 45 VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence having 100% identity with respect to the amino acid sequence of SEQ ID NO: 45; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 46.

[0023] In certain non-limiting embodiments, the multispecific binding molecule includes: (1) The binding domain that specifically binds to LTBR includes a heavy chain variable region (VH) and a light chain variable region (VL), and comprises a BHA10 antibody or CBE11 antibody or its fragment or derivative, for example, a single-chain antibody fragment (scFv), wherein VH comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL comprise any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (ii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (iii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44. For example, VH includes an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence of SEQ ID NO: 44 VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43; VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 43 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43 VL contains an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 44, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; or (v)VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48. For example, VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 48. In contrast, VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 47 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47 VL contains an amino acid sequence having at least 99% identity with respect to the amino acid sequence of SEQ ID NO: 48, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 48; or (vi) Sequence ID 22; or (vii) Sequence ID 23; or (viii) Sequence ID 25; and, (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof includes HCDR1, HCDR2, and HCDR3, respectively, which include the amino acid sequences of SEQ ID NOs. 72, 73, and 74, and LCDR1, LCDR2, and LCDR3, respectively, which include the amino acid sequences of SEQ ID NOs. 75, 76, and 77.

[0024] In certain non-limiting embodiments, the multispecific binding molecule includes: (1) The binding domain that specifically binds to LTBR includes a heavy chain variable region (VH) and a light chain variable region (VL), and comprises a BHA10 antibody or CBE11 antibody or its fragment or derivative, for example, a single-chain antibody fragment (scFv), wherein VH comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL comprise any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (ii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (iii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44. For example, VH includes an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence of SEQ ID NO: 44 VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43; VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 43 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43 VL contains an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 44, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; or (v) VH includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 47, and VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48. For example, here, VH includes an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 47, and VL includes an amino acid sequence of SEQ ID NO: 48. VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 47 VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47 VL contains an amino acid sequence having at least 99% identity with respect to the amino acid sequence of SEQ ID NO: 48, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 48; or (vi) Sequence ID 22; or (vii) Sequence ID 23; or (viii) Sequence ID 25; and, (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45. VH contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 45; VL contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45 VL contains an amino acid sequence that has at least 96% identity with the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 45; VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 45 VL contains an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45; VH contains an amino acid sequence that has at least 99% identity with the amino acid sequence of SEQ ID NO: 45; VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46;VH contains an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46.

[0025] In certain non-limiting embodiments, the multispecific molecules include: (1) A binding domain that specifically binds to LTBR containing sequence number 22; and, (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof includes any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46.

[0026] In certain non-limiting embodiments, the multispecific molecules include: (1) A binding domain that specifically binds to LTBR containing sequence number 23; and, (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof includes any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46.

[0027] In certain non-limiting embodiments, the multispecific molecules include: and (1) A binding domain that specifically binds to LTBR containing sequence number 25; (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof includes any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46.

[0028] In certain non-limiting embodiments, the multispecific binding molecule includes any of the following: (a)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 1, which forms a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO: 2, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA14121), which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO: 5; or (b) A first heavy chain containing the amino acid sequence of SEQ ID NO: 9, which forms a binding domain with (i) a first light chain containing the amino acid sequence of SEQ ID NO: 10, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA14122), which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO: 5.

[0029] In certain further non-limiting embodiments, the multispecific binding molecule includes any of the following: (c)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 30, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA1480); or (d)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 31, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with the light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain together with the light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA1481); or (e)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 32, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA1482), in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (f)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 33, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with the light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA1483), in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with the light chain containing the amino acid sequence of SEQ ID NO: 5; or (g)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 34, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA14107), in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (h)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 35, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA14108), in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (j)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, in which the heavy chain portion (SEQ ID NO: 3) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA14133); or (k)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 39, in which the heavy chain portion (SEQ ID NO: 3) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA14174); or (l)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 56, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA1456).

[0030] In some embodiments, the multispecific molecule comprises (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5.

[0031] One or more nucleic acid molecules encoding the multispecific binding molecules disclosed herein are also provided. One or more vectors comprising one or more nucleic acid molecules disclosed herein are also provided. Isolated host cells comprising one or more isolation vectors disclosed herein are also provided.

[0032] Pharmaceutical compositions comprising the multispecific binding molecules disclosed herein and a pharmaceutically acceptable carrier are also provided.

[0033] Methods for treating cancer in subjects requiring treatment are also provided. These methods include administering to a subject a multispecific binding molecule disclosed herein, one or more nucleic acid molecules disclosed herein, one or more vectors disclosed herein, or a pharmaceutical composition disclosed herein.

[0034] Also provided is the use of multispecific binding molecules, one or more nucleic acid molecules, one or more vectors, or pharmaceutical compositions disclosed herein for activating LTBR in tumor tissue.

[0035] A method for producing multispecific binding molecules disclosed herein is also provided, comprising expressing one or more nucleic acid molecules or one or more vectors disclosed herein in a host cell, and collecting the multispecific binding molecules.

[0036] In the multispecific binding molecule of the present invention, the first antigen-binding domain binds to LTBR on cells present in tumors (e.g., tumor cells, fibroblasts, monocytes, etc.). The second antigen-binding domain binds to EDB, which is a tumor-associated antigen (TAA) in the extracellular matrix present in tumors.

[0037] In certain embodiments, a multispecific binding molecule, such as a bispecific antibody or its antigen-binding fragment, comprises two heavy chains (HC) and two light chains (LC) that form two binding domains with respect to the EDB.

[0038] In certain embodiments, the scFv is fused to the carboxyl (C) or amino (N) terminus of one HC. In certain embodiments, the scFv fused to the HC contains an amino acid sequence selected from SEQ ID NOs. 30, 31, 32, 33, 34, 35, 38, 39, or 56.

[0039] Also provided are isolated nucleic acids encoding scFv fused to the HC or antigen-binding fragment of an isolated anti-LTBR bispecific antibody, as disclosed herein. Also provided are isolated nucleic acids encoding the HC and LC or antigen-binding fragments of an anti-LTBR bispecific antibody, as disclosed herein.

[0040] In certain embodiments, heavy chains, light chains, and / or their functional fragments, such as antigen-specific binding domains, are human or humanized.

[0041] Nucleic acids encoding heavy chains, light chains, and / or functional fragments thereof of multispecificity binding molecules, as disclosed herein, are also provided.

[0042] Vectors containing nucleic acid molecules disclosed herein are also provided.

[0043] Host cells containing nucleic acid molecules or vectors disclosed herein are also provided.

[0044] In preferred embodiments, the multispecificity binding molecule, bispecificity antibody, nucleic acid, vector, or host cell according to the present invention are, respectively, isolated multispecificity binding molecules, isolated bispecificity antibodies, isolated nucleic acids, isolated vectors, or isolated host cells.

[0045] Also provided are pharmaceutical compositions comprising a multispecific binding molecule, such as a bispecific antibody or its antigen-binding fragment, as disclosed herein, and a pharmaceutically acceptable carrier.

[0046] A method for treating cancer in a subject requiring treatment is also provided. This method comprises (a) identifying a subject requiring cancer treatment, and (b) administering the multispecific binding molecule of the present invention to the subject requiring it, for example in the form of a pharmaceutical composition, thereby treating cancer in the subject by administering the pharmaceutical composition to the subject requiring it.

[0047] A method for activating LTBR-expressing cells is also provided. This method involves contacting LTBR-expressing cells with a multispecific binding molecule, for example, in the form of the pharmaceutical composition of the present invention, and contacting LTBR-expressing cells with the multispecific binding molecule or pharmaceutical composition results in increased expression of RANTES, IL-6, IL-8, MIP-3b, ICAM-1, I-TAC, IP-10, IL-12p70, TNF-a, MIP-3a, and / or SDF-1a compared to cells expressing LTBR in an environment without EDB.

[0048] A method for inhibiting the growth or proliferation of cancer cells in tumors expressing EDB is also provided. This method involves contacting cancer cells and / or cells in the tumor microenvironment with a multispecific binding molecule, for example in the form of the pharmaceutical composition of the present invention, thereby inhibiting the growth or proliferation of cancer cells by contacting cancer cells and / or cells in the tumor microenvironment with the pharmaceutical composition.

[0049] Methods for producing the pharmaceutical compositions disclosed herein are also provided. These methods include combining an isolated multispecific binding molecule of the present invention, such as a bispecific antibody or its antigen-binding fragment, with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0050] A method for producing multispecific binding molecules, such as bispecific antibodies or their antigen-binding fragments, is also provided. This method involves culturing host cells containing nucleic acids disclosed herein under conditions for producing multispecific binding molecules, such as bispecific antibodies or their antigen-binding fragments, and for recovering the multispecific binding molecules, such as bispecific antibodies or their antigen-binding fragments. [Brief explanation of the drawing]

[0051] The above summary and the following detailed description of preferred embodiments of this application will be better understood in conjunction with the accompanying drawings. However, it should be understood that this application is not limited to the embodiments shown in the drawings. [Figure 1A]A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The control monoclonal antibody IgG1, which possesses the silencing Fc mutation IgG1σ, is also shown. [Figure 1B] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The control monoclonal antibody IgG1, possessing the silencing Fc mutation IgG1σ, is also shown. [Figure 1C] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The control monoclonal antibody IgG1, possessing the silencing Fc mutation IgG1σ, is also shown. [Figure 1D] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The control monoclonal antibody IgG1, possessing the silencing Fc mutation IgG1σ, is also shown. [Figure 1E] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. It shows a 1:1 knob-into-hole (KiH) heterodimer containing a targeted arm (B21M or EDBmAb1) and human LIGHT fused to Fc. A series of mutations were introduced into the Fc fused to human LIGHT to suppress binding to protein A, facilitating the purification of the heterodimer. [Figure 1F] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. It shows a 1:1 knob-into-hole (KiH) heterodimer containing a targeted arm (B21M or EDBmAb1) and human LIGHT fused to Fc. A series of mutations were introduced into the Fc fused to human LIGHT to suppress binding to protein A, facilitating the purification of the heterodimer. [Figure 1G] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The human LTα1β2 antibody fusion is also shown. [Figure 1H] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The human LTα1β2 antibody fusion is also shown. [Figure 1I] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The human LTα1β2 antibody fusion is also shown. [Figure 1J] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The human LTα1β2 antibody fusion is also shown. [Figure 1K] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The 1:1 KiH heterodimer is shown. [Figure 1L] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The 1:1 KiH heterodimer is shown. [Figure 1M] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The 1:1 KiH heterodimer is shown. [Figure 1N] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The 1:1 KiH heterodimer is shown. [Figure 10] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The 1:1 KiH heterodimer is shown. [Figure 1P] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The diagram shows a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1, and an isotype control antibody. [Figure 1Q] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The diagram shows a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1, and an isotype control antibody. [Figure 1R] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The diagram shows a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1, and an isotype control antibody. [Figure 1S] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. The diagram shows a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1, and an isotype control antibody. [Figure 1T] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. EDBmAb1, a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1, is shown. [Figure 1U] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. EDBmAb1, a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1, is shown. [Figure 1V]A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. EDBmAb1, a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1, is shown. [Figure 1W] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. EDBmAb1, a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1, is shown. [Figure 1X] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. EDBmAb1 is shown, a 2:1 heterodimer fused to a stapled scFv derived from a lower affinity variant of LTBRmAb1. [Figure 1Y] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. EDBmAb1 is shown, a 2:1 heterodimer fused to a stapled scFv derived from a lower affinity variant of LTBRmAb1. [Figure 1Z] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. It shows a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1 that does not contain the protein A mutation in the Fc region, either EDBmAb1 or B21M. [Figure 1A1] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. It shows a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1 that does not contain the protein A mutation in the Fc region, either EDBmAb1 or B21M. [Figure 1A2] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. It represents a 2:1 heterodimer, EDBmAb1, or B21M fused to a disulfide-stabilized scFv derived from LTBRmAb1. [Figure 1A3] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. It represents a 2:1 heterodimer, EDBmAb1, or B21M fused to a disulfide-stabilized scFv derived from LTBRmAb1. [Figure 1A4]A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. It represents a 2:1 heterodimer, EDBmAb1, or B21M fused to a disulfide-stabilized scFv derived from LTBRmAb1. [Figure 1A5] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. It represents a 2:1 heterodimer, EDBmAb1, or B21M fused to a disulfide-stabilized scFv derived from LTBRmAb1. [Figure 1A6] A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. MSLNmAb1, a 2:1 heterodimer fused to a stapled scFv derived from LTBRmAb1, is shown. [Figure 2A] This shows the size exclusion chromatogram (SEC) of COVA1418, which consists of 3xhmLIGHT-Fc containing the heavy and light chains of the anti-RSV antibody B21M. [Figure 2B] This shows the size exclusion chromatogram (SEC) of COVA1454, which consists of 3xhmLIGHT-Fc containing the heavy and light chains of the anti-EDB antibody EDBmAb1. [Figure 2C] This shows the size exclusion chromatogram (SEC) of COVA14133, which consists of an anti-EDB antibody EDBmAb1 heavy chain and a C-terminal stapled scFv BHA10 (VH-VL oriented) fusion with the heavy and light chains of the anti-EDB antibody EDBmAb1. [Figure 2D] This shows the size exclusion chromatogram (SEC) of COVA14113, which consists of the EDBmAb1 heavy chain carrying a C-terminal LTα1β2 fusion with the light chain of the anti-EDB antibody EDBmAb1. [Figure 2E] This shows the size exclusion chromatogram (SEC) of COVA14114, which consists of an anti-RSV B21M antibody heavy chain carrying a C-terminal LTα1β2 fusion with the light chain of the anti-RSV B21M antibody. [Figure 2F] This shows the size exclusion chromatogram (SEC) of COVA14116, which consists of the EDBmAb1 heavy chain carrying the C-terminal LTα1β2 fusion with the heavy and light chains of the anti-EDB antibody EDBmAb1. [Figure 2G] This shows the size exclusion chromatogram (SEC) of COVA14117, which consists of an anti-RSV B21M antibody heavy chain carrying a C-terminal LTα1β2 fusion with the heavy and light chains of the anti-RSV B21M antibody. [Figure 3A] The graph demonstrates the results of the A549 NF-κB reporter assay. Tumor-associated antigen (TAA)-dependent activation of LTBR by COVA1454 compared to COVA1418 and recombinant human LIGHT. [Figure 3B] The graph demonstrates the results of the A549 NF-κB reporter assay. It shows TAA-independent activation of LTBR by COVA1454 compared to COVA1418 and recombinant human LIGHT. [Figure 3C] The graph shows the results of the A549 NF-κB reporter assay. TAA-dependent activation of LTBR by COVA14113 and COVA14116 compared to recombinant human LIGHT and recombinant human LTα1β2. [Figure 3D] The graph demonstrates the results of the A549 NF-κB reporter assay. It shows TAA-independent activation of LTBR by COVA14113 and COVA14116 compared to recombinant human LIGHT and recombinant human LTα1β2. [Figure 4A] The graph illustrates the results of an A549 NF-κB reporter assay using a 1:1 heterodimer consisting of EDBmAb1 and LTBRmAb1 or LTBRmAb2. It shows tumor-associated antigen (TAA)-dependent activation of LTBR by COVA14121 compared to COVA14120, COVA14124, COVA1413, COVA1440, and recombinant human LIGHT. [Figure 4B]The graph demonstrates the results of the A549 NF-κB reporter assay using a 1:1 heterodimer consisting of EDBmAb1 and LTBRmAb1 or LTBRmAb2. It shows TAA-independent activation of LTBR by COVA14121 compared to COVA14120, COVA14124, COVA1413, COVA1440, and recombinant human LIGHT. [Figure 4C] The graphs illustrate the results of the A549 NF-κB reporter assay using a 1:1 heterodimer consisting of EDBmAb1 and LTBRmAb1 or LTBRmAb2. TAA-dependent activation of LTBR by COVA14122 compared to COVA14123, COVA14124, COVA1402, COVA1440, and recombinant human LIGHT. [Figure 4D] The graph demonstrates the results of the A549 NF-κB reporter assay using a 1:1 heterodimer consisting of EDBmAb1 and LTBRmAb1 or LTBRmAb2. It shows TAA-independent activation of LTBR by COVA14122 compared to COVA14123, COVA14124, COVA1402, COVA1440, and recombinant human LIGHT. [Figure 5A] The graph illustrates the results of an A549 NF-κB reporter assay using a 2:1 bispecific antibody. It shows efficient activation of LTBR by COVA1456 (2:1 EDBmAb1×LTBR mAB1) in the presence of EDB-containing fibronectin. No LTBR activation was observed with the isotype control molecule COVA1462 (2:1B21M×LTBR mAb1). [Figure 5B] The graph illustrates the results of the A549 NF-κB reporter assay using a 2:1 bispecific antibody. Measurements were taken in the absence of EDB-containing fibronectin with COVA1456 or its isotype control molecule COVA1462, without LTBR activation. [Figure 5C]The graphs illustrate the results of an A549 NF-κB reporter assay using 2:1 bispecific antibodies. They show a comparison of TAA-dependent LTBR activation by COVA1456 with COVA1482, their respective control molecules COVA1462 and COVA1486, and recombinant human LIGHT. [Figure 5D] The graphs illustrate the results of the A549 NF-κB reporter assay using 2:1 bispecific antibodies. The graphs compare TAA-dependent LTBR activation by COVA1482, as well as bispecific antibodies COVA14107 and COVA14108 containing lower affinity variants of LTBRmAb1, and COVA1486. [Figure 5E] The graphs illustrate the results of an A549 NF-κB reporter assay using 2:1 bispecific antibodies. They compare TAA-dependent LTBR activation by COVA1482 and COVA14133 (constructs without protein A mutations), as well as their respective control molecules, COVA1486 and COVA14136. [Figure 5F] Efficient activation of LTBR by COVA14133 (2:1 EDBmAb1×LTBR mAB1) and COVA14116 (2:1 EDBmAb1×LTα1β2) in the presence of EDB-containing fibronectin. No LTBR activation was observed with the isotype control molecule COVA14136 (2:1 B21M×LTBR mAb1). TAA-independent activation of LTBR by COVA14117 (2:1 B21M×LTα1β2). [Figure 5G] No LTBR activation was observed in the absence of EDB-containing fibronectin, as measured by COVA14133 or its isotype control molecule COVA14136. TAA-independent activation of LTBR was observed by COVA14116 and COVA14117. [Figure 6] The results of flow cytometry staining of A375 cells after co-culture experiments for ICAM-1 are shown. COVA1482 and its control molecule COVA1486 are compared with recombinant human LIGHT. [Figure 7A]The graph demonstrates the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Human RANTES concentration. [Figure 7B] The graph demonstrates the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Human IL-6 concentration. [Figure 7C] The graph demonstrates the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Human IL-8 concentration. [Figure 7D] The graph demonstrates the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Human MIP-3b concentration. [Figure 7E] A graph is shown demonstrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Furthermore, concentrations of human IP-10 are shown, including 2:1 antibody × LTα1β2 fusions COVA14116 and COVA14117, as well as EDBmAb1 COVA1452. [Figure 7F] A graph is shown demonstrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Furthermore, concentrations of human SDF-1a are shown, including 2:1 antibody × LTα1β2 fusions COVA14116 and COVA14117, as well as EDBmAb1 COVA1452. [Figure 7G] A graph is shown demonstrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Furthermore, concentrations of human IL-12p70 are shown, including 2:1 antibody × LTα1β2 fusions COVA14116 and COVA14117, as well as EDBmAb1 COVA1452. [Figure 7H] A graph is shown demonstrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Furthermore, concentrations of human I-TAC are shown, including 2:1 antibody × LTα1β2 fusions COVA14116 and COVA14117, as well as EDBmAb1 COVA1452. [Figure 7I] A graph is shown demonstrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Furthermore, concentrations of human MIP-3a are shown, including 2:1 antibody × LTα1β2 fusions COVA14116 and COVA14117, as well as EDBmAb1 COVA1452. [Figure 7J] A graph is shown demonstrating the measurement of cytokines in the supernatant of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440. The assay is performed using the MSD platform. Furthermore, the concentrations of human TNFα, including 2:1 antibody × LTα1β2 fusions COVA14116 and COVA14117, as well as EDBmAb1 COVA1452, are shown. [Figure 8A]This study demonstrates LTBR activation by MSLN / LTBR bispecificity (dual singularity) in A549 NF-κB reporter / CHOK1MSLN or A549 NF-κB reporter / H226 co-culture cell assays. It also shows LTBR activation in the A549 NF-κB reporter / H226 co-culture assay. COVA14146 (2:1 MSLNmAb1 × LTBRmAb1) is compared with the LIGHT and isotype control 2:1 construct COVA1486. [Figure 8B] This shows LTBR activation by MSLN / LTBR bispecificity (bispecificity) in A549 NF-κB reporter / CHOK1MSLN or A549 NF-κB reporter / H226 co-culture cell assays. The concentration of RANTES secreted upon LTBR activation in the A549 NF-κB reporter / H226 co-culture assay is also shown. COVA14146 (2:1 MSLNmAb1×LTBRmAb1) is compared with the LIGHT and isotype control 2:1 construct COVA1486. [Figure 9A] A schematic diagram of possible LTBR activation mechanisms is shown. In the presence of EDB (tumor-associated antigen (TAA)) in the extracellular matrix, bispecific antibodies can cluster LTBRs on the cell surface via binding to EDB. LTBR activation leads to the secretion of chemotactic cytokines and chemokines. [Figure 9B] A schematic diagram of possible LTBR activation mechanisms is shown. In the absence of EDB in the extracellular matrix, LTBR clustering does not occur. As a result, LTBR activation cannot occur. [Figure 10]This shows the migration of PBMCs to cytokines induced by LTBR activation. Supernatants of co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 compared to COVA14136 and COVA1440, and the anti-EDB / LTα1β2 fusion COVA14116 compared to COVA14117 (shown in Figure 7), acted as PBMC attractants in the Transwell migration assay. The number of PBMCs that migrated toward the co-culture supernatant was counted and is shown in the graph. PBMC migration was induced in a dose-dependent manner from the supernatant of co-cultures stimulated with COVA14133 and COVA14116, down to the slightly smaller COVA14117. Supernatants from co-cultures incubated with the untargeted control molecule COVA14136 did not induce PBMC migration. [Figure 11A] This shows the adhesion and migration of monocytes to HUVEC monolayers stimulated with the anti-EDB / anti-LTBR bispecific antibody COVA14133, compared to the control COVA14136. The number of adherent monocytes was counted over time in an image-based assay consisting of a continuous stream of monocytes traversing HUVEC monolayers grown in the presence of EDB and stimulated with 50 nM COVA14133 or COVA14136. Student's t-test analysis of COVA14133 against COVA14136 was performed, and the following marks were made: *P<0.05, **P<0.01, ***P<0.005. [Figure 11B] This shows the adhesion and migration of monocytes to HUVEC monolayers stimulated with the anti-EDB / anti-LTBR bispecific antibody COVA14133, compared to the control COVA14136. The number of migrating monocytes was counted over time in an image-based assay consisting of a continuous stream of monocytes traversing HUVEC monolayers grown in the presence of EDB and stimulated with 50 nM COVA14133 or COVA14136. Student's t-test analysis of COVA14133 against COVA14136 was performed, and the following marks were made: *P<0.05, **P<0.01, ***P<0.005. [Modes for carrying out the invention]

[0052] In the background art and throughout this specification, various publications, articles, and patents are cited or referenced, and each of these references is incorporated herein by reference in its entirety. The considerations of documents, operations, materials, devices, articles, etc., included herein are for the purpose of providing context for the present invention. Such considerations do not constitute an endorsement that any or all of these things constitute part of the prior art to any invention disclosed or claimed.

[0053] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Otherwise, certain terms used herein have the meanings set forth herein.

[0054] When used herein and in the appended claims, it should be noted that the singular forms "a," "an," and "the" refer to multiple objects unless otherwise clearly indicated by the context.

[0055] Unless otherwise specified, all numerical values, such as concentrations or concentration ranges, described herein should be understood in all cases as being modified by the term “approximately.” Therefore, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). When used herein, the use of numerical ranges explicitly includes all possible subranges, including integers and fractions of values ​​within that range, and all individual numerical values ​​within that range, unless the context explicitly indicates otherwise.

[0056] Unless otherwise stated, the term “at least” preceding a set of elements should be understood to refer to all of those elements. Those skilled in the art will recognize or confirm many equivalents to the specific embodiments of the invention described herein by simply using ordinary experimental procedures. Such equivalents are intended to be encompassed by the invention.

[0057] When used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, are intended to include the integer or set of integers mentioned, but not to exclude other integers or sets of integers, and are intended to be non-exclusive or non-restrictive. For example, a composition, mixture, process, method, article, or apparatus comprising a set of elements is not necessarily limited to those elements alone, and may include other elements not expressly enumerated or not inherently present in such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly otherwise indicated, “or” refers to an inclusive “or” and not an exclusive “or.” For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0058] When used herein, the connecting term "and / or" between multiple enumerated elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is included in the meaning and therefore satisfies the requirements of the term "and / or" when used herein. The simultaneous applicability of two or more of the options is also included in the meaning and therefore satisfies the requirements of the term "and / or."

[0059] When used herein, variations of the term "consists of," "consist of," or "consisting of" include, when used throughout the specification and claims, any enumerated integer or set of integers, but no additional integers or sets of integers are added to the specified method, structure, or composition.

[0060] When used herein, variations of the term "consists essentially of," "consist essentially of," or "consisting essentially of" include, when used throughout the specification and claims, any enumerated integer or set of integers, and optionally any enumerated integer or set of integers that does not substantially alter the basic or novel properties of the specified method, structure, or composition. See MPEP §2111.03.

[0061] As used herein, “subject” means any animal, preferably a mammal, most preferably a human. As used herein, the term “mammal” encompasses all mammals. Examples of mammals, but not limited to, include cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, preferably humans.

[0062] The terms “about,” “approximately,” “generally,” and “substantially,” as used herein when referring to the dimensions or features of preferred components of an invention, should also be understood, as those skilled in the art will understand, to indicate that the described dimensions / features are not strict boundaries or parameters and do not exclude slight differences from them that are functionally the same or similar. At a minimum, such references involving numerical parameters will include variations in which the minimum significant figures do not change when using mathematical and industrial principles accepted in the art (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, etc.).

[0063] The term “identical” or “identity” percentage refers to two or more sequences or subsequences that are identical or have a specific percentage of identical amino acid residues or nucleotides when compared and aligned to maximize the match, when measured using one of the following sequence comparison algorithms or by visual inspection, in relation to two or more nucleic acid or polypeptide sequences (e.g., anti-LTBR bispecific antibody and the polynucleotide encoding it, anti-LTBR / anti-EDB bispecific antibody and the polynucleotide encoding it, LTBR polypeptide and the LTBR / polynucleotide encoding it, EDB polypeptide and the EDB polynucleotide encoding it).

[0064] For sequence comparison, typically one sequence acts as the reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and sub-sequence coordinates and sequence algorithm program parameters are specified, if necessary. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence to the reference sequence based on the specified program parameters.

[0065] The optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv.Appl.Math.2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J.Mol.Biol.48:443 (1970), the similarity search method of Pearson & Lipman, Proc.Nat'l.Acad.Sci.USA85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (generally, Current Protocols in Molecular Biology, FMAusubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995) This can be done by (see Supplement)(Ausubel).

[0066] Examples of suitable algorithms for determining sequence identity percentage and sequence similarity are the BLAST and BLAST2.0 algorithms described in Altschul et al., (1990) J.Mol.Biol.215:403~410 and Altschul et al., (1997) Nucleic Acids Res.25:3389~3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match when aligned with words of the same length in the database sequence, or satisfy a threshold score T of some positive value. The above T is referred to as the adjacent word score threshold (Altschul et al., above). These initial adjacent word hits serve as seeds to initiate a search to find longer HSPs that contain them. Next, extend the word hits along each sequence in both directions, as long as the cumulative alignment score can be increased.

[0067] For nucleotide sequences, the cumulative score is calculated using parameter M (reward score for matching residue pairs, always greater than 0) and parameter N (penalty score for mismatched residues, always less than 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Word hit extension in each direction is stopped when the cumulative alignment score falls by amount X from its maximum value, when the cumulative score becomes zero or less due to the accumulation of alignments of one or more negative scoring residues, or when the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, word length (W)=11, expected value (E)=10, M=5, N=-4, and comparison of both strands. For amino acid sequences, the BLASTP program uses a default word length (W) of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0068] In addition to calculating the sequence identity percentage, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide sequences or two amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0069] A further indicator that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example, the two peptides differ only by conserved substitutions. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions.

[0070] As used herein, the term “polynucleotide” is synonymous with “nucleic acid molecule,” “nucleotide,” or “nucleic acid,” and refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA, or modified RNA or DNA. “Polynucleotides” include, but are not limited to, single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. In addition, “polynucleotide” refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA having a backbone modified for stability or other reasons. “Modified” bases include, for example, tritylated bases and unusual bases, such as inosine. Various modifications can be made to DNA and RNA. Therefore, "polynucleotides" encompass chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms that possess the characteristics of viral and cellular DNA and RNA. "Polynucleotides" also include relatively short nucleic acid chains (often called oligonucleotides).

[0071] As used herein, the term "vector" refers to a replicon that can replicate or express another nucleic acid segment by functionally inserting that segment.

[0072] As used herein, the term “host cell” refers to a cell containing the nucleic acid molecule of the present invention. A “host cell” may be any type of cell, such as a primary cell, a cell in culture, or a cell line-derived cell. In one embodiment, the “host cell” is a cell transfected with the nucleic acid molecule of the present invention. In another embodiment, the “host cell” is a descendant or potential descendant of such a transfected cell. A descendant of a cell may not be identical to the parent cell, for example, due to mutations that may occur in subsequent generations, environmental influences, or the incorporation of the nucleic acid molecule into the host cell genome.

[0073] As used herein, the term “expression” refers to the biosynthesis of a gene product. Such expression includes the transcription of a gene into RNA, the translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications. The expressed multispecificity binding molecule, such as a bispecific antibody, may be present in the cytoplasm of a host cell, in the extracellular environment such as a cell culture growth medium, or immobilized on the cell membrane. Preferably, the multispecificity binding molecule is secreted from the producing host cell into the culture medium.

[0074] As used herein, the terms “peptide,” “polypeptide,” or “protein” may refer to a molecule composed of amino acids that can be recognized as a protein by those skilled in the art. Conventional one- or three-letter codes for amino acid residues are used herein. The terms “peptide,” “polypeptide,” and “protein” may be used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also encompass amino acid polymers that are naturally modified or modified by intervention. Examples of interventions include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as conjugate with a labeling component. The definition also includes, for example, polypeptides containing one or more analogues of amino acids (including, for example, non-natural amino acids), and other modifications known in the art.

[0075] The peptide sequences described herein are written according to common convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although the isomer forms of amino acids are known, unless otherwise explicitly indicated, only the L-form of the amino acids is shown.

[0076] As used herein, "multispecific binding molecule" means a molecule that specifically binds to at least two different molecules. Preferably, the molecule is a protein, including, for example, an antibody or a fragment or derivative thereof. The multispecific binding molecule or antibody of the present invention has at least one binding domain that specifically binds to LTBR and at least one binding domain that specifically binds to the EDB of fibronectin, and in consideration of the presence of binding specificity to LTBR, ​​it may be referred to herein as an "anti-LTBR" binding molecule or antibody.

[0077] As used herein, the term "binding domain" means a functional portion of a binding molecule, such as from an antibody, that confers specific binding of the binding molecule to a target molecule. Examples of binding domains are the variable regions of antibodies that confer specific binding to a target molecule, which may be formed by two or more chains of an antibody, such as the variable domain of a heavy chain paired with the variable domain of a light chain, or by a single chain such as an scFv molecule, or by a single domain such as a VHH derived from camelids, such as a nanobody. The target molecule of the present invention is LTBR or fibronectin, particularly the EDB of fibronectin.

[0078] As used herein, the term "specific binding" refers to the binding of an antibody to a given antigen with a higher affinity than to other antigens. Typically, an antibody binds to a given antigen with a dissociation constant (K D M) of about 1×10 -8 M or less, such as about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, about 1×10 -12 M or less, about 1×10 -13 M or less, or about 1×10 -14 M or less. With respect to binding to a non-specific antigen or epitope (e.g., BSA, casein), typically, its K D is at least 10-fold lower than its K D for binding to the non-specific antigen or epitope. The dissociation constant can be measured using standard procedures. However, an antibody that specifically binds to a given antigen may cross-react with the same given antigen (homolog) from other species, such as human or monkey, such as Macaca fascicularis (cynomolgus monkey, cyno) or Pan troglodytes (chimpanzee, chimp).

[0079] ​​The terms “tumor-associated antigen” or “TAA,” as used herein, mean an antigen present on tumor cells or in the extracellular matrix of a tumor, which is not qualitatively different in form from antigens found on normal cells or in the extracellular matrix of normal tissues, but differs quantitatively in several respects, for example, being present on tumor cells or in the extracellular matrix of a tumor in significantly greater amounts, at higher densities, at different sites of expression, and / or differentially accessible to the immune system, etc. In certain embodiments, the tumor-associated antigen is present on tumor cells or in the extracellular matrix of a tumor in amounts at least twice as much as on non-tumor cells or in the extracellular matrix, more preferably at least five times, e.g., at least ten times more, even more preferably at least 100 times more, e.g., at least 1000 times more, most preferably at least 10,000 times more. EDB is present in fibronectin in the extracellular matrix of tumor tissue, whereas typically it is undetectable in the fibronectin form present in normal tissues (i.e., the same tissue under normal conditions and not in a tumor environment).

[0080] The term “extracellular matrix,” as used herein, refers to the non-cellular components present in all tissues and organs, in the form of a three-dimensional network of extracellular macromolecules such as collagen, enzymes, and glycoproteins, which provide structural and biochemical support to the surrounding cells. Its exact composition varies by tissue, but it generally consists of proteoglycans, water, minerals, and fibrous proteins. Proteoglycans are composed of a protein core surrounded by long chains of starch-like molecules called glycosaminoglycans. The two main classes of extracellular matrix molecules constitute the matrix: proteoglycans and fibrous proteins (e.g., collagen, elastin, fibronectin, and laminin).

[0081] antibody The present invention generally relates to anti-LTBR multispecificity binding molecules, nucleic acids and expression vectors encoding the multispecificity binding molecules, recombinant cells containing the vectors, and compositions comprising the multispecificity binding molecules. In preferred embodiments, the anti-LTBR multispecificity binding molecule is an anti-LTBR multispecific antibody, such as an anti-LTBR bispecific antibody or its antigen-binding fragment. In certain embodiments, the anti-LTBR multispecificity binding molecule may include a binding domain that specifically binds to LTBR in a form different from that of the antibody or its functional fragment, for example, these may include anti-LTBR Fynomers, anti-LTBR Affimers, anti-LTBR Darrpins, and / or other protein scaffolds screened for candidates that specifically bind to LTBR. In the multispecificity binding molecules of the present invention, the binding domain specific to LTBR is not provided by LIGHT or LTα1β2 (the native ligand of LTBR), nor by its functional fragment or derivative, such as 3xhmLIGHT. In preferred embodiments, the LTBR-specific binding domain in the multispecific binding molecule of the present invention comprises an antibody against LTBR, ​​preferably an agonist antibody against LTBR, ​​or a functional fragment thereof such as scFv or a derivative thereof. The agonist antibodies against LTBR themselves have been described, and non-limiting examples include BHA10 (e.g., International Publication No. 2004002431) and CBE11 (e.g., International Publication No. 0230986), or can be produced according to known methods for antibody production such as mouse immunization or phage display.

[0082] Fyn SH3-derived polypeptides or "Fynomers" are well known in the art, for example, Grabulovski et al. (2007), "JBC", Vol. 282, pp. 3196-3204; International Publication No. 2008 / 022759; Bertschinger et al. (2007), "Protein Eng Des Sel", Vol. 20 (No. 2): pp. 57-68; and Gebauer and Skerra (2009), "Curr Opinion in Chemical Biology", Vol. 13: pp. 245-255. The term "Fynomer" and the term "Fyn SH3-derived polypeptide," as used interchangeably herein, refer to non-immunoglobulin-derived conjugated polypeptides derived from the human Fyn SH3 domain (e.g., so-called scaffolds, as described, e.g., Gebauer and Skerra (2009), *Curr Opinion in Chemical Biology*, Vol. 13: pp. 245-255). Fynomers are small, spherical polypeptides of approximately 7 kDa. The SH3 domain of human Fyn kinase has been successfully used as a scaffold for manipulating proteins (Fyn SH3-derived binding proteins, referred to as Fynomers) that bind with high affinity and specificity to different target proteins (International Publication Nos. 2008 / 022759, 2011 / 023685, 2013 / 135588, 2014 / 170063; Grabulovski D. et al. (2007), "J Biol Chem," Vol. 282, pp. 3196-3204; Bertschinger J. et al. (2007), "Protein Eng Des Sel," Vol. 20, pp. 57-68; and Schlatter et al. (2012), "mAbs," Vol. 4 (No. 4), pp. 497-4950).

[0083] Affimer molecules are small proteins (12-14 kDa) that bind to target molecules with similar specificity and affinity to antibodies. These engineered non-antibody-binding proteins are designed to mimic the molecular recognition properties of monoclonal antibodies in different applications (e.g., Tiede et al., "eLife 2017", DOI:10.7554 / eLife.24903).

[0084] DARPin (in the case of engineered ankyrin repeat proteins) are genetically engineered antibody-mimicking proteins derived from the natural ankyrin protein, typically exhibiting highly specific protein binding. DARPin consists of at least three repeat motifs, and their molecular weights are typically about 14 kDa or 18 kDa, respectively, for DARPin with four or five repeats. DARPin designs are described, for example, by Binz et al., 2003, "J.Mol.Biol.", Vol. 332, pp. 489-503.

[0085] Other binding protein formats, such as protein scaffolds, are known in the art and can also be used to provide one or more binding domains for certain embodiments of the multispecific binding molecule of the present invention.

[0086] In preferred embodiments of the present invention, the binding domain that binds to LTBR is derived from an antibody, preferably an agonist antibody, that activates LTBR upon binding and specifically binds to LTBR. In specific embodiments, the binding domain that binds to LTBR is a single-chain variable domain (scFv) of the antibody, which can be stabilized in any available format, for example, by the methods described above and / or herein.

[0087] In certain embodiments, the present invention relates to an anti-LTBR / anti-EDB bispecific antibody or its antigen-binding fragment, a nucleic acid and expression vector encoding this antibody, recombinant cells containing this vector, and a composition comprising this bispecific antibody. Methods for producing multispecific conjugating molecules and / or antibodies, and methods for using multispecific conjugating molecules and / or antibodies to treat diseases, including cancer, are also provided. The multispecific conjugating molecules and / or antibodies disclosed herein possess one or more desirable functional properties, including, but not limited to, specific binding to LTBR and EDB, high specificity to LTBR and EDB, and / or the ability to treat or prevent cancer when administered alone or in combination with other anticancer therapies.

[0088] As used herein, the term “antibody” is used broadly and includes immunoglobulins, or human, humanized, complex, and chimeric antibodies, which are monoclonal or polyclonal, and immunoglobulin or antibody molecules containing an antigen-binding domain. In general, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen. The structure of antibodies is known. Immunoglobulins can be assigned to five main classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided as isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibodies of the present invention can be any of the five main classes or the corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. The antibody light chains of vertebrate species can be assigned to one of two distinct types, namely κ and λ, based on the amino acid sequence of their constant domain. Therefore, the antibodies of the present invention can contain a κ or λ light chain constant domain. According to certain embodiments, the antibody of the present invention comprises heavy and / or light chain constant regions derived from a rat or human antibody. In addition to the heavy and light constant domains, the antibody contains an antigen-binding region comprising a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0089] As used herein, the term “isolated antibody” refers to an antibody that substantially does not contain other antibodies having different antigen specificities (for example, an isolated bispecific antibody that specifically binds to LTBR substantially does not contain antibodies that do not bind to LTBR; an isolated bispecific antibody that specifically binds to LTBR and / or EDB substantially does not contain bispecific antibodies that do not bind to LTBR and / or EDB). Furthermore, an isolated antibody substantially does not contain other cellular material and / or chemical substances.

[0090] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for trace amounts of spontaneous variation. The monoclonal antibodies of the present invention can be produced by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or recombinant DNA methods. For example, monoclonal antibodies may be produced by hybridomas containing B cells obtained from transgenic non-human animals, e.g., transgenic mice or rats, and having a genome containing human heavy chain and light chain transgenes. In certain embodiments, monoclonal antibodies are produced by recombinant host cells expressing a nucleic acid sequence encoding the antibody. Such recombinant host cells can be obtained, for example, by transfection of a nucleic acid sequence into parental cells, e.g., CHO cells. Recombinant host cells can be cultured under conditions that contribute to antibody expression within the host cell, and the antibody can be isolated from the host cell, the culture medium, or both.

[0091] In certain embodiments, the multispecific binding molecule of the present invention comprises an antibody, or one or more antigen-binding fragments thereof. As used herein, the term “antigen-binding fragment” refers to antibody fragments such as, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), single-domain antibody (sdab)scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragments that bind to an antigen but do not contain a complete antibody structure. The antigen-binding fragment can bind to the same antigen to which the parent antibody or parent antibody fragment binds. According to certain embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and a heavy chain Fd fragment. According to other specific embodiments, the antigen-binding fragment comprises Fab and F(ab'). In some embodiments, the antigen-binding fragment may be an IgG-like molecule having a complementary CH3 domain that forms a heterodimer, a recombinant IgG-like bitargeting molecule (where each of the two sides of the molecule contains a Fab fragment or portion of a Fab fragment of at least two different antibodies), an IgG fusion molecule (where a full-length IgG antibody is fused to an extra Fab fragment or portion of a Fab fragment), an Fc fusion molecule (where a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a portion thereof), a Fab fusion molecule (where different Fab fragments are fused together), an ScFv and diabody base and a heavy chain antibody (e.g., a domain antibody, a nanobody) (where different single-chain Fv molecules, different diabodies, or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule).In some embodiments, IgG-like molecules having complementary CH3 domain molecules include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-in-Holes (Genentech), CrossMAbs (Roche) and electrostatically modified (Amgen), LUZ-Y (Genentech), strand-exchanged and manipulated domain bodies (SEEDbody) (EMD Serono), Biclonic (Merus), or DuoBody (Genmab A / S, see, e.g., Labrijn et al., 2013, PNAS 110:5145-5150). In some embodiments, the antigen-binding fragment comprises a "stapled single-chain Fv" or "spFv," and refers to an scFv containing one or more disulfide bonds between VH and the linker or between VL and the linker. Typically, an spFv may contain one disulfide bond between VH and the linker, one disulfide bond between VL and the linker, or two disulfide bonds, one between VH and the linker and one between VL and the linker. scFv molecules containing a disulfide bond between VH and VL are excluded from the definition of "spFv".

[0092] As used herein, the term "single-chain antibody" refers to a single-chain antibody conventionally known in the art, comprising a heavy-chain variable region and a light-chain variable region linked by, for example, a short peptide of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to a single-domain antibody conventionally known in the art, comprising a heavy-chain variable region and a heavy-chain constant region, or comprising only the heavy-chain variable region.

[0093] In certain embodiments, the multispecific binding molecule of the present invention comprises an antibody having one or more mutations in Fc that inhibit binding to protein A. Such mutations facilitate the purification of heterodimers, as described, for example, in International Publication No. 2010151792.

[0094] As used herein, the term “human antibody” means an antibody produced by a human, or an antibody prepared using any technique known in the art that has an amino acid sequence corresponding to a human-produced antibody. This definition of a human antibody includes an intact or full-length antibody, its antigen-binding fragment, and / or an antibody comprising at least one human heavy chain and / or light chain polypeptide.

[0095] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology to a human antibody, while retaining the antigen-binding properties of the antibody, thereby reducing the antigenicity of the antigen in the human body.

[0096] As used herein, the term “chimeric antibody” refers to an antibody whose immunoglobulin molecule’s amino acid sequence originates from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit) that possesses the desired specificity, affinity, and capability, while the constant region corresponds to the sequence of an antibody derived from another species of mammal (e.g., human) to avoid inducing an immune response in that species.

[0097] As used herein, the term “multispecific antibody” refers to an antibody comprising multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence has binding specificity to a first epitope, and a second immunoglobulin variable domain sequence has binding specificity to a second epitope. In some embodiments, the first and second epitopes do not overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, for example, on different proteins (or different subunits of a polymer protein). In certain embodiments, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain, or even more immunoglobulin variable domains. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a triplicate antibody molecule, or a quadruplicate antibody molecule.

[0098] As used herein, the term “bispecific antibody” refers to a multispecific antibody that binds to two or fewer epitopes, preferably two or fewer antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain having binding specificity to a first epitope (e.g., an epitope on an LTBR antigen) and a second immunoglobulin variable domain having binding specificity to a second epitope (e.g., an epitope on an EDB). In one embodiment, the bispecific antibody comprises a first heavy chain variable domain and a first light chain variable domain forming a binding domain having binding specificity to a first epitope, and a second heavy chain variable domain and a second light chain variable domain forming a binding domain having binding specificity to a second epitope. In another embodiment, the bispecific antibody comprises a half-antibody or fragment thereof having binding specificity to a first epitope and a half-antibody or fragment thereof having binding specificity to a second epitope. In one embodiment, the bispecific antibody comprises an scFv or fragment thereof having binding specificity to a first epitope and an scFv or fragment thereof having binding specificity to a second epitope. In another embodiment, the bispecific antibody comprises an scFv or fragment thereof having binding specificity to a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a second epitope. In a preferred embodiment of the present invention, the first epitope is located on the LTBR and the second epitope is located on fibronectin, particularly on its EDB.

[0099] In certain embodiments, the multispecific binding molecule according to the present invention comprises an antibody, for example, IgG having an scFv fused to the antibody. In certain embodiments, the scFv may have binding specificity to LTBR. In certain embodiments, both arms of the antibody (including the variable region) may be bound to the EDB of fibronectin. The scFv may be fused to the light chain or the heavy chain of the antibody, or to the N-terminus or C-terminus of the heavy chain or light chain. In certain embodiments, the scFv is fused to the N-terminus of the heavy chain. In other embodiments, the scFv is fused to the C-terminus of the heavy chain. It will be apparent to those skilled in the art based on this disclosure that other forms are also possible, for example, in which a bispecific antibody comprising one arm that specifically binds to LTBR and the other arm that specifically binds to EDB is supplemented by fusing an scFv that specifically binds to EDB to one of the antibody chains.

[0100] As used herein, the term “LTBR” refers to a polypeptide that is a cell surface receptor for lymphotoxins involved in apoptosis and cytokine release, and is a member of the tumor necrosis factor receptor superfamily. LTBR may also be referred to as “tumor necrosis factor receptor superfamily member 3 (TNFRSF3).” LTBR is expressed on the surface of many cell types, including epithelial and myeloid cells. LTBR can specifically bind to lymphotoxin membrane forms (lymphotoxin-alpha and lymphotoxin-beta complexes). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5, which may lead to the release of interleukin-8. Unless otherwise specified, LTBR is preferably human LTBR. The amino acid sequence of human LTBR is provided by UniProt number P36941.

[0101] The term "EDB" or "extra domain B" refers to a domain of fibronectin that may be included in the fibronectin molecule based on the splicing pattern of fibronectin premRNA. Extra domain B is a complete fibronectin (FN) type III repeat containing 91 amino acid residues. Generally, EDBs are undetectable in normal adult tissues but show greater expression in fetal and tumor tissues in the extracellular matrix and accumulate around new vascular systems during the angiogenesis process, making EDBs a potential marker and target of angiogenesis. Unless otherwise stated, EDB is preferably human EDB. Human EDBs containing fibronectin isoform amino acid sequences are available under UniProt number P02751.

[0102] The term "fibronectin" refers to polypeptides, which are high molecular weight glycoproteins of the extracellular matrix. Fibronectin can bind to transmembrane receptor proteins called integrins. Fibronectin can also bind to other extracellular matrix proteins such as collagen, fibrin, and heparan sulfate proteoglycans. Fibronectin can exist as a protein dimer consisting of two nearly identical monomers linked by a pair of disulfide bonds. Although fibronectin is produced from a single gene, alternative splicing of the fibronectin premRNA molecule creates several isoforms of fibronectin, one of which is EDB fibronectin. Fibronectin can play a role in cell adhesion, growth, migration, and differentiation, and may be important for processes such as wound healing and embryonic development. The amino acid sequence of human fibronectin is provided by UniProt number P02751, which contains extradomain B, and NCBI accession numbers NP_001263337 (isoform B), NP_001263338 (isoform c), NP_001263339 (isoform d), NP_001263340 (isoform e), and NP_001263341 (isoform f), NP_001293058 (isoform 8), NP_001293059 (isoform 9), NP_001293060 (isoform 10), NP_001293061 (isoform 11), and NP_002017 (isoform 3).

[0103] When used herein, an antibody or binding molecule that "specifically binds to LTBR" is defined as an antibody or binding molecule that binds to LTBR, ​​preferably human LTBR, ​​with a KD of 1 × 10⁻¹⁶. -7 M or less, preferably 1 × 10 -8 M or less, more convenient 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 × 10 -10The term "KD" refers to an antibody or molecule containing an antigen-binding domain that binds at a M or less. The term "KD" refers to a dissociation constant obtained from the Kd to Ka (i.e., Kd / Ka) ratio and expressed as molar concentration (M). The KD value of an antibody can be determined using methods in the art in consideration of this disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, for example by using a biosensor system such as the BIACORE® system, or by using biolayer interferometry techniques such as the Octet RED96 system. In preferred embodiments, the binding domain specific to LTBR in the multispecific binding molecule of the present invention includes an antibody against LTBR, ​​preferably an agonist antibody against LTBR, ​​or a functional fragment thereof such as scFv or a derivative thereof. As used herein, "agonist antibody against LTBR" means an antibody that binds to LTBR and can induce downstream signaling, either directly or in the context of higher-order clustering, for example by immobilization on a solid support or the use of a crosslinking antibody. Agonist antibodies against LTBR themselves have been described, and non-limiting examples include BHA10 (e.g., International Publication No. 2004002431), CBE11 (e.g., International Publication No. 0230986), REA412 (commercially available from Miltenyi Biotec), 31G4D8 (commercially available from BioLegend), and 71319 / MAB629 (commercially available from Novus Biologicals), or they can be produced according to known methods of antibody production such as mouse immunization and phage display.

[0104] When used herein, the antigen-binding domain or antigen-binding fragment that "specifically binds to EDB" has a KD of 1 × 10⁻¹⁴. -7 M or less, preferably 1 × 10 -8 M or less, more convenient 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 × 10 -10This refers to an antigen-binding domain or antigen-binding fragment with a magnitude of M or less that binds to EDB (for example, in the formation of EDB fibronectin).

[0105] In preferred embodiments, the EDB-specific binding domain in the multispecific binding molecule of the present invention includes an antibody against EDB, or a functional fragment thereof such as scFv or a derivative thereof. The agonist antibody against EDB itself has been described, and non-limiting examples include L19 (e.g., International Publication No. 9745544) and other antibodies that bind to ED-B or adjacent domains (e.g., Carnemolla et al., "Int. J. Cancer", Vol. 68, pp. 397-405 (1996)), or which can be produced according to known methods for antibody production such as mouse immunization or phage display.

[0106] The lower the KD value of an antibody, the higher its affinity for binding to the target antigen.

[0107] According to a particular aspect of the present invention, a multispecific binding molecule is provided herein. The multispecific binding molecule comprises (i) a first binding domain that specifically binds to a lymphotoxin beta receptor (LTBR), and (ii) a second binding domain that specifically binds to an EDB, wherein the multispecific binding molecule activates the LTBR upon binding to the EDB.

[0108] In certain embodiments, the multispecific binding molecule activates LTBR in a tumor-specific manner. Activating LTBR in a tumor-specific manner means that, when used herein, LTBR is activated upon simultaneous binding of the multispecific binding molecule to LTBR and EDB, both present in the tumor microenvironment either on the cell surface or in the extracellular matrix, triggering signaling via the standard and / or non-standard NF-κB pathway. Activation of the NF-κB pathway can lead to the establishment of a pro-inflammatory tumor microenvironment through the secretion of pro-inflammatory chemokines and cytokines, as well as the expression of adhesion molecules on the cell surface. Simultaneous binding of the multispecific binding molecule results in LTBR activation in the tumor. If EDB is not present in normal tissue, i.e., normal cells, or in the extracellular matrix adjacent to normal tissue, the multispecific binding molecule can only bind to LTBR on normal tissue and does not result in LTBR activation. This represents a significant advantage over molecules described in the prior art, such as those based on natural LTBR ligands, e.g., LIGHT antibody fusions, which can activate LTBR independently of TAA and therefore produce far fewer tumors specific to LTBR activation compared to the molecules of the present invention, as shown in the examples herein.

[0109] In certain embodiments, the multispecific binding molecule comprises a bispecific antibody having two binding domains, for example, two antigen-binding domains, one of which binds to LTBR and the other to EDB. In preferred embodiments, the multispecific binding molecule comprises three or more antigen-binding domains, for example, one binding to LTBR and two bindings to EDB. In certain embodiments, the multispecific binding molecule comprises three binding domains. In certain embodiments, all three binding domains are different and bind to three different antigens. In certain preferred embodiments, the three antigen-binding domains comprise one binding domain that binds to a first antigen and two binding domains that bind to a second antigen. In this embodiment, the three antigen-binding domains exist in a 2:1 stoichiometric ratio. The three antigen-binding domains may include, for example, one first binding domain that specifically binds to LTBR on LTBR-expressing cells. The three antigen-binding domains may include, for example, two second binding domains that specifically bind to EDB. In certain embodiments, the two second binding domains may have identical binding specificity to EDB, for example, the two second binding domains may be identical. It is shown herein that a multispecific binding molecule of the present invention having two or more binding domains specific to EDB has even more advantageous properties than a multispecific binding molecule of the present invention having only one binding domain specific to EDB. In certain embodiments, LTBR is activated upon binding to LTBR and EDB (EDB is a portion of fibronectin present in the extracellular matrix within tumor tissue).

[0110] In certain embodiments, isolated anti-lymphophotoxin beta receptor (LTBR) bispecific antibodies or antigen-binding fragments thereof are provided herein. In certain non-limiting embodiments, the binding domain that specifically binds to LTBR comprises an agonist anti-LTBR antibody or fragment or derivative thereof, such as a single-chain antibody fragment (scFv), comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the VH and VL comprise any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (ii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (iii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44. For example, VH includes an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence of SEQ ID NO: 44 VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43; VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 43 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43 VL contains an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 44, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; or (v)VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48. For example, VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 48. In contrast, VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 47 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47 VL contains an amino acid sequence having at least 99% identity with respect to the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence having 100% identity with respect to the amino acid sequence of SEQ ID NO: 47; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 48; or (vi) Sequence ID 22; or (vii) Sequence ID 23; or (viii) Sequence ID 25.

[0111] In certain non-limiting embodiments, the second binding domain that specifically binds to EDB includes, for example, an antibody that binds to EDB or a fragment or derivative of such an antibody, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof comprises any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46. For example, VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 46 VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence having at least 97% identity with respect to the amino acid sequence of SEQ ID NO: 45 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 45; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 45 VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence having 100% identity with respect to the amino acid sequence of SEQ ID NO: 45; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 46.

[0112] In certain non-limiting embodiments, the multispecific binding molecule includes: (1) The binding domain that specifically binds to LTBR includes a heavy chain variable region (VH) and a light chain variable region (VL), and comprises a BHA10 antibody or CBE11 antibody or its fragment or derivative, for example, a single-chain antibody fragment (scFv), wherein VH comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL comprise any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (ii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (iii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44. For example, VH includes an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence of SEQ ID NO: 44 VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43; VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 43 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43 VL contains an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 44, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; or (v)VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48. For example, VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 48. In contrast, VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 47 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47 VL contains an amino acid sequence having at least 99% identity with respect to the amino acid sequence of SEQ ID NO: 48, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 48; or (vi) Sequence ID 22; or (vii) Sequence ID 23; or (viii) Sequence ID 25; and, (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof includes HCDR1, HCDR2, and HCDR3, respectively, which include the amino acid sequences of SEQ ID NOs. 72, 73, and 74, and LCDR1, LCDR2, and LCDR3, respectively, which include the amino acid sequences of SEQ ID NOs. 75, 76, and 77.

[0113] In certain non-limiting embodiments, the multispecific binding molecule includes: (1) The binding domain that specifically binds to LTBR includes a heavy chain variable region (VH) and a light chain variable region (VL), and comprises a BHA10 antibody or CBE11 antibody or its fragment or derivative, for example, a single-chain antibody fragment (scFv), wherein VH comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL comprise any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (ii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (iii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44. For example, VH includes an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence of SEQ ID NO: 44 VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43; VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 43 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43 VL contains an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 44, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; or (v)VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48. For example, VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 48. In contrast, VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 47 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47 VL contains an amino acid sequence having at least 99% identity with respect to the amino acid sequence of SEQ ID NO: 48, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 48; or (vi) Sequence ID 22; or (vii) Sequence ID 23; or (viii) Sequence ID 25; and, (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45. VH contains an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 46, and VL contains an amino acid sequence that is identical to at least 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 46; for example, VH contains an amino acid sequence that is identical to at least 95% of the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that is identical to at least 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 45 VL contains an amino acid sequence that has at least 96% identity with the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 45; VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 46; VH6% identity with the amino acid sequence of SEQ ID NO: 45; VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence that has at least 96% identity with the amino acid sequence of SEQ ID NO: 4 VL contains an amino acid sequence that has at least 98% identity with the amino acid sequence of 5, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence that has at least 99% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46;VH contains an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46.

[0114] In certain non-limiting embodiments, the multispecific molecules include: (1) A binding domain that specifically binds to LTBR containing sequence number 22; and, (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof includes any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46.

[0115] In certain non-limiting embodiments, the multispecific molecules include: (1) A binding domain that specifically binds to LTBR containing sequence number 23; and, (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof includes any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46.

[0116] In certain non-limiting embodiments, the multispecific molecules include: (1) A binding domain that specifically binds to LTBR containing sequence number 25; and, (2) The second binding domain that specifically binds to EDB includes, for example, an L19 antibody or a fragment or derivative thereof, which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof includes any of the following: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46.

[0117] In certain non-limiting embodiments, the multispecific binding molecule includes any of the following: (a)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 1, which forms a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO: 2, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA14121), which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO: 5; or (b) A first heavy chain containing the amino acid sequence of SEQ ID NO: 9, which forms a binding domain with (i) a first light chain containing the amino acid sequence of SEQ ID NO: 10, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA14122), which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO: 5.

[0118] In certain further non-limiting embodiments, the multispecific binding molecule includes any of the following: (c)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 30, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA1480); or (d)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 31, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with the light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain together with the light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA1481); or (e)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 32, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA1482), in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (f)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 33, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with the light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA1483), in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with the light chain containing the amino acid sequence of SEQ ID NO: 5; or (g)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 34, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA14107), in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (h)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 35, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4 (a multispecific binding molecule referred to as COVA14108), in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (j)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, in which the heavy chain portion (SEQ ID NO: 3) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA14133); or (k)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 39, in which the heavy chain portion (SEQ ID NO: 3) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA14174); or (l)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 56, in which the heavy chain portion (SEQ ID NO: 84) forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule referred to as COVA1456).

[0119] In some embodiments, the multispecific molecule comprises (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5.

[0120] In some embodiments, multispecific (e.g., bispecific) molecules induce NF-κB signaling in the presence of EDB that is at least twice, at least three times, and for example at least four times greater than NF-κB signaling induced in the absence of EDB (under the same conditions). In some cases, the assay is an NF-κB luciferase reporter assay. The NF-κB luciferase reporter assay may be carried out using the protocol of Example 2.

[0121] In some embodiments, multispecific (e.g., bispecific) molecules induce cell surface ICAM-1 expression in the presence of EDB at least twice, at least three times, and for example at least four times greater than ICAM-1 expression induced in the absence of EDB (under the same conditions). In some cases, the assay is an in vitro LTBR activation assay, such as the A375 / WI38A sublineage 2RA co-culture cell assay. The A375 / WI38A sublineage 2RA co-culture cell assay may be performed using the protocol of Example 3.

[0122] In certain embodiments, the heavy and light chains are humanized.

[0123] In some embodiments, the bispecific antibodies of the present invention include diabodies, crossbodies, scFv, duobody, spFv, or bispecific antibodies obtained by controlled Fab arm exchange, such as those described in the present invention.

[0124] In some embodiments, bispecific antibodies include IgG-like molecules having complementary CH3 domains that force heterodimerization, recombinant IgG-like bitargeting molecules (where each of the two sides of the molecule contains Fab fragments or portions of Fab fragments of at least two different antibodies), IgG fusion molecules (where a full-length IgG antibody is fused to an extra Fab fragment or portion of a Fab fragment), Fc fusion molecules (where a single-chain Fv molecule or stabilized diabody is fused to a heavy chain constant domain, Fc region, or portion thereof), Fab fusion molecules (where different Fab fragments are fused together), ScFv and diabody bases and heavy chain antibodies (e.g., domain antibodies, nanobodies) (where different single-chain Fv molecules, different diabodies, or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule).

[0125] In some embodiments, IgG-like molecules having complementary CH3 domain molecules include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche) and electrostatically modified versions (Amgen), LUZ-Y (Genentech), strand-exchanged and manipulated domain bodies (SEEDbody) (EMD Serono), Biclonic (Merus), or DuoBody (Genmab A / S).

[0126] In some embodiments, recombinant IgG-like dual-targeting molecules include Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), or CovX-body (CovX / Pfizer).

[0127] In some embodiments, the IgG fusion molecule may be Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ), and BsAb (Zymogenetics), HERCULES (Biogen Idec), or TvAb (Roche).

[0128] In some embodiments, the Fc fusion molecule may include ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), or Dual(ScFv)2-Fab (National Research Center for Antibody Medicine--China).

[0129] In some embodiments, examples of Fab-fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), or Fab-Fv (UCB-Celltech). Examples of ScFv-, diabody-based, and domain antibodies include, but are not limited to, Bispecific T Cell Engager (BITE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack), or COMBODY (Epigen Biotech), dual-targeting nanobody (Ablynx), and dual-targeting heavy chain only domain antibody.

[0130] The full-length bispecific antibodies of the present invention can be generated, for example, by using Fab arm exchange (or half-body exchange) between two monospecific bivalent antibodies, in vitro, either in a cell-free environment or under co-expression, by introducing substitutions at the heavy chain CH3 interface in each half to favor heterodimer formation of two antibody halves with distinct specificities. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-association of the CH3 domain. The heavy chain disulfide bond in the hinge region of the monospecific parent antibody is reduced. The resulting free cysteine ​​from one of the parent monospecific antibodies forms an intra-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while simultaneously, the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm can be manipulated to favor heterodimer formation over homodimer formation. The resulting product is a bispecific antibody having two Fab arms or halves, each binding to a distinct epitope, namely the epitope in LTBR and the epitope in fibronectin EDB.

[0131] As used herein, “homodimerization” refers to the interaction of two heavy chains having the same CH3 amino acid sequence. As used herein, “homodimer” refers to an antibody having two heavy chains having the same CH3 amino acid sequence.

[0132] As used herein, “heterodimerization” refers to the interaction of two heavy chains having non-identical CH3 amino acid sequences. As used herein, “heterodimer” refers to an antibody having two heavy chains having non-identical CH3 amino acid sequences.

[0133] Full-length bispecific antibodies can be generated using the "knob-in-hole" strategy (see, for example, International Publication No. 2006 / 028936). Briefly, selected amino acids forming the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (the hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (the knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of preferential interaction between the heavy chain containing the "hole" and the heavy chain containing the "knob". Exemplary CH3 substitution pairs that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, or T366W / T366S_L368A_Y407V (expressed using Kabat numbering as modification position in the first CH3 domain of the first heavy chain / modification position in the second CH3 domain of the second heavy chain).

[0134] Other strategies, such as promoting heavy chain heterodimer formation by using electrostatic interactions through the substitution of a positively charged residue on one CH3 surface and a negatively charged residue on a second CH3 surface, may be used, as described, for example, in U.S. Patent Application Publication 2010 / 0015133, U.S. Patent Application Publication 2009 / 0182127, U.S. Patent Application Publication 2010 / 028637, or U.S. Patent Application Publication 2011 / 0123532. In other strategies, heterodimer formation is achieved by substitutions such as those described in U.S. Patent Application Publication 2012 / 0149876 or U.S. Patent Application Publication 2013 / 0195849: L351Y_F405A Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A This can be facilitated by Y407V / T350V_T366L_K392L_T394W (expressed as modification position in the first CH3 domain of the first heavy chain / modification position in the second CH3 domain of the second heavy chain).

[0135] In addition to the above method, the bispecific antibodies of the present invention can be generated in vitro in a cell-free environment by introducing asymmetric mutations into the CH3 region of two monospecific homodimer antibodies and forming a bispecific heterodimer antibody from the two parent monospecific homodimer antibodies under reducing conditions that isomerize the disulfide bond, according to the method described in International Publication No. 2011 / 131746. In this method, the first monospecific bivalent antibody and the second monospecific bivalent antibody are manipulated to have a specific substitution in the CH3 domain that promotes the stability of the heterodimer, but these antibodies are incubated together under reducing conditions sufficient to isomerize the disulfide bond at the cysteine ​​in the hinge region, thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions can be optimally returned to non-reducing conditions. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol at pH 5 to 8, for example pH 7.0 or pH 7.4, for at least 90 minutes may be used.

[0136] In some embodiments described herein, the immunoeffector properties of multispecific binding molecules, such as the bispecific antibodies of the present invention, can be modified, and preferably silenced, for example, by modifying Fc using techniques known to those skilled in the art. For example, Fc effector functions, such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptors, BCRs), can be provided and / or controlled by modifying the residues in Fc that are responsible for these activities. For example, see: the N297 mutation in Nose et al., "PNAS" (1983); the LALA mutation in Xu et al., "Cell Immunol.", Vol. 200 (No. 1): pp. 16-26 (2000); and the DANA mutation in Wilson et al., "Cancer Cell", Vol. 19 (No. 1): pp. 101-113 (2011); or mutations in, for example, aspartic acid (D) at position 265, asparagine (N) at position 297, and proline (P) at position 329. The numbering here is indicated by an EU index such as Kabat, for example, relative to alanine (A), to obtain so-called DANAPA variants, as detailed in International Publication No. 2019 / 068632.

[0137] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing the Fc receptor (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells, subsequently causing lysis of the target cells.

[0138] In certain embodiments, the multispecificity binding molecule of the present invention includes a chimeric bispecificity antibody.

[0139] In certain embodiments, the multispecificity binding molecule of the present invention comprises a human or humanized bispecificity antibody.

[0140] In another general aspect, the present invention relates to one or more isolated nucleic acids encoding the multispecific binding molecule of the present invention, for example, a bispecific antibody, or its antigen-binding fragment. In a non-limiting example, the heavy chain of a bispecific antibody may be encoded by one nucleic acid, and the light chain may be encoded by a second nucleic acid. In another example, the heavy and light chains of a bispecific antibody may be encoded on a single nucleic acid molecule. Considering the degeneracy of the genetic code, it will be understood by those skilled in the art that the coding sequence of a protein can be altered (e.g., by substitution, deletion, insertion, etc.) without altering the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that the nucleic acid sequence encoding the monoclonal antibody and / or bispecific antibody of the present invention can be altered without altering the amino acid sequence of the protein. In addition, one or more nucleic acids of the present invention may be isolated nucleic acids. Accordingly, the present invention relates to any nucleic acid molecule or combination of nucleic acid molecules encoding the molecule of the present invention.

[0141] In another general aspect, the present invention relates to one or more vectors comprising one or more nucleic acids of the present invention. In view of the present disclosure, any vector known to those skilled in the art, such as plasmids, cosmids, phage vectors, or viral vectors, can be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements for establishing the conventional function of an expression vector, such as a promoter, a ribosome-binding element, a terminator, an enhancer, a selection marker, and / or an origin of replication. The promoter may be constitutively expressed, inducible, or reconfigurable. Numerous expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to generate antibodies or their antigen-binding fragments in cells. Recombinant expression vectors according to embodiments of the present invention can be generated using conventional cloning techniques or artificial gene synthesis methods. Such techniques are well known to those skilled in the art from the perspective of the present disclosure.

[0142] In another general aspect, the present invention relates to a host cell comprising one or more vectors comprising one or more nucleic acids encoding a multispecific binding molecule, such as the bispecific antibody or its antigen-binding fragment. In consideration of this disclosure, any host cell known to those skilled in the art can be used for recombinant expression of the bispecific antibody or its antigen-binding fragment, or other multispecific binding molecule, of the present invention. In some embodiments, the host cell is Escherichia coli TG1 or BL21 cell (e.g., in the case of expression of scFv or Fab antibody), CHO-DG44 or CHO-K1 cell, or HEK293 cell (e.g., in the case of expression of full-length IgG antibody). According to a particular embodiment, the recombinant expression vector is transformed into a host cell by a conventional method such as chemical transfection, heat shock, or electroporation, so that the recombinant nucleic acid can be stably incorporated into the host cell genome so as to be effectively expressed.

[0143] In another general aspect, the present invention relates to a method for producing multispecific binding molecules, such as bispecific antibodies or their antigen-binding fragments, as disclosed herein. The method comprises culturing cells containing nucleic acids encoding multispecific binding molecules, such as bispecific antibodies or their antigen-binding fragments, under conditions that produce multispecific binding molecules, such as bispecific antibodies or their antigen-binding fragments, as disclosed herein, and recovering the multispecific binding molecules, such as bispecific antibodies or their antigen-binding fragments, from the cells or cell culture (e.g., supernatant). The expressed multispecific binding molecules, such as bispecific antibodies or their antigen-binding fragments, can be collected from the cells and purified according to the prior art known in the art and as described herein.

[0144] Pharmaceutical composition In another general aspect, the present invention relates to a pharmaceutical composition comprising the multispecificity conjugating molecule of the present invention (e.g., a bispecific antibody or its antigen-binding fragment) and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutical composition” means a product comprising the multispecificity conjugating molecule of the present invention together with a pharmaceutically acceptable carrier. The multispecificity conjugating molecule of the present invention (e.g., a bispecific antibody) and compositions comprising the same are also useful in the manufacture of pharmaceuticals for therapeutic uses as referred herein.

[0145] As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the properties of a carrier, excipient, or diluent are determined by the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effects of the composition according to the present invention or the biological activity of the composition according to the present invention. Depending on the particular embodiment, any pharmaceutically acceptable carrier suitable for use in a pharmaceutical composition of an antibody may be used herein in consideration of this disclosure.

[0146] Formulations of pharmaceutically active ingredients having pharmaceutically acceptable carriers are known in the art, for example, as described in Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent revisions). Non-limiting examples of additional components include buffers, diluents, solvents, tonicity modifiers, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used in the formulation of the pharmaceutical composition of the present invention.

[0147] In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation containing water. The liquid formulation may include a solution, suspension, emulsion, microemulsion, gel, etc. The aqueous formulation typically contains at least 50% by weight of water, or at least 60% by weight, 70% by weight, 75% by weight, 80% by weight, 85% by weight, 90% by weight, or at least 95% by weight of water.

[0148] In one embodiment, the pharmaceutical composition may be formulated as an injectable agent that can be injected, for example, via an injection device (e.g., a syringe or infusion pump). The injection may be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreously, or intravenously.

[0149] In another embodiment, the pharmaceutical composition is a solid formulation, such as a lyophilized or spray-dried composition that can be used as is or to which a solvent and / or diluent is added by a physician or patient before use. Examples of solid dosage forms include tablets such as compressed tablets and / or coated tablets, and capsules (e.g., hard or soft gelatin capsules). The pharmaceutical composition may also be in the form of, for example, sachets, sugar-coated tablets, powders, granules, lozenges, or powders for reconstitution.

[0150] The dosage form may be immediate-release and may contain a water-soluble or water-dispersible carrier, or the dosage form may be delayed-release, sustained-release, or controlled-release and may contain a water-insoluble polymer to control the dissolution rate of the dosage form in the gastrointestinal tract or subcutaneously.

[0151] In other embodiments, the pharmaceutical composition may be delivered into the nasal cavity, into the mouth, or under the tongue.

[0152] The pH of the aqueous formulation can range from pH 3 to pH 10. In one embodiment of the present invention, the pH of the formulation is approximately 7.0 to approximately 9.5. In another embodiment of the present invention, the pH of the formulation is approximately 3.0 to approximately 7.0.

[0153] In certain embodiments, the pharmaceutical composition includes a buffer. Non-limiting examples of buffers include arginine, aspartic acid, bicine, citrate, disodium monohydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, or tris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffers may be present individually or in whole at concentrations ranging from about 0.01 mg / mL to about 50 mg / mL, for example, from about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition containing one of each of these specific buffers constitutes an alternative embodiment of the present invention.

[0154] In certain embodiments, the pharmaceutical composition includes a preservative. Non-limiting examples of preservatives include benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidourea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. The preservatives may be present individually or in whole at concentrations ranging from about 0.01 mg / mL to about 50 mg / mL, for example, from about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition containing one of each of these specific preservatives constitutes an alternative embodiment of the present invention.

[0155] In certain embodiments, the pharmaceutical composition includes an isotonic agent. Non-limiting examples of isotonic agents include salts (such as sodium chloride), amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, or threonine), algitols (such as glycerol, 1,2-propanediol, propylene glycol, 1,3-propanediol, or 1,3-butanediol), polyethylene glycol (e.g., PEG400), and mixtures thereof. Another example of an isotonic agent is sugar. Non-limiting examples of sugars may include monosaccharides, disaccharides, or polysaccharides, or water-soluble glucans, such as fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch, or sodium carboxymethylcellulose. Another example of isotonic agents is sugar alcohols, the term “sugar alcohol” is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, or arabitol. Isotonic agents may be present individually or in whole at concentrations ranging from about 0.01 mg / mL to about 50 mg / mL, for example, from about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition containing one of each of these specific isotonic agents constitutes an alternative embodiment of the present invention.

[0156] In certain embodiments, the pharmaceutical composition includes a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA) salts, and mixtures thereof. The chelating agents may be present individually or in combination at concentrations ranging from about 0.01 mg / mL to about 50 mg / mL, for example, from about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition containing one of each of these specific chelating agents constitutes an alternative embodiment of the present invention.

[0157] In certain embodiments, the pharmaceutical composition includes a stabilizer. Non-limiting examples of stabilizers include one or more flocculation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0158] In certain embodiments, the pharmaceutical composition includes a stabilizer, which is carboxy- / hydroxycellulose and their derivatives (such as HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances (e.g., monothioglycerol), or thioglycolic acid. The stabilizer may be present individually or in whole at concentrations ranging from about 0.01 mg / mL to about 50 mg / mL, for example, from about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition containing one of each of these specific stabilizers constitutes an alternative embodiment of the present invention.

[0159] In certain embodiments, the pharmaceutical composition comprises one or more surfactants. The term "surfactant" refers to any molecule or ion composed of a water-soluble (hydrophilic) part and a lipid-soluble (lipophilic) part. Surfactants may be selected from the group consisting of, for example, anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitter surfactants. Surfactants may be present individually or collectively at concentrations ranging from about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions comprising one of each of these specific surfactants constitute alternative embodiments of the present invention.

[0160] In certain embodiments, the pharmaceutical composition comprises one or more protease inhibitors, such as EDTA and / or benzamidine hydrochloride (HCl). The protease inhibitors may be present individually or collectively at concentrations ranging from about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition comprising each of these specific protease inhibitors constitutes an alternative embodiment of the present invention.

[0161] In another general aspect, the present invention relates to a method for producing a pharmaceutical composition comprising a multispecific binding molecule, such as a bispecific antibody or an antigen-binding fragment thereof, comprising combining the multispecific binding molecule, such as a bispecific antibody or an antigen-binding fragment thereof, with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0162] How to use In another general aspect, the present invention relates to a method for targeting LTBRs on cells present in a tumor (e.g., tumor cells, fibroblasts, monocytes, etc.), the method comprising exposing cells present in the tumor to the multispecific binding molecule or pharmaceutical composition of the present invention.

[0163] The functional activity of multispecific binding molecules that bind to LTBR and / or EDB (e.g., bispecific antibodies and their antigen-binding fragments) can be characterized by methods known in the art and as described herein. Methods for characterizing multispecific binding molecules that bind to LTBR and / or EDB include, but are not limited to, affinity and specificity assays including Biacore, ELISA, and / or OctetRed analysis; and binding assays for detecting the binding of multispecific binding molecules to LTBR on cancer cells and other cells by FACS. According to certain embodiments, methods for characterizing multispecific binding molecules that bind to LTBR and / or EDB are described below.

[0164] In another general aspect, the present invention relates to a method for establishing a pro-inflammatory tumor microenvironment. The method comprises contacting LTBR-expressing cells in the tumor microenvironment with the multispecific binding molecules of the present invention, wherein contact of LTBR-expressing cells with the multispecific binding molecules results in the secretion of pro-inflammatory chemokines and cytokines, as well as the expression of adhesion molecules on the cell surface.

[0165] In another general aspect, the present invention relates to a method for treating cancer in a subject requiring such treatment, comprising administering to the subject a multispecific binding molecule of the present invention (e.g., a bispecific antibody or its antigen-binding fragment) that specifically binds to fibronectin LTBR and EDB, or a pharmaceutical composition disclosed herein. The cancer is preferably an EDB-expressing cancer. The cancer may be, for example, an LTBR-expressing cancer. Cancers can be selected from the group consisting of, for example, prostate cancer, lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0166] According to embodiments of the present invention, the pharmaceutical composition comprises an effective amount of an anti-LTBR multispecific binding molecule (e.g., an anti-LTBR / anti-EDB bispecific antibody or its antigen-binding fragment). As used herein, the term "effective amount" refers to the amount of an active ingredient or component that elicits a desired biological or pharmaceutical response in a subject.

[0167] According to a particular embodiment, an effective dose refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, disorder, or condition being treated or the symptoms associated therewith; (ii) shortening the duration of the disease, disorder, or condition being treated or the symptoms associated therewith; (iii) preventing the progression of the disease, disorder, or condition being treated or the symptoms associated therewith; (iv) causing regression of the disease, disorder, or condition being treated or the symptoms associated therewith; (v) preventing the progression or onset of the disease, disorder, or condition being treated or the symptoms associated therewith. (vi) preventing recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reducing hospitalizations of subjects with the disease, disorder or condition being treated, or symptoms associated therewith; (viii) shortening the length of hospitalization of subjects with the disease, disorder or condition being treated, or symptoms associated therewith; (ix) increasing the survival rate of subjects with the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibiting or reducing the disease, disorder or condition being treated, or symptoms associated therewith; and / or (xii) enhancing or improving the preventive or therapeutic effect of another treatment.

[0168] In some embodiments, the effective amount of the multispecific binding molecule of the present invention may be administered in doses ranging from about 0.1 mg / kg to about 25 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 15 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 5 mg / kg.

[0169] The effective dose or dosage may vary depending on various factors, including the disease, disorder, or condition being treated, the means of administration, the target site, the physiological state of the subject (e.g., age, weight, and health status), whether the subject is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. The therapeutic dose is selectively escalated to optimize safety and efficacy.

[0170] In certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to the subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration. In some embodiments, the compositions disclosed herein can be administered to the subject by various routes, such as topical, oral, or parenteral. Methods of parenteral delivery include intra-arterial (directly to tissue), intramedullary, intrathecal, intraventricular, intraperitoneal, or intranasal administration.

[0171] As used herein, the terms “treat,” “treating,” and “treatment” all refer to the improvement or restoration of at least one measurable physical parameter related to cancer, which may or may not be recognizable in the subject. The terms “treat,” “treating,” and “treatment” may also refer to regression, prevention of progression, or at least slowing of progression of a disease, disorder, or condition. In certain embodiments, “treat,” “treating,” and “treatment” refer to the alleviation, prevention of progression, or onset of one or more symptoms associated with a disease, disorder, or condition such as a tumor or, more preferably, cancer, or to a reduction in the duration thereof. In certain embodiments, “treat,” “treating,” and “treatment” refer to the prevention of recurrence of a disease, disorder, or condition. In certain embodiments, “treat,” “treating,” and “treatment” refer to the improvement of survival in a subject having a disease, disorder, or condition. In certain embodiments, “to treat,” “to heal,” and “treatment” refer to the disappearance of a disease, disorder, or pathological condition in the subject.

[0172] According to certain embodiments, compositions for use in the treatment of cancer are provided. For the treatment of cancer, the compositions can be used in combination with other treatments, including but not limited to chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-CTLA-4 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, PD-1 / PD-L1 therapy, indoleamine-2,3-dioxygenase (IDO), anti-OX40 antibody, anti-GITR antibody, anti-CD40 antibody, anti-CD38 antibody, cytokines, oncolytic viruses, TLR agonists, STING agonists, other immunocancer drugs, anti-angiogenic agents, radiotherapy, antibody-drug conjugates (ADCs), targeted therapies, or other anticancer agents.

[0173] As used herein, the term “combined use” refers to the use of two or more therapeutic agents in relation to the administration of two or more therapeutic agents to a subject. The use of the term “combined use” is not limited to the order in which the therapeutic agents are administered to the subject. For example, the first therapeutic agent (e.g., a composition described herein) may be administered to the subject before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior) the administration of the second therapeutic agent to the subject.

[0174] Embodiment The present invention provides the following non-limiting embodiments.

[0175] Embodiment 1 is a multispecific binding molecule, and is described below: (i) A first binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), (ii) comprising a second binding domain that specifically binds to the extra domain B (EDB) of fibronectin, The multispecific binding molecule is a multispecific binding molecule that activates LTBR upon binding to EDB.

[0176] Embodiment 2 is the multispecific binding molecule described in Embodiment 1, wherein the multispecific binding molecule activates LTBR in a tumor-specific manner.

[0177] Embodiment 3 is the multispecificity binding molecule according to Embodiment 1 or 2, wherein the multispecificity binding molecule is a bispecificity antibody.

[0178] Embodiment 4 is a multispecificity binding molecule according to any one of Embodiments 1 to 3, wherein the multispecificity binding molecule comprises two antigen-binding domains.

[0179] Embodiment 5 is a multispecificity binding molecule according to any one of Embodiments 1 to 3, wherein the multispecificity binding molecule comprises three antigen-binding domains.

[0180] Embodiment 6 is the multispecific binding molecule described in Embodiment 5, wherein the three antigen-binding domains include one binding domain that specifically binds to LTBR.

[0181] Embodiment 7 is a multispecific binding molecule according to Embodiment 5 or 6, wherein the three antigen-binding domains include two binding domains that specifically bind to EDB.

[0182] Embodiment 8 is a multispecific binding molecule according to any one of Embodiments 5 to 7, wherein the binding domain that specifically binds to LTBR includes the single-chain variable domain of the antibody.

[0183] Embodiment 9 is a multispecific binding molecule according to any one of Embodiments 1 to 8, wherein the first binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH) and a light chain variable region (VL), where VH comprises heavy chain complementarity determination region 1 (HCDR1), HCDR2, and HCDR3, and VL comprises light chain complementarity determination region 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL are as follows ((i) to (viii)): (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (ii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (iii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44. For example, VH includes an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 43, and VL includes an amino acid sequence of SEQ ID NO: 44 VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43; VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence that has at least 97% identity with the amino acid sequence of SEQ ID NO: 43 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 43 VL contains an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 44, and VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44; or (v)VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48. For example, VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 48. In contrast, VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 47 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 48; VH includes an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 47 VL contains an amino acid sequence having at least 99% identity with respect to the amino acid sequence of SEQ ID NO: 48; VH contains an amino acid sequence having 100% identity with respect to the amino acid sequence of SEQ ID NO: 47; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 48; or (vi) Sequence ID 22; or (vii) Sequence ID 23; or (viii) Sequence ID 25 It is a multispecific binding molecule that contains one or more of the following:

[0184] Embodiment 10 is a multispecific binding molecule according to any one of Embodiments 1 to 9, wherein the second binding domain that specifically binds to EDB includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH includes heavy chain complementarity determination region 1 (HCDR1), HCDR2, and HCDR3, and VL includes light chain complementarity determination region 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL are as follows ((i) to (ii)): (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 46. For example, VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 46 VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence having at least 97% identity with respect to the amino acid sequence of SEQ ID NO: 45 VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 45; VL includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH includes an amino acid sequence that has at least 98% identity to the amino acid sequence of SEQ ID NO: 45 VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence having 100% identity with respect to the amino acid sequence of SEQ ID NO: 45; VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the amino acid sequence of SEQ ID NO: 46.

[0185] Embodiment 11 is a multispecific binding molecule according to any one of Embodiments 1 to 10, and [(a) to (l)]: (a)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 1, forming a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO: 2, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO: 5; or (b) A multispecific binding molecule comprising one or more of the following: (i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 9, which forms a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO: 10; and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO: 5.

[0186] Embodiment 12 is a multispecific binding molecule according to any one of Embodiments 1 to 10, and is as follows: (c)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 30, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (d)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 31, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (e)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 32, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (f)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 33, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (g)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 34, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (h)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 35, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (j)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, wherein the heavy chain portion forms a binding domain with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (k)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 39, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (l)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 56, in which the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, in which the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5. This is a multispecific binding molecule.

[0187] Embodiment 13 is a multispecific binding molecule according to any one of Embodiments 1 to 10, comprising (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, in which the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, in which the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5.

[0188] Embodiment 14 is one or more nucleic acid molecules encoding the multispecific binding molecule according to any one of Embodiments 1 to 13.

[0189] Embodiment 15 is one or more vectors comprising the one or more nucleic acid molecules according to Embodiment 14.

[0190] Embodiment 16 is an isolated host cell comprising the one or more vectors according to Embodiment 15.

[0191] Embodiment 17 is a pharmaceutical composition comprising the multispecific binding molecule according to any one of Embodiments 1 to 13 and a pharmaceutically acceptable carrier.

[0192] Embodiment 18 is a method of treating cancer in a subject in need thereof, comprising administering to the subject the multispecific binding molecule according to any one of Embodiments 1 to 14, the one or more nucleic acid molecules according to Embodiment 15, the one or more vectors according to Embodiment 16, or the pharmaceutical composition according to Embodiment 17.

[0193] Embodiment 19 is the use of the multispecific binding molecule according to any one of Embodiments 1 to 14, the one or more nucleic acid molecules according to Embodiment 15, the one or more vectors according to Embodiment 16, or the pharmaceutical composition according to Embodiment 17, for activating LTBR in tumor tissue.

[0194] Embodiment 20 is a method of producing the multispecific binding molecule according to any one of Embodiments 1 to 13, comprising expressing the one or more nucleic acid molecules according to Embodiment 14 or the one or more vectors according to Embodiment 15 in a host cell and collecting the multispecific binding molecule.

[0195] Embodiment 21 is a multispecific molecule according to any one of Embodiments 1 to 10, which induces NF-κB signaling in the presence of EDB that is at least twice, at least three times, and for example at least four times greater than the NF-κB signaling induced in the absence of EDB.

[0196] Embodiment 22 is a multispecific molecule according to any one of Embodiments 1 to 10 or 21, which induces cell surface ICAM-1 expression in the presence of EDB that is at least twice, at least three times, and for example at least four times greater than the ICAM-1 expression induced in the absence of EDB. [Examples]

[0197] Example 1: Production of EDB / LTBR bispecific antibody and control molecule The bispecific antibodies and control molecules were derived from the target binding sequences shown in Table 1, transiently expressed in serum-free / animal component-free medium in CHO suspension culture medium, and purified using Akta Pure instrument (GE Healthcare) by protein A affinity chromatography followed by preparative size exclusion chromatography (SEC) on a Superdex 200 10 / 300GL column (GE Healthcare). The heavy chain contained a knob-in-hole (KiH) mutation that promotes heterodimerization (Ridgway et al., "Protein Eng.", Vol. 9 (No. 7): 617-21 (1996); Atwell et al., "J.Mol.Biol.", Vol. 270 (No. 1): pp. 26-35 (1997); Merchant et al., "Nat.Biotechnol.", Vol. 16 (No. 7): pp. 677-81 (1998)). The antibody contained IgG1 sigma Fc, including a set of seven Fc mutations—L234A, L235A, G237A, P238S, H268A, A330S, and P331S—compared to wild-type IgG1, which reduces Fc receptor interaction (Tam et al., "Antibodies" (2017)).

[0198] Symmetric monoantibodies and bispecific antibodies were generated with IgG1 sigma mutations but without KiH mutations.

[0199] [Table 1] * The EDBmAb1 used herein (International Publication No. 9745544) is an anti-ED-B antibody tested in a clinical setting. Other antibodies that bind to ED-B or its adjacent domain have been previously described (Carnemolla et al., "Int. J. Cancer", Vol. 68, pp. 397-405 (1996)).

[0200] Protein concentration was determined by absorbance measurement at 280 nm (OD280), and the purification yield was determined. Analytical SEC was performed using a Bio SEC-5 column (Agilent, 5 μm particle size, 300 Å) in a Thermo Vanquish HPLC system. 10 μl of purified protein was packed into the column, and elution was recorded by OD280.

[0201] Table 2 outlines the structural characteristics of the bispecific antibodies and control molecules described in this embodiment. Molecules in bold are those of the present invention, while others are controls in different embodiments.

[0202] Table 3 shows the structural characteristics of another comparable bispecific antibody that targets LTBR and mesothelin (tumor-associated antigens not present in the extracellular matrix), as discussed in Comparative Example 4.

[0203] [Table 2]

[0204] [Table 3]

[0205] [Table 4] * Mutations in the Fc portion suppress binding to Protein A and facilitate purification of the heterodimers described in WO 2010 / 151792.

[0206]

Table 5

[0207] How the different constructs were generated is described below.

[0208] Asymmetric antibodies with a 1:1 stoichiometry (all IgG1 sigma; all with knob-in-hole (KiH) mutations): i. COVA14121 was generated by co-expression of the heavy chain (HC; SEQ ID NO: 1) and light chain (LC; SEQ ID NO: 2) of agonist LTBR antibody LTBRmAb1 and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of anti-EDB antibody EDBmAb1 (Figure 1L). ii. COVA14120 was generated by co-expression of the heavy chain (HC; SEQ ID NO: 1) and light chain (LC; SEQ ID NO: 2) of agonist LTBR antibody LTBRmAb1 and the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of anti-RSV antibody B21M (Figure 1K). iii. COVA14122 was generated by co-expression of the heavy chain (HC; SEQ ID NO: 9) and light chain (LC; SEQ ID NO: 10) of agonist LTBR antibody LTBRmAb2 and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of anti-EDB antibody EDBmAb1 (Figure 1M). iv. COVA14123 was generated by co-expression of the heavy chain (HC; SEQ ID NO: 9) and light chain (LC; SEQ ID NO: 10) of agonist LTBR antibody LTBRmAb2 and the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of anti-RSV antibody B21M (Figure 1N). v.COVA14124 was generated by co-expression of the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 and the heavy chain (HC; SEQ ID NO: 6) and light chain (LC; SEQ ID NO: 8) of the anti-RSV antibody B21M (Figure 1O). vi.COVA1454 was generated by co-expression of 3xhmLIGHT-Fc (SEQ ID NO: 15) with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1F). 3xhmLIGHT-Fc is a single-chain trimer LIGHT fused to the N-terminus of IgG1 sigma-Fc, engineered for better stability and human-mouse cross-reactivity (Tangr et al., "Cancer Cell", Vol. 29: pp. 285-2896 (2016)). vii.COVA1418 was generated by co-expression of 3xhmLIGHT-Fc (SEQ ID NO: 15) with the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV antibody B21M (Figure 1E). 3xhmLIGHT-Fc is a single-chain trimer LIGHT fused to the N-terminus of IgG1 sigma Fc (SEQ ID NO: 58) for improved stability and human-mouse cross-reactivity (Tangr et al., "Cancer Cell", Vol. 29: pp. 285-2896 (2016)).

[0209] Symmetric antibodies (all are IgG1 sigma and do not contain KiH mutations): viii.COVA14114 was generated by expressing an anti-RSV B21M antibody heavy chain carrying a C-terminal LTα1β2 fusion (SEQ ID NO: 18) together with the B21M antibody light chain (LC; SEQ ID NO: 8) (Figure 1G). ix.COVA14113 was generated by expressing the EDBmAb1 heavy chain carrying a C-terminal LTα1β2 fusion (SEQ ID NO: 20) together with the light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1H). x.COVA1413 was generated by co-expression of the heavy chain (HC; SEQ ID NO: 11) and light chain (LC; SEQ ID NO: 2) of the agonist LTBR antibody LTBRmAb1 (Figure 1B). xi.COVA1402 was generated by co-expression of the heavy chain (HC; SEQ ID NO: 13) and light chain (LC; SEQ ID NO: 10) of the agonist LTBR antibody LTBRmAb2 (Figure 1A). xii.COVA1440 was generated by co-expression of the heavy chain (HC; SEQ ID NO: 14) and light chain (LC; SEQ ID NO: 8) of the anti-RSV antibody B21M (Figure 1C). xiii.COVA1452 was generated by co-expression of the heavy chain (HC; SEQ ID NO: 12) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1D).

[0210] Asymmetric antibodies with a 2:1 stoichiometry (all are IgG1 sigma and all are accompanied by KiH mutations) xiv.COVA14116 was generated by co-expression of the EDBmAb1 heavy chain carrying a C-terminal LTα1β2 fusion (including SEQ ID NOs. 21 and 84) with the heavy chain (HC; SEQ ID NO. 4) and light chain (LC; SEQ ID NO. 5) of the anti-EDB antibody EDBmAb1 (Figure 1I). xv.COVA14117 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying a C-terminal LTα1β2 fusion (including SEQ ID NOs. 19 and 85) with the heavy chain (HC; SEQ ID NOs. 7) and light chain (LC; SEQ ID NOs. 8) of the anti-RSV B21M antibody (Figure 1J). xvi.COVA1484 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying an N-terminally stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 22) fusion (including SEQ ID NOs: 26 and 85) with the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV B21M antibody (Figure 1P). xvii.COVA1485 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying an N-terminally stapled scFv BHA10 (VL-VH oriented SEQ ID NO: 23) fusion (including SEQ ID NOs: 27 and 85) with the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV B21M antibody (Figure 1Q). xviii.COVA1486 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying a C-terminally stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 22) fusion (including SEQ ID NOs: 28 and 85) with the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV B21M antibody (Figure 1R). xix.COVA1487 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying a C-terminally stapled scFv BHA10 (VL-VH oriented SEQ ID NO: 23) fusion (including SEQ ID NOs: 29 and 85) with the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV B21M antibody (Figure 1S). xx.COVA1480 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying an N-terminally stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 22) fusion (including SEQ ID NOs: 30 and 84) with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1T). xxi.COVA1481 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying an N-terminally stapled scFv BHA10 (VL-VH oriented SEQ ID NO: 23) fusion (including SEQ ID NOs: 31 and 84) with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1U). xxii.COVA1482 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminal stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 22) fusion (including SEQ ID NOs: 32 and 84) with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1V). xxiii.COVA1483 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminal stapled scFv BHA10 (VL-VH oriented SEQ ID NO: 23) fusion (including SEQ ID NOs: 33 and 84) with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1W). xxiv.COVA14107 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminally stapled scFv BHA10 (VH-VL orientation, VL3 Y36F_S49Y_F87Y SEQ ID NO: 53) fusion (including SEQ ID NOs: 34 and 84) with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1X). xxv.COVA14108 was generated by co-expression of an anti-EDB antibody EDBmAb1 heavy chain carrying an anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminally stapled scFv BHA10 (VH-VL orientation, VH_CDR1_Y33A SEQ ID NO: 54) fusion (including SEQ ID NOs: 35 and 84), and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1Y). xxvi.COVA14133 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminally stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 22) fusion (including SEQ ID NO: 38 and SEQ ID NO: 3) with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1Z). xxvii.COVA14136 was generated by co-expression of the heavy chain of an anti-RSV B21M antibody carrying a C-terminally stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 22) fusion (including SEQ ID NOs: 41 and 6) with the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-EDB antibody EDBmAb1 (Figure 1A1). xxviii.COVA14174 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminal disulfide-stabilized scFv BHA10 (VH-VL oriented SEQ ID NO: 25) fusion (including SEQ ID NO: 39 and SEQ ID NO: 3) with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1A2). xxix.COVA14175 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying a C-terminal disulfide-stabilized scFv BHA10 (VH-VL oriented SEQ ID NO: 25) fusion (including SEQ ID NO: 40 and SEQ ID NO: 6) with the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV B21M antibody (Figure 1A3). xxx.COVA1456 was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminal disulfide-stabilized scFv BHA10 (VH-VL oriented SEQ ID NO: 25) fusion (including SEQ ID NOs: 56 and 84) with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 (Figure 1A4). xxxi.COVA1462 was generated by co-expression of an anti-RSV B21M antibody heavy chain carrying a C-terminal disulfide-stabilized scFv BHA10 (VH-VL oriented SEQ ID NO: 25) fusion (including SEQ ID NOs: 57 and 85) with the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV B21M antibody (Figure 1A5).

[0211] Mesothelin / LTBR bispecificity: Asymmetric antibody, 2:1 stoichiometric ratio (IgG1 sigma, associated with KiH mutations) xxxii.COVA14146 was generated by co-expression of the anti-mesothelin antibody MSLNmAb1 heavy chain carrying a C-terminal stapled scFv BHA10 (VH-VL oriented SEQ ID NO: 22) fusion (including SEQ ID NOs: 80 and 86) with the heavy chain (HC; SEQ ID NO: 81) and light chain (LC; SEQ ID NO: 82) of the anti-mesothelin antibody MSLNmAb1 (Figure 1A6 and Table 3).

[0212] result While all of the above constructs could be expressed and purified, surprisingly, the LIGHT and LTα1β2-containing constructs (COVA1418, COVA1454, COVA14113, COVA14114, COVA14116, and COVA14117; Table 2) showed a purification yield up to 10 times lower compared to the EDB / LTBR bispecific constructs containing stapled scFv derived from the agonist anti-LTBR antibody (e.g., COVA1482 and COVA14133; see Table 4). Furthermore, the LIGHT-containing construct (e.g., COVA1454) showed a tendency for decreasing monomer content, as can be seen from the size exclusion chromatograms shown in Figure 2 and Table 4. In summary, these facts (up to 10 times higher purification yield and higher monomer content) indicate that the bispecific constructs of the present invention may have better biophysical properties than constructs containing LIGHT or LTα1β2-Fc fusion.

[0213] [Table 6]

[0214] Example 2: EDB-dependent in vitro LTBR-activated NF-κB luciferase reporter assay To demonstrate that the EDB / LTBR bispecific compound can activate LTBR in an EDB-dependent manner, the compound's activity was tested in an A549 cell NF-κB luciferase reporter assay in the presence or absence of EDB-containing fibronectin (EDB + fibronectin). NF-κB signaling plays a central role in regulating cell development and immunohomeostasis. Activation of NF-κB via tumor necrosis factor receptor (TNFR) or TNFR superfamily members (e.g., LTBR) occurs in conjunction with their respective ligands. The A549 lung epithelial cell line spontaneously expresses LTBR, ​​and the NF-κB luciferase reporter construct is stably integrated into the genome of the A549 lung epithelial cell line. Following activation by the stimulant, the endogenous NF-κB transcription factor binds to DNA response elements to induce transcription of the luciferase gene.

[0215] To demonstrate EDB-dependent activation of LTBR, ​​high-binding 96-well μClear flat-bottom plates (Greiner;Monroe, NC) were coated overnight with 150 ng / well of human recombinant EDB+ fibronectin domain 7-B-8-9 (EDB, SEQ ID NO: 51) or 150 ng / well of human recombinant fibronectin domain 7-8-9 (EDB-, SEQ ID NO: 52).

[0216] After incubation overnight, the coated plates were washed with PBS and blocked in assay medium (DMEM + 10% thermally inactivated FBS) at 37°C for 2 hours. A 1:5 dilution series of the compound to be tested was prepared in assay medium as 2x concentration stocks (the final concentrations tested ranged from 200 nM to 2.6 pM). After removing the blocking solution by aspirate, 50 μL of the diluted compound was added to the pre-blocked plates. 50 μL of A549 cell suspension (cell suspension concentration = 0.4 MiO cells / ml assay medium) was added to each well (20,000 cells / well). A549 cells were pre-separated from the cell culture flask using Accutase / EDTA and then transferred to assay medium. The cells were incubated with the compound at 37°C / 5% CO2 for 18–20 hours.

[0217] After 18 hours of incubation, luciferase activity was detected using the Bio-Glo® luciferase assay system (Promega, Madison, WI). Luminescence was measured using a Tecan M 1000Pro instrument with an integration time of 500 milliseconds. From the obtained relative light units (RLU), the induction factor of LTBR signaling was calculated as follows: Induction Factor = RLU 刺激済み cells / average RLU 非刺激 Cells (non-stimulated cells were included as a control in each plate being tested).

[0218] Dose-response curves, including standard deviations, were plotted using GraphPad Prism, and nonlinear fitting was applied where applicable (logarithmic (agonist) versus response (variable gradient - 3 parameters)). To fit the data, x values ​​(compound concentration) were transformed using the x=Log(x) function in GraphPad Prism.

[0219] result Antibody-LIGHT fusion Similar to the study published by Tang et al. ("Cancer Cell", Vol. 29: pp. 285-2896 (2016)) which described the bispecific molecule anti-EGFR-LIGHT fusion as having antitumor activity, a bispecific molecule consisting of COVA1454, one EDB-binding arm, and one LIGHT trimer-Fc fusion (Figure 1F and Table 2) was designed, expressed, and tested in an A549 cell NF-κB luciferase reporter assay in the presence or absence of EDB-containing fibronectin. The activity of COVA1454 was tested using soluble recombinant human LIGHT (catalog number 664-LI-025 / CF; R&D This was compared with Lukashev et al., "Cancer," (Lukashev et al., "Cancer"), and untargeted LIGHT (COVA1418; Figure 1E and Table 2). Figure 3A shows that in the presence of EDB-containing fibronectin, COVA1454 activates LTBR only slightly more than untargeted LIGHT (COVA1418) or soluble recombinant human LIGHT. Interestingly, Figure 3B shows that in the absence of EDB-containing fibronectin, COVA1454, COVA1418, and soluble recombinant LIGHT activate LTBR to the same or nearly the same extent as in the presence of EDB (Figure 3A). These findings, along with the widespread expression of LTBR in normal tissues (Lukashev et al., "Cancer"), are relevant. "Res.", Vol. 66 (No. 19): pp. 9617-9624 (2006), shows that antibody LIGHT fusion is not suitable for achieving tumor-specific activation of LTBR. In fact, activation of LTBR in normal tissue may lead to unwanted off-tumor toxicity.

[0220] Antibody-LTα1β2 fusion Next, LTα1β2 antibody fusions containing one or two LTα1β2 moieties fused to the anti-EDB antibody EDBmAb1 were generated (Figures 1G-1J and Table 2) and tested using a reporter assay.

[0221] These constructs were designed similarly to those used in the study by Gurney et al., which reported in vitro and in vivo studies using a bispecific fusion construct consisting of a heterotrimeric single-chain LTα1β2 moiety fused to a B7-H4 specific tumor-targeting antibody (International Publication No. 2018 / 119118). Importantly, unlike LIGHT used in the previous section, the LTα1β2 fusion construct is a specific agonist of LTBR and does not activate HVEM.

[0222] Figures 3C and 3D show the results obtained with the fusion of two LTα1β2 molecules to COVA14113, the EDBmAb1 antibody (Figure 1H and Table 2); the fusion of one LTα1β2 molecule to COVA14116, the EDBmAb1 antibody (Figure 1I and Table 2) compared to COVA14114; the fusion of two LTα1β2 molecules to the isotype control antibody B21M (Figure 1G and Table 2), which acts as a non-targeted LTα1β2 control, soluble recombinant human LIGHT, and soluble LTα1β2 (recombinant human lynphotoxin α1β2; catalog number 8884-LY / CF; R&D Systems). In the presence of EDB (Figure 3C), both COVA14113 and COVA14116 achieved more potent LTBR activation than the soluble intrinsic ligands LIGHT and LTα1β2, while the untargeted LTα1β2 control COVA14114 showed activation levels comparable to COVA14113 and COVA14116. In the absence of EDB (Figure 3D), the activity of COVA14113 and COVA14114 (carrying two LTα1β2 moieties) remained unchanged, while the activity of COVA14116 (carrying one LTα1β2 moiety) decreased to a level slightly below the activation achieved by soluble LTα1β2. These data indicate that tumor antigen-dependent activation of LTBR is extremely difficult to achieve with such antibody-LTα1β2 constructs. In fact, the activation levels achieved in the absence of EDB-containing fibronectin (Figure 3D) could be problematic due to the widespread expression of LTBR in normal tissues (Lukashev et al., "Cancer Res.", Vol. 66 (No. 19): pp. 9617-9624 (2006).

[0223] Bispecific antibody based on agonist anti-LTBR antibody To achieve tumor antigen-dependent activation of LTBR, ​​the inventors initiated the production of a bispecific antibody (1:1 heterodimer; the inventors included a KiH mutation in the Fc region to promote correct pairing) consisting of an anti-EDB antibody EDBmAb1 and anti-LTBR agonist antibodies LTBRmAb1 and LTBRmAb2 (Figures 1K to 1M), with the aim of activating LTBR only upon binding to a tumor antigen (a tumor antigen present in the extracellular matrix, which is the EDB of fibronectin). A corresponding control antibody consisting of an isotype control antibody B21M paired with LTBRmAb1 and LTBRmAb2 was also produced (Figures 1N to 1O).

[0224] Figures 4A and 4C show that COVA14121 (1:1 heterodimer EDBmAb1 and LTBRmAb1; Figure 1L and Table 2) and COVA14122 (1:1 heterodimer EDBmAb1 and LTBRmAb2; Figure 1M and Table 2) were able to activate LTBR in an EDB-dependent manner. In contrast to the molecules described above, in the above examples shown in Figures 4B and 4D, COVA14121 and COVA14122 demonstrated only minimal LTBR activation in the absence of EDB. This residual activity may have been due to residual impurities in the purified material. Table 5 shows a comparison of the maximum induction ratios (of NF-κB signaling, in the presence or absence of ED-B-containing fibronectin) obtained using heterodimers COVA14121 and COVA14122, or LIGHT-(COVA1454) or LTα1β2- antibody fusions (COVA14113 and COVA14116). The comparison clearly shows that the use of agonist antibodies makes the LTBR bispecific molecule more specific. In fact, the ratio of the maximum induction ratio achieved in the presence or absence of ED-B is in the range of 4.4 to 5.4 for COVA14121 and COVA14122, while it is in the range of 1.1 to 1.6 for ligand antibody fusions. This demonstrates that these ligand antibody fusions do not achieve specific TAA-dependent LTBR activation, in contrast to the bispecific antibodies of the present invention.

[0225] [Table 7]

[0226] In summary, these results suggest that it is possible to activate LTBR in a tumor-dependent manner using bispecific antibodies based on agonist LTBR antibodies, minimizing or completely eliminating activation in the absence of tumor antigens. With such bispecific molecules, the LTBR-binding antibody activates LTBR only upon binding to the tumor antigen, in this case, EDB-containing fibronectin.

[0227] To further enhance tumor antigen-dependent LTBR activation, a 2:1 bispecific format was designed, featuring two binding sites to EDB (to increase antigen-mediated clustering) or two nonspecific binding sites, and one binding site to LTBR (see Figures 1P-1W and 1A2-1A5). For the LTBR binding site, scFv fragments were used. Because scFv fragments had potential stability issues, two different methods were employed to stabilize them: scFv fragments derived from LTBRmAb1 were stabilized using either a further disulfide bond between VH and VL (Reiter et al., "Nat Biotechnol.", Vol. 14 (No. 10): pp. 1239-1245 (1996)), or a stapled scFv platform (VH-VL; VL-VH), and then fused to either EDBmAb1 or B21M (isotype control antibody) via a (G4S)3 linker.

[0228] Figure 5A demonstrates that the 2:1 bispecific EDB / LTBR antibody COVA1456 (Figure 1A4 and Table 2) potently activated LTBR, ​​while the control bispecific antibody COVA1462 (Figure 1A5 and Table 2) failed to activate LTBR. This indicates that clustering via binding to TAA (in this case, immobilized EDB-containing fibronectin) was a prerequisite for potent LTBR activation by the 2:1 bispecific antibody COVA1456. In the absence of EDB-containing fibronectin (Figure 5B), COVA1456 failed to activate LTBR, ​​supporting the fact that the presence of EDB is essential for LTBR activation, and that tumor-specific activation of LTBR was achieved by bispecific antibodies targeting LTBR and EDB in the extracellular matrix.

[0229] To demonstrate that the ability to activate LTBR in a TAA-dependent manner was not an inherent characteristic of the disulfide-stabilized scFv derived from LTBRmAb1 used to construct COVA1456, COVA1456 was compared to COVA1482 in the same A549 NF-κB reporter assay. COVA1482 differs from COVA1456 only in the stabilization method used for the scFv. The scFv in COVA1482, also derived from LTBRmAb1, was stabilized using a stapled platform. Figure 5C shows that both COVA1482 and COVA1456 potently activated LTBR in an EDB-dependent manner. The corresponding isotype controls COVA1486 and COVA1462 did not activate LTBR (Figure 5C). These results suggest that the method used to stabilize the scFv fragment did not affect its bispecific ability to activate LTBR in a TAA-dependent manner. Surprisingly, 2:1 bispecific EDB / LTBR antibodies (COVA1482 or COVA1456) showed increased potency in inducing NF-κB signaling in this reporter assay. The mean EC2 was calculated for COVA1482 across several assays in the same experimental setup. 50 The EC2 concentration was approximately 30 pM ± 10 pM, and COVA14121 (1:1 heterodimer) had an EC2 concentration of approximately 3 nM in the assay shown in Figure 4A. 50 This demonstrates that 2:1 bispecificity can be 100 times stronger than 1:1 bispecificity. This can be explained by the increased clustering of LTBR binding sites achieved by the two binding sites to TAA.

[0230] To study the effect of affinity to LTBR on the ability of such a bispecific compound to activate LTBR in a TAA-dependent manner, lower affinity variants of the scFv fragment derived from LTBRmAb1 (SEQ ID NO: 53, KD ≈ 60 nM and SEQ ID NO: 54, KD ≈ 600 nM) were generated and used to construct 2:1 bispecific compounds (COVA14107, Figure 1X and Table 2; and COVA14108, Figure 1Y and Table 2). The resulting bispecific compounds were tested using the A549 NF-κB reporter assay to confirm the effect of affinity on LTBR activation. Figure 5D shows that lower affinity to LTBR corresponded to a decrease in the bispecific compound's ability to activate LTBR in a TAA-dependent manner in this assay.

[0231] As described in Example 1, mutations that suppress binding to protein A (used for antibody purification) (International Publication No. 2010 / 151792) were introduced into the Fc region of several constructs to facilitate the purification of the desired heterodimer. COVA14133 was generated without these mutations, and its activity, compared to COVA 1482, showed that the mutations in the Fc region did not affect the activity of the bispecific compound. COVA14133 and COVA1482, as well as their respective isotype controls COVA14136 and COVA1486, were compared in the A549 NF-κB reporter assay. Figure 5E shows that COVA14133 activates LTBR in a TAA-dependent manner with similar efficiency to COVA1482, demonstrating that the mutations in the Fc region did not affect the bispecific ability to activate LTBR.

[0232] In the presence of EDB (Figure 5F), both COVA14133 (2:1 EDBmAb1×LTBR mAb1) and COVA14116 (2:1 EDBmAb1×LTα1β2) achieved potent LTBR activation. No LTBR activation was observed with the untargeted isotype control molecule COVA14136 (2:1 B21M×LTBR mAb1), but the untargeted LTα1β2 control COVA14117 (2:1 B21M×LTα1β2) showed activation independently of TAA binding. In the absence of EDB (Figure 5G), LTBR activation could not be detected by COVA14133 or its isotype control molecule COVA14136. In contrast, when TAA-independent activation of LTBR by COVA14116 and COVA14117 was measured in the absence of EDB, it was shown that achieving tumor antigen-dependent activation of LTBR with such antibody-LTα1β2 constructs is extremely difficult.

[0233] In conclusion, COVA14133 demonstrated excellent ability to activate LTBR in a TAA-dependent manner.

[0234] Example 3: EDB-dependent in vitro LTBR activation-A375 / WI38VA sub-lineage 2RA co-culture cell assay To investigate whether activation of LTBR in the presence of EDB + fibronectin (produced and deposited in the extracellular matrix by WI38VA cells (Zardi, L. et al., "EMBO J", Vol. 6, pp. 2337-2342 (1987)) results in the release of cytokines and chemokines, as well as the upregulation of the adhesion molecule ICAM-1 on A375 cells), an A375 / WI38VA sublineage 2RA co-culture assay was performed. WI38VA sublineage 2RA (ATCC® CCL75.1®) cells were seeded at a density of 5000 cells / well in 96-well plates and cultured at 37°C / 5% CO2 in growth medium (MEM). The cells were incubated for 48 hours in (w / o glutamine + 10% heat-inactivated FBS + 0.1 mM NEAA + 2 mM L-Gln + 1 mM sodium pyruvate). Three 1:5 dilution series of the compounds to be tested were prepared as 2-fold stocks in assay medium (DMEM + 10% heat-inactivated FBS) (the final concentrations tested ranged from 40 nM to 0.5 pM). Prior to incubation in co-culture with WI38VA sublineage 2RA cells, A375 cells (ATCC® CRL-1619®) were labeled with CellTrace violet (CTV, Invitrogen, Carlsbad, CA). For labeling, 10 × 10⁶ cells were used in 5% FBS in PBS. 6 Cell suspensions with a concentration of cells / mL and 2.5 μM CTV were incubated at room temperature for 5 minutes while protected from light. Then the cells were washed and 0.4 × 10⁶ cells were removed. 6 The cells were resuspended in assay medium at a density of cells / mL. The culture medium was carefully removed from the plate containing the 48-hour WI38VA sub-lineage 2RA culture, and then 50 μL of A375 cell suspension (20,000 cells / well, CTV+ or CTV-) was added to each well. 50 μL of serially diluted compound (final volume 100 μL per well) was added to the cells, and the mixture was incubated at 37°C / 5% CO2 for 24 hours.

[0235] After incubation for 24 or 72 hours, the supernatant was removed by centrifugation and stored for measurement of cytokines and chemokines using the MSD assay, or for use in the PBMC migration assay (24-hour incubation, Example 5). The cells were further processed by flow cytometry for ICAM-1 measurement (24-hour incubation).

[0236] Detection of ICAM-1 by flow cytometry All remaining culture medium in the 96-well plate was carefully removed, cells were isolated with Accutase and transferred to a DeepWell 96-well plate (pooled in one well in three different ways), washed, resuspended in 100 μL of FACS buffer (PBS + 1% FBS + 0.1% NaN3), and transferred to a round-bottom 96-well plate. Antibodies, i.e., labeled anti-human ICAM-1 PE (clone 1H4, Thermo;Waltham, MA) or labeled isotype control antibody PE (MPC-11, BioLegend, San Diego, CA) and LIVE / DEAD fixable near-IR staining (Invitrogen), single staining or combination staining were diluted as shown in Table 6.

[0237] [Table 8]

[0238] Cells were centrifuged at 400×g for 4 minutes at 4°C, the supernatant was discarded, and 50 μL of antibody solution was prepared as shown in Table 6. Cells and antibody were incubated in the dark at 4°C for 30 minutes. After incubation, 120 μL was added to each well, and then the cells were centrifuged at 400×g for 4 minutes at 4°C. The cells were washed once with FACS buffer, centrifuged, and resuspended in 90 μL of FACS buffer. The cells were then fixed by adding 90 μL of 3.7% formalin solution in PBS and incubated on ice in the dark for 15 minutes. After fixation, the cells were centrifuged at 400×g for 4 minutes at 4°C and resuspended in 100 μL of FACS buffer. Cells were measured at high flow rate using a MACS Quant instrument in screen mode, and 49 μL / well was obtained. The data were analyzed using Flowlogics Software (version 700.2A) and plotted in Graphpad Prism.

[0239] Cytokine measurement of supernatant from treated cells using the MSD platform Several cytokines known to be regulated by NF-κB signaling were measured using the MSD platform and multiplex MSD plates. Some examples of the measured cytokines are listed here. ■RANTES: R-Plex antibody set using human RANTES (MSD) ■Using I-TAC, IP-10, and MIP-3b:3-PLEX cytokine release assay (MSD), ■IL-8, IP-10, MIP-3b:3-PLEX cytokine release assay (MSD) was used, and ■IL-12p70, IL-6, TNF-α, MIP-3α, SDF-1α: 5-PLEX cytokine release assay (MSD) was used.

[0240] The cytokine concentrations in the supernatant of treated cells were measured using the MSD platform according to the manufacturer's instructions. Briefly, the protocol involved the following steps: (1) Plate preparation involved coating the provided plates with linker-bound capture antibodies. The plates were incubated overnight at 2–8°C with shaking. The following day, the plates were washed with PBST (PBS + 0.05% Tween-20) using a plate washer (Biotek; Winooski, VT). (2) Calibration standards and detection antibody solutions were prepared. (3) Depending on the availability of the material, the supernatant was diluted to 1:3 or 1:5. The supernatant was measured after incubation for 24 hours or 72 hours (for I-TAC, MIP-3a, and TNFα).

[0241] Assay protocol: Step 1: The sample or calibration standard was added to the plate, and the plate was incubated at room temperature for 1 hour while shaking. Step 2: Wash the plate and add the detection antibody. Incubate the plate at room temperature for 1 hour with shaking. Step 3: The plate was washed and 2x read buffer T was added. The plate was analyzed using an MSD instrument. -The data was analyzed using Mesoscale software (MSD Exploration Workbench Program v 4.0.12.1). Dose-response curves, including standard deviations from three different approaches, were plotted using GraphPad Prism, and nonlinear fitting was applied where applicable (logarithmic (agonist) vs. response (variable gradient - 4 parameters)). To fit the data, the x-values ​​(compound concentration) were transformed using the x=Log(x) function in GraphPad Prism.

[0242] Results - Detection of ICAM-1 by flow cytometry It had been previously shown that NF-κB signaling may lead to the upregulation of ICAM-1 on the cell surface (da Silva Antunes et al., "Front Immunol", Vol. 9, p. 576 (2018)). Therefore, we measured the level of ICAM-1 expression on the surface of A375 cells after co-culture incubation with EDB / LTBR bispecific molecules. As an example, Figure 6 shows the upregulation of ICAM-1 after incubation with EDB / LTBR bispecific COVA1482. In contrast, the isotype control molecule COVA1486 did not induce upregulation of ICAM-1. These findings indicate that the ability to cluster LTBR scFv via binding to EDB is a prerequisite for LTBR activation, and consequently, a prerequisite for ICAM-1 upregulation.

[0243] Results - Measurement of cytokines in the supernatant of treated cells Several cytokines and chemokines expressed as a result of LTBR activation were measured in the supernatant of co-cultures treated with EDB / LTBR bispecific compounds and control molecules, as described above. Figures 7A to 7J show representative examples of cytokine readout upregulated by LTBR activation by COVA14133 (Figure 7A: RANTES, Figure 7B: IL-6, Figure 7C: IL-8, Figure 7D: MIP-3b, Figure 7E: IP-10, Figure 7F: SDF-1a, Figure 7G: IL-12p70, Figure 7H: I-TAC, Figure 7I: MIP-3a, Figure 7J: TNFα). Untargeted LTBRmAb1-derived scFv in COVA14136 did not activate LTBR, ​​and as a result, the cytokine concentration in the supernatant did not increase beyond the background. Background levels were expressed at the levels achieved with B21M (COVA1440) and EDBmAb1 (COVA1452) antibodies (shown as single concentrations in the plots). Figures 7E–7J show that both COVA14133 (2:1 EDBmAb1 × LTBR mAb1) and COVA14116 (2:1 EDBmAb1 × LTα1β2) achieved potent activation of LTBR, ​​as measured by the induction of cytokine release. No LTBR activation was observed with the untargeted isotype control molecule COVA14136 (2:1 B21M × LTBR mAb1), while the untargeted LTα1β2 control COVA14117 (2:1 B21M × LTα1β2) showed cytokine induction independent of TAA binding. These data again illustrate the extreme difficulty in achieving tumor antigen-dependent activation of LTBR with such antibody-LTα1β2 constructs.

[0244] In summary, the upregulation of ICAM-1 and cytokine secretion during LTBR activation confirmed the expected effects that LTBR activation may have on cells.

[0245] In this example, the molecule of the present invention was demonstrated to achieve efficient tumor-associated antigen (EDB-containing fibronectin)-dependent activation of LTBR. Due to the widespread expression of LTBR in normal tissue (Lukashev et al., "Cancer Res.", Vol. 66 (No. 19): pp. 9617-9624 (2006)), the molecule of the present invention has a clear advantage over the previously described LIGHT and LTα1β2 antibody fusion. This is because such aforementioned fusions efficiently activate LTBR even in the absence of tumor-associated antigens and, as shown herein, lack the desired tumor specificity for LTBR activation. In contrast, the multispecificity binding molecule of the present invention surprisingly possesses this desired tumor specificity.

[0246] Comparative Example 4: Co-culture cell assay using mesothelin-dependent in vitro LTBR-activated A549 NF-κB reporter cells and CHOK1-huMSLN or H226 Examples 2 and 3 demonstrated that a bispecific antibody, targeted EDB (tumor-associated antigen in the extracellular matrix), and LTBR activated LTBR very efficiently in a tumor antigen-dependent manner. To confirm whether this finding applies to any tumor antigen, regardless of its location (deposited in the extracellular matrix or on the cell surface of tumor cells), a bispecific 2:1 antibody-targeted mesothelin (MSLN), tumor-associated antigens expressed in different types of tumors (Hassan and Ho, "European Journal of Cancer," Vol. 44: pp. 46-53 (2008)), and LTBR were designed and produced as described in Example 1. COVA14146 is a 2:1 MSLN / LTBR bispecific antibody consisting of an anti-mesothelin antibody (MSLNmAb1) fused to an scFv fragment derived from LTBRmAb1. To demonstrate whether LTBR bispecific antibody-targeted LTBR and tumor-associated antigens present on the cell surface of tumor cells (e.g., mesothelin) can efficiently activate LTBR in a tumor-dependent manner, co-culture cell assays were used. The co-culture assays used were the A549 cell NF-κB luciferase reporter cell assay (described in Example 2), H226 cells (mesothelioma cell line; ATCC® CRL-5826) (known to express mesothelin (Fan et al., "Mol. Canc. Ther.", Vol. 1, pp. 595-600 (2002))), and LTBR.

[0247] Preparation of H226 cells 10,000 cells per well of a suspension of H226 cells (expressing approximately 200,000 copies of mesothelin and 10,000 copies of LTBR) were seeded in 75 μL of assay medium (DMEM + 10% FBS-HI) into a 96-well tissue culture plate. These cells were incubated for 6 hours at 37°C / 5% CO2 in growth medium (MEM + 2 mM glutamine + 10% FBS-HI + 10 μg / ml puromycin and RPMI-1640 + 10% FBS + 1 mM Na-pyruvate, respectively) to allow the cells to adhere to the plate.

[0248] Preparation of compounds The compounds were tested at concentrations ranging from 100 nM to 1.3 pM. Four-fold 1:5 serial dilutions of the compounds were prepared in assay medium (DMEM + 10% FBS-HI) and stored at 4°C until use.

[0249] Preparation and addition of A549 reporter cells A549 reporter cells were isolated from cell culture flasks using Accutase / EDTA and transferred to assay medium (DMEM + 10% FBS-HI). A total of 20,000 A549 reporter cells per well were added to plates containing H226 cells, and then 50 μL of pre-diluted compound was added to each well. The mixture was incubated at 37°C / 5% CO2 for 20 hours.

[0250] Measurement of luminescence in treated co-cultures After 20 hours of incubation, luciferase activity was detected using the Bio-Glo® luciferase assay system (Promega; Madison, WI) according to the manufacturer's instructions. Luminescence was measured using a Tecan M 1000Pro instrument with an integration time of 500 milliseconds. From the obtained relative luminous units (RLU), the induction factor of LTBR signaling was calculated as follows: Induction Factor = RLU 刺激済み cells / average RLU 非刺激 Cells (non-stimulated cells were included as a control in each plate being tested).

[0251] Dose-response curves, including standard deviations, were plotted using GraphPad Prism, and nonlinear fitting was applied where applicable (logarithmic (agonist) versus response (variable gradient - 3 parameters)). To fit the data, x values ​​(compound concentration) were transformed using the x=Log(x) function in GraphPad Prism.

[0252] Cytokine measurement of supernatant from treated cells using the MSD platform Several cytokines known to be regulated by NF-κB signaling can be measured using the MSD platform and multiplexed MSD plates. As an example, a method for measuring RANTES using the R-Plex antibody set Human RANTES (MSD) is described herein.

[0253] The concentration of RANTES in the supernatant of treated cells was measured using an MSD platform according to the manufacturer's instructions. Briefly, the protocol involved the following steps: (1) Plate preparation involved coating the provided plates with linker-bound capture antibodies. The plates were incubated overnight at 2–8°C with shaking. The following day, the plates were washed with PBST (PBS + 0.05% Tween-20) using a plate washer (Biotek; Winooski, VT). (2) Calibration standards and detection antibody solutions were prepared. (3) Depending on the availability of the material, the supernatant was diluted to a ratio of 1:3 or 1:5.

[0254] Assay protocol: Step 1: Add the sample or calibration standard to the plate and incubate at room temperature for 1 hour while shaking the plate. Step 2: Wash the plate and add the detection antibody. Incubate the plate at room temperature for 1 hour with shaking. Step 3: The plate was washed and 2x read buffer T was added. The plate was analyzed using an MSD instrument. - The data was analyzed using Mesoscale software (MSD Exploration Workbench Program v 4.0.12.1) and plotted using GraphPad Prism.

[0255] Results - Mesothelin-dependent activation of LTBR in A549 reporter cell / H226 co-culture assay Co-culture assays using A549 reporter cells and H226 cells were performed to investigate whether COVA14146 could activate LTBR in a more physiological system when LTBR and mesothelin (another tumor-associated antigen on the surface of tumor cells, e.g., EGFR) are expected to be co-expressed on the surface of tumor cells due to their broad expression (Lukashev et al., "Cancer Res.", Vol. 66, No. 19, pp. 9617-9624 (2006)). Figure 8A shows that COVA14146 did not efficiently activate LTBR under these conditions. The concentration of RANTES secreted into the supernatant of treated cells was measured to confirm that COVA14146 could not efficiently activate LTBR. As expected, Figure 8B shows that RANTES was secreted by cells treated with COVA14146 to the same extent as by cells treated with the isotype control molecule COVA1486, confirming that LTBR could not be activated under these conditions.

[0256] In summary, the data presented in Examples 2 to 4 suggest that EDB-containing fibronectin (a tumor-associated antigen deposited in the extracellular matrix of tumors; Figure 9A) was able to efficiently cluster and activate LTBR, ​​unlike antigens co-expressed with LTBR on the surface of tumor cells (e.g., mesothelin). Activation of LTBR by the bispecific antibody of the present invention under the conditions shown in Figure 9A resulted in the secretion of chemokines and cytokines, as well as the overexpression of adhesion molecules (e.g., ICAM-1) on treated cells. In the absence of EDB-containing fibronectin (Figure 9B), the bispecific antibody of the present invention could not activate LTBR, ​​and consequently, no expression of chemokines and cytokines, or overexpression of adhesion molecules, was observed. Furthermore, tumor-associated antigens co-expressed with LTBR on tumor cells were not suitable for tumor-dependent activation of LTBR by the bispecific antibody.

[0257] In short, as shown herein, targeting LTBR, ​​and TAAs co-expressed with LTBR on tumor cells, with bispecific antibodies that bind to both LTBR and such tumor-associated antigens (TAAs), failed to activate LTBR in a tumor-specific manner (Example 4), and targeting LTBR via a fusion protein containing a TAA-binding moiety and one of the native LTBR ligands, LIGHT or LTα1β2, resulted in LTBR activation, but not in a tumor-specific manner (Example 2). However, remarkably, bispecific antibodies that bind to one binding domain for LTBR and another binding domain of fibronectin to EDB (TAAs present in the extracellular matrix) were able to activate LTBR in a tumor-specific manner (Examples 2 and 3). Particularly favorable results were observed when such bispecific antibodies contained three binding domains, e.g., two binding domains targeting EDB and one binding domain targeting LTBR. Thus, the bispecific antibodies of the present invention are interesting candidates for cancer immunotherapy in terms of their tumor specificity.

[0258] Those skilled in the art will understand that modifications can be made to the embodiments described above without departing from the broader concept of the invention. Therefore, it is understood that the present invention is not limited to any specific embodiment disclosed, but is intended to encompass modifications that fall within the spirit and scope of the invention as defined herein.

[0259] Example 5: Transwell migration of PBMCs from A375 / WI38VA sub-lineage 2RA co-culture cell assay to conditioned medium. In Example 3, it was demonstrated that a bispecific antibody, targeted EDB (tumor-associated antigen in the extracellular matrix), and LTBR activated LTBR very efficiently in a tumor antigen-dependent manner, leading to the production of pro-inflammatory cytokines.

[0260] The purpose of this assay is to study whether cytokines and chemokines produced in the co-culture assay can attract PBMCs and induce their migration. Human PBMCs were isolated from the buffy coat by Ficoll Paque density gradient centrifugation, and A375 / WI38VA co-cultures were prepared and stimulated with EDB / LTBR bispecific and control molecules as described in Example 3.

[0261] After incubation at 37°C / 5% CO2 for 24 hours, the supernatant of the stimulated co-culture was transferred to a 96-well DeepWell plate and diluted 1:1 with assay medium (RPMI1640 + 10% FBS + 1 mM sodium pyruvate). After dilution, the supernatant was centrifuged (500xg / 5 min) and transferred to a fresh 96-well DeepWell plate to remove any cells or cell debris.

[0262] Recombinant SDF-1a, a potent chemotactic, was used as a positive control to stimulate PBMC migration at a concentration of 40 ng / mL in the assay medium. 235 μL / well of acclimatization medium, SDF-1a control, or assay medium was transferred for migration assays (three variations) to carrier plates of HTS Transwell®-96 Permeable Supports with a 5 μm pore polycarbonate membrane (Corning), which had been pre-equilibrated in assay medium (RPMI 1640 + 10% FBS + 1 mM sodium pyruvate) at 37°C / 5% CO2 for at least 1 hour. After returning the membrane insert to the carrier plate, 4.67 × 10⁶ units were transferred. 6 A 75 μL / well PBMC suspension containing cells / mL was added to all wells of a migration assay plate, yielding 350,000 cells / well. The plate was incubated at 37°C / 5% CO2 for 2 hours to allow PBMC migration into the acclimatization medium.

[0263] After 2 hours of incubation, the plate insert was removed, the migrating cells from the carrier plate were carefully resuspended, and transferred to a fresh U-bottom 96-well plate. The migrating cells were centrifuged (400xg / 4 min), resuspended in 50 μL / well of FACS buffer (PBS containing 1% FBS-HI, 0.1% sodium azide, and 1 mM EDTA), and measured directly using a MACS Quant instrument (high flow, fast mode). Data were analyzed using Flowlogics Software (version 700.2A). Dose-response curves, including standard deviations from three different values, were plotted using GraphPad Prism. To fit the data, the x-values ​​(compound concentration) were transformed using the x=Log(x) function in GraphPad Prism.

[0264] Results - Transwell migration of PBMCs from A375 / WI38VA sub-lineage 2RA co-culture cell assay to conditioned medium. In this example, we tested whether a cocktail of cytokines and chemokines expressed upon LTBR activation in a co-culture assay (see Example 3) could induce migration of PBMCs toward the cytokine and chemokine gradient. A Transwell migration assay was established by placing the supernatant of co-cultures stimulated with different concentrations of EDB / LTBR bispecific antibodies into the lower chamber and adding newly isolated human PBMCs to the upper chamber of a Transwell plate. After 2 hours of incubation, the migrated cells were counted and phenotyped by flow cytometry. Figure 10 shows representative results of the migration assay. PBMC migration was induced in a dose-dependent manner from the supernatant of co-cultures stimulated with COVA14133 (EDB / LTBR bispecific), whereas the supernatant from co-cultures incubated with the untargeted control molecule COVA14136 (isotype control / LTBR) did not induce PBMC migration. -LTα1β2 antibody-fused COVA14116 (EDBmAb1-LTα1β2;2:1) and to some extent COVA14117 (B21M-LTα1β2;2:1) also induced migration of PBMCs. Migration of different immune cell subpopulations was confirmed by staining with immunocytomarkers to phenotypologically determine the migrating cells. Migration of monocytes, eosinophils / neutrophils, basophils, NK cells, NKT cells, dendritic cells, and T cells was confirmed (data not shown). This example confirms that EDB-dependent activation of LTBR leads to the secretion of cytokines and chemokines, which may act as chemotactic factors for immune cells (Figure 10).

[0265] Furthermore, this embodiment again demonstrates that the molecule of the present invention has clear advantages over the previously described LTα1β2 antibody fusions. This is because such aforementioned fusions efficiently activate LTBR even in the absence of tumor-associated antigens, leading to PBMC migration, and thus lacking the desired tumor specificity for LTBR activation, as has been shown herein. In contrast, the multispecificity binding molecule of the present invention surprisingly possesses this desired tumor specificity.

[0266] Example 6: Effect of EDB-dependent LTBR-mediated endothelial activation on monocyte transport through the endothelial monolayer. In Examples 3 and 5, we demonstrated that cytokines produced during EDB-dependent activation of LTBRs could induce migration of PBMCs toward the cytokine gradient. Following this, the objective of the assay described herein was to verify whether EDB-dependent activation of LTBRs on endothelial cells increases monocyte transport across the endothelial layer.

[0267] The monocytes used in this assay were purified from EDTA-treated blood collected from healthy donors using their respective negative selection kits (Miltenyi Biotec), and measured in 1.5 × 10⁶ units. 6 Cells / mL were used. Human umbilical vein endothelial cells (HUVECs) were cultured for 48 hours in M199 supplement medium (M199 medium, 20% FCS, hydrocortisone (0.1 μM), heparin (100 μg / mL), ECGS 15 μg / mL, vitamin C (10 μg / mL), penicillin / streptomycin (1% / 1%)) on chamber slides coated with recombinant EDB+ fibronectin domain 7-B-8-9 (EDB+; SEQ ID NO: 51). Subsequently, HUVECs were stimulated with TNF (500 U / mL; positive control), EDB / LTBR bispecificity (COVA14133; 50 nM), or untargeted LTBRmAb1-derived scFv (COVA14136; 50 nM) and incubated for 2 days.

[0268] The flow assay setup consisted of a heated microscope chamber (37°C) and a calibrated pump, which could generate flow on a HUVEC monolayer by perfusing with wash buffer (M199 medium, 0.1% BSA) ± monocyte suspension. The flow rate was representative of venule / capillary flow (0.05-Pa). Next, the wash buffer was pumped onto the HUVEC for 10 minutes to remove the activated medium. This corresponds to 20 minutes of total HUVEC exposure. Then, the monocytes were perfused onto the HUVEC for 6 minutes (Step 2), followed by perfusing with wash buffer for 50 minutes (Step 3). This was done at 0.1 Pa, which is standard for all monocyte recruitment protocols. Throughout Steps 2 and 3, images of the captured monocytes were created using a phase-contrast microscope and camera. Individual images were recorded every 30 seconds in a single fixed field of view and combined into short video sequences to enable analysis of individual monocytes over a wide area. Monocytes adhered to the surface of HUVEC have a white / gray appearance, while monocytes that escape have a black appearance.

[0269] Adherent and migratory cells were identified by playing back a video sequence over the course of the experiment, measuring 0.19 mm. 2 Cells were counted within a fixed grid on each image within the defined region. Time points for each cell count are taken at the fixed point throughout the experiment.

[0270] The total number of adherent cells represents the sum of captured cells at each time point; the percentage of cells released (gray phase + black phase). The release event (black phase) is the percentage of total monocytes (gray phase + black phase) captured from the flow per unit field of view. Monocytes typically remain adherent due to very few separation events over the duration of co-culture.

[0271] All experiments were conducted using three different fields of view, and the mean values ​​were shown using standard error measurement (+SEM). Statistical analysis was performed using a Student's t-test, assuming a parametric distribution. The p-values ​​from the significance scores are shown in the figure below: * P<.05, ** P<.01, ***P<.005 (Bradfield PF et al., "Blood.", Oct 1, 2007; Vol. 110 (No. 7): pp. 2545-2555).

[0272] Results - EDB-dependent LTBR-mediated endothelial activation effect on monocyte transport through the endothelial monolayer. This example investigates the effect of EDB-dependent LTBR activation on endothelial cell monolayers on monocyte adhesion and migration, as it has been previously shown that endothelial cells express LTBR on their surface (Lukashev et al., "Cancer Res.", Vol. 66 (No. 19): pp. 9617-9624 (2006)).

[0273] Figure 11A shows that more monocytes can adhere to HUVEC monolayers grown in the presence of EDB-containing fibronectin activated with COVA14133 (EDB / LTBR bispecificity) compared to monolayers activated with the non-targeting control molecule COVA14136 (isotype control / LTBR).

[0274] Figure 11B shows that not only adhesion, but also monocyte evacuation through the HUVEC monolayer after activation by COVA14133 is increased compared to HUVEC incubated with the untargeted control COVA14136.

[0275] In conclusion, the results of this embodiment further confirm that the molecule of the present invention, unlike the LTα1β2 antibody fusion described previously, can activate LTBR only in the presence of EDB-containing fibronectin, and has the clear advantage of conferring the desired tumor specificity to LTBR activation. The following embodiments may be included. [1] A multispecific binding molecule, the following: (i) A first binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), (ii) comprising a second binding domain that specifically binds to the extra domain B (EDB) of fibronectin, The multispecific binding molecule is a multispecific binding molecule that activates LTBR upon binding of EDB. [2] The multispecific binding molecule described in [1] above, wherein the multispecific binding molecule activates LTBR in a tumor-specific manner. [3] The multispecificity binding molecule according to [1] or [2] above, wherein the multispecificity binding molecule is a bispecificity antibody. [4] The multispecificity binding molecule according to any one of the above [1] to [3], wherein the multispecificity binding molecule comprises two antigen-binding domains. [5] The multispecificity binding molecule according to any one of the above [1] to [3], wherein the multispecificity binding molecule comprises three antigen-binding domains. [6] The multispecific binding molecule according to [5] above, wherein the three antigen-binding domains each include one binding domain that specifically binds to LTBR. [7] The multispecific binding molecule according to [5] or [6] above, wherein the three antigen-binding domains include two binding domains that specifically bind to EDB. [8] The multispecific binding molecule according to any one of [5] to [7] above, wherein the binding domain that specifically binds to LTBR includes the single-chain variable domain of the antibody. [9] The first binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determination region 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity determination region 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL are as follows: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (ii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (iii) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 44; or (v) VH contains an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 48: or (vi) Sequence ID 22; or (vii) Sequence ID 23; or (viii) Sequence ID 25 A multispecific binding molecule according to any one of the above [1] to [8], comprising any of the above.

[10] The second binding domain that specifically binds to the EDB comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determination region 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity determination region 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL are as follows: (i) HCDR1, HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 46. A multispecific binding molecule according to any one of the above [1] to [9], which includes any of the above.

[11] Below: (a)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO: 1, forming a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO: 2, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO: 4, forming a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO: 5; or (b) (i) A first heavy chain containing the amino acid sequence of SEQ ID NO: 9, which forms a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO: 10, and (ii) A second heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO: 5. A multispecific binding molecule according to any one of the above [1] to

[10] , comprising any of the above.

[12] Below: (a)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 30, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (b)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 31, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (c)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 32, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (d)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 33, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (e)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 34, wherein the heavy chain portion forms a binding domain with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (f)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 35, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (g)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (h)(i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 39, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5; or (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 56, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5. A multispecific binding molecule according to any one of the above items [1] to [3] or [5] to

[10] , comprising any of the above.

[13] A multispecific binding molecule according to any one of the above [1] to [3] or [5] to

[10] , comprising (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, wherein the heavy chain portion forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain containing the amino acid sequence of SEQ ID NO: 5.

[14] One or more nucleic acid molecules encoding a multispecific binding molecule as described in any one of the above items [1] to

[13] .

[15] One or more vectors comprising one or more nucleic acid molecules as described in

[14] above.

[16] An isolated host cell containing one or more nucleic acid molecules as described in

[14] above or one or more vectors as described in

[15] above.

[17] A pharmaceutical composition comprising a multispecific binding molecule described in any one of the above items [1] to

[13] and a pharmaceutically acceptable carrier.

[18] A method for treating cancer in a subject requiring treatment, comprising administering to the subject a multispecific binding molecule described in any one of the above items [1] to

[13] , one or more nucleic acid molecules described in

[14] , one or more vectors described in

[15] , or a pharmaceutical composition described in

[17] .

[19] Use of a multispecific binding molecule according to any one of [1] to

[13] above, one or more nucleic acid molecules according to

[14] above, one or more vectors according to

[15] above, or a pharmaceutical composition according to

[17] above, for activating LTBR in tumor tissue.

[20] A method for producing a multispecific binding molecule as described in any one of the above [1] to

[13] , comprising expressing one or more nucleic acid molecules as described in

[14] or one or more vectors as described in

[15] in a host cell, and collecting the multispecific binding molecule.

[0276] Sequence List Sequence ID 1 (HC BHA10 IgG1 knob) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0277] Sequence ID 2 (LC BHA10) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0278] Sequence ID 3 (HC L19 IgG1 knob) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0279] Sequence ID No. 4 (HC L19 IgG 1 hole) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0280] Sequence ID 5 (LC L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0281] Sequence ID 6 (HC B21M(RSV)IgG1 knob) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPPVDTATYYCARLYGFTYGFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0282] Sequence ID 7 (HC B21M(RSV)IgG 1 hole) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPPVDTATYYCARLYGFTYGFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVCTLPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0283] Sequence ID 8 [LC B21M(RSV)] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0284] Sequence ID 9 HC (CBE11 IgG1 knob) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMYWFRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCAREENGNFYYFDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0285] Sequence ID 10 (LC CBE11) DIQMTQSPSSLSASVGDRVTITCKAGQDIKSYLSWYQQKPGKAPKLLIYYATRLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQHGESPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0286] Sequence ID 11 (HC BHA10 IgG1) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0287] SEQ ID NO:12(HC L19 IgG1) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0288] Sequence ID 13 (HC CBE11 IgG1) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMYWFRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCAREENGNFYYFDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0289] Sequence ID 14 (HC B21M(RSV)IgG1) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPPVDTATYYCARLYGFTYGFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0290] Sequence ID No. 15 (IgG1, knob, and 3xhmLIGHT fusion to Fc with pA mutation) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGGSGGGGSGGGGSGGGGSRRSHEVNPAAHL TGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGGSGGGGSGGGGSGGGGSRRSHEVNPAAHLTGANSSLTGSGG PLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTC VVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPC REEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0291] Sequence ID No. 16 (3xhmLIGHT single chain used in fusion) RRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEA GEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGGSGGGGSGGGGSGGGGSRRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTIT HGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGGSGGGGSGGGGSGGGGSRRSHEVNPAAHLTGANSSLTGSGGPLLW ETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMV

[0292] Sequence ID 17 (LTa1b2 used in fusion) KPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQG DQLSTHTDGIPHLVLSPSTVFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGP GTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLAL PQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0293] Sequence ID 18 (HC B21M fusion to LTa1b2 IgG1) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAHSTLKPAAHL IGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGDPQGRSVTLRSSLYRAGGAYGPGTP ELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0294] Sequence ID 19 (HC B21M fusion to LTa1b2, IgG1, knob, pA mutation present) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMPDPVDTATYYCARLYGFTYGFAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQ PREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKAHSTLKPAAHL IGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLS THTDGIPHLVLSPSTVFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTP ELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALP QDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0295] Sequence ID No. 20 (HC L19 fusion to LTa1b2 IgG1) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAHSTLKPAAHLIG DPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTH TDGIPHLVLSPSTVFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPE LLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQ DGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0296] Sequence ID No. 21 (HC L19 fusion to LTa1b2, IgG1, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKAHSTLKPAAHLIG DPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTH TDGIPHLVLSPSTVFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPE LLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQ DGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0297] Sequence ID 22 [Staple processing scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0298] Sequence ID 23 [Staple processing scFv BHA10(VL-VH)] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPC GSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0299] Sequence ID 24 [Staple-processed linker (VH-VL)] GGGSGGGSGCPPCGGGG

[0300] Sequence ID 25 [Disulfide-stabilized scFv BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0301] Sequence ID No. 26 (HC B21M N-terminal stapled BHA10 (VH-VL), IgG1, knob, pA mutation present) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPK SLISSASYRYSGVPSRFSGGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDT ATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSA EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0302] Sequence ID No. 27 (HCB21M N-terminal stapled BHA10 (VL-VH), IgG1, knob, pA mutation present) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGN VHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSSGGGGSGGGGSGGGGGSGGGGSGGGGSQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTA TYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSA EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0303] Sequence ID No. 28 (HC B21M C-terminal stapled BHA (VH-VL), IgG1, knob, pA mutation present) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP

[0304] Sequence ID No. 29 (HC B21M C-terminal stapled BHA (VL-VH), IgG1, knob, pA mutation present) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFS GSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0305] Sequence ID No. 30 (HC L19 N-terminal stapled BHA10 (VH-VL), IgG1, knob, pA mutation present) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAP KSLISSASYRYSGVPSRFSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0306] Sequence ID No. 31 (HC L19 N-terminal stapled BHA10 (VL-VH), IgG1, knob, pA mutation present) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPG NVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP AVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0307] Sequence ID 32 (HC L19 C-terminal stapled BHA10 (VH-VL), IgG1, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0308] Sequence ID 33 (HC L19 C-terminal stapled BHA10 (VL-VH), IgG1, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSG SGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0309] Sequence ID 34 (HC L19 C-terminal stapled (VL3_Y36F S49Y_F87Y) BHA (VH-VL), IgG1, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0310] Sequence ID 35 (HC L19 C-terminal stapled (VH_CDR1_Y33A) BHA10 (VH-VL), IgG1, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0311] Sequence ID 36 (HC B21M C-terminal stapled (VL3_Y36F S49Y_F87Y) BHA (VH-VL), IgG1, knob, pA mutation present) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP

[0312] Sequence ID 37 (HC B21M C-terminal stapled (VH_CDR1_Y33A) BHA10 (VH-VL), IgG1, knob, pA mutation present) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP

[0313] Sequence ID No. 38 (HC L19 C-terminal stapled BHA10 (VH-VL), IgG1, knob, no pA mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0314] Sequence ID No. 39 (HC L19 C-terminal disulfide stabilized, BHA (VH-VL), IgG1, knob, no pA mutation) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPP CREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0315] Sequence ID No. 40 (HC B21M C-terminal disulfide stabilized, BHA10 (VH-VL), IgG1, knob, no pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0316] Sequence ID No. 41 (HC B21M C-terminal stapled BHA10 (VH-VL), IgG1, knob, no pA mutation) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGP

[0317] Sequence ID 42 [(GGGGS) 4-linker (used to connect Fv fragments in disulfide-stabilized scFv)] GGGGSGGGGSGGGGSGGGGS

[0318] Sequence ID 43 (VH BHA10) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSS

[0319] Sequence ID 44 (VL BHA10) DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIK

[0320] Sequence ID 45 (VH L19) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

[0321] Sequence ID 46 (VL L19) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

[0322] Sequence ID 47 (VH CBE11) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMYWFRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCAREENGNFYYFDYWGQGTTVTVSS

[0323] Sequence ID 48 (VL CBE11) DIQMTQSPSSLSASVGDRVTITCKAGQDIKSYLSWYQQKPGKAPKLLIYYATRLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQHGESPWTFGGGTKLEIK

[0324] Sequence ID 49 (VH B21M) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSS

[0325] Sequence ID 50 (VL B21M) DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIK

[0326] Sequence ID 51 (fibronectin domain 7B89) PLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGNSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTA VPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLIGQQSTHHHHHH

[0327] Sequence ID 52 (fibronectin domain 789) PLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGNSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVA ELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREEESPLLIGQQSTHHHHHH

[0328] Sequence ID 53 [Staple processing scFv(VL3_Y36F_S49Y_F87Y)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0329] Sequence ID 54 [Staple processing scFv(VH_CDR1_Y33A)BHA10(VH-VL)] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGS GGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0330] Sequence ID 55 [Staple-processed linker (VL-VH)] GGSGGSGGCPPCGSGG

[0331] Sequence ID No. 56 (HC L19 C-terminal disulfide stabilized, BHA10 (VH-VL), IgG1, knob, pA mutation present) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPP CREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0332] Sequence ID No. 57 (HC B21M C-terminal disulfide stabilized, BHA10 (VH-VL), IgG1, knob, pA mutation present) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0333] Sequence ID 58 (IgG1 Sigma Fc) DKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0334] Sequence ID 59 [(GGGGS)3 linker scFv vs Fc] GGGGSGGGGSGGGGS

[0335] Sequence ID 60 (HCDR1 of VH BHA10) TYYLH

[0336] Sequence ID 61 (HCDR2 of VH BHA10) WIYPGNVHAQYNEKFKG

[0337] Sequence ID 62 (HCDR3 of VH BHA10) SWEGFPY

[0338] Sequence ID 63 (LCDR1 of VL BHA10) KASQNVGINVA

[0339] Sequence ID 64 (LCDR2 of VL BHA10) SASYRYS

[0340] Sequence ID 65 (LCDR3 of VL BHA10) QQYDTYPFT

[0341] Sequence ID 66 (HCDR1 of VH CBE11) DYYMY

[0342] Sequence ID 67 (HCDR2 of VH CBE11) TISDGGSYTYYPDSVK

[0343] Sequence ID 68 (HCDR3 of VH CBE11) EENGNFYYFDY

[0344] Sequence ID 69 (LCDR1 of VL CBE11) KAGQDIKSYLS

[0345] Sequence ID 70 (LCDR2 of VL CBE11) YATRLAD

[0346] Sequence ID 71 (LCDR3 of VL CBE11) LQHGESPWT

[0347] Sequence ID 72 (HCDR1 of VH L19) SFSMS

[0348] Sequence ID 73 (HCDR2 of VH L19) SISGSSGTTYYADSVKG

[0349] Sequence ID 74 (HCDR3 of VH L19) PFPYFDY

[0350] Sequence ID 75 (LCDR1 of VL L19) RASQSVSSSFLA

[0351] Sequence ID 76 (LCDR2 of VL L19) YASSRAT

[0352] Sequence ID 77 (LCDR3 of VL L19) QQTGRIPPT

[0353] Sequence ID 78 (VH MSLNmAb1) QVQLQQSGPELEKPGASVKISKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS

[0354] Sequence ID 79 (VL MSLNmAb1) DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK

[0355] Sequence ID 80 (MSLNmAb1 HC C-terminal stapled BHA10 (VH-VL), IgG1, knob, pA mutation present) QVQLQQSGPELEKPGASVKISKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVDVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYT LPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0356] Sequence ID 81 (HC MSLNmAb1 IgG1 hole) QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSG TPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVS...

Claims

1. A multispecific binding molecule, which is as follows: (i) A first binding domain that specifically binds to the lymphotoxin beta receptor (LTBR), wherein the first binding domain that specifically binds to the LTBR (i-1) (a) comprising a heavy chain variable region (VH) including HCDR1 containing the amino acid sequence of SEQ ID NO: 60, HCDR2 containing the amino acid sequence of SEQ ID NO: 61, and HCDR3 containing the amino acid sequence of SEQ ID NO: 62, and (b) comprising a light chain variable region (VL) including LCDR1 containing the amino acid sequence of SEQ ID NO: 63, LCDR2 containing the amino acid sequence of SEQ ID NO: 64, and LCDR3 containing the amino acid sequence of SEQ ID NO: 65, or (i-2) (a) A heavy chain variable region (VH) comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 66, HCDR2 containing the amino acid sequence of SEQ ID NO: 67, and HCDR3 containing the amino acid sequence of SEQ ID NO: 68, and (b) A light chain variable region (VL) comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 69, LCDR2 containing the amino acid sequence of SEQ ID NO: 70, and LCDR3 containing the amino acid sequence of SEQ ID NO: 71, (ii) A second binding domain that specifically binds to the extra domain B (EDB) of fibronectin, wherein the second binding domain that specifically binds to the EDB comprises (a) a heavy chain variable region (VH) comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 72, HCDR2 containing the amino acid sequence of SEQ ID NO: 73, and HCDR3 containing the amino acid sequence of SEQ ID NO: 74, and (b) a light chain variable region (VL) comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 75, LCDR2 containing the amino acid sequence of SEQ ID NO: 76, and LCDR3 containing the amino acid sequence of SEQ ID NO:

77. Includes, The multispecific binding molecule is a multispecific binding molecule that activates LTBR upon binding of EDB.

2. (i) The first binding domain includes a VH containing an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 43 or 47, and a VL containing an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 44 or 48, and / or (ii) The second binding domain includes VH, which contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 45, and VL, which contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:

46. The multispecific binding molecule according to claim 1.

3. The multispecific binding molecule according to claim 1, wherein the first binding domain comprises a single-chain antibody fragment (scFv) or a stapled single-chain antibody fragment (spFv).

4. The multispecific binding molecule according to claim 3, wherein the spFv includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 22, 23, 53, or 54.

5. (i) The first binding domain comprises (a) a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 1, and (b) a light chain (LC) containing the amino acid sequence of SEQ ID NO: 2, (ii) The second binding domain comprises (a) an HC containing the amino acid sequence of SEQ ID NO: 4, and (b) an LC containing the amino acid sequence of SEQ ID NO: 5 The multispecific binding molecule according to claim 1.

6. (i) The first binding domain comprises (a) an HC containing the amino acid sequence of SEQ ID NO: 9, and (b) an LC containing the amino acid sequence of SEQ ID NO: 10, (ii) The second binding domain comprises (a) an HC containing the amino acid sequence of SEQ ID NO: 4, and (b) an LC containing the amino acid sequence of SEQ ID NO: 5 The multispecific binding molecule according to claim 1.

7. (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 32, and an LC containing the amino acid sequence of SEQ ID NO: 5, wherein the heavy chain portion forms a binding domain together with the LC, and (ii) HC containing the amino acid sequence of SEQ ID NO: 4, and LC containing the amino acid sequence of SEQ ID NO: 5 A multispecific binding molecule according to claim 1, comprising:

8. (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, and an LC containing the amino acid sequence of SEQ ID NO: 5, wherein the heavy chain portion forms a binding domain together with the LC, and (ii) HC containing the amino acid sequence of SEQ ID NO: 4, and LC containing the amino acid sequence of SEQ ID NO: 5 A multispecific binding molecule according to claim 1, comprising:

9. (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 56, and an LC containing the amino acid sequence of SEQ ID NO: 5, wherein the heavy chain portion forms a binding domain together with the LC, and (ii) HC containing the amino acid sequence of SEQ ID NO: 4, and LC containing the amino acid sequence of SEQ ID NO: 5 A multispecific binding molecule according to claim 1, comprising:

10. It is a multispecific binding molecule, It comprises a first binding domain that specifically binds to the lymphotoxin beta receptor (LTBR) and a second binding domain that specifically binds to the extradomain B (EDB) of fibronectin, (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO: 38, and an LC containing the amino acid sequence of SEQ ID NO: 5, wherein the heavy chain portion forms a binding domain together with the LC, and (ii) HC containing the amino acid sequence of SEQ ID NO: 4, and LC containing the amino acid sequence of SEQ ID NO: 5 Includes, The multispecific binding molecule is a multispecific binding molecule that activates LTBR upon binding of EDB.

11. The multispecific binding molecule according to any one of claims 1 to 10, wherein the multispecific binding molecule activates LTBR in a tumor-specific manner.

12. (a) The multispecific binding molecule is a bispecific antibody. (b) The multispecific binding molecule comprises two antigen-binding domains, or (c) The multispecific binding molecule comprises three antigen-binding domains, A multispecific binding molecule according to any one of claims 1 to 11.

13. The multispecific binding molecule according to claim 12, wherein the three antigen-binding domains include one binding domain that specifically binds to LTBR and / or two binding domains that specifically bind to EDB.

14. The multispecific binding molecule according to any one of claims 1 to 13, wherein the binding domain that specifically binds to LTBR includes the single-chain variable domain of the antibody.

15. One or more nucleic acid molecules encoding the multispecific binding molecule described in any one of claims 1 to 14.

16. One or more vectors comprising one or more nucleic acid molecules as described in claim 15.

17. An isolated host cell comprising one or more nucleic acid molecules according to claim 15 or one or more vectors according to claim 16.

18. A pharmaceutical composition comprising a multispecific binding molecule according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18 for use in a method of treating cancer in a subject requiring treatment.

20. A pharmaceutical composition according to claim 18 or 19 for activating LTBR in tumor tissue.

21. A method for producing a multispecific binding molecule according to any one of claims 1 to 14, comprising expressing one or more nucleic acid molecules according to claim 15 or one or more vectors according to claim 16 in a host cell, and collecting the multispecific binding molecule.

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