Multispecific binding molecules comprising LTBR and EDB binding domains and uses thereof

Abstract

To provide anti-LTBR multispecific binding molecules, nucleic acids encoding the anti-LTBR multispecific binding molecules, vectors comprising the nucleic acids, host cells comprising the vectors, and pharmaceutical compositions comprising the anti-LTBR multispecific binding molecules.SOLUTION: Provided is a multispecific binding molecule comprising: (i) a first binding domain that specifically binds to lymphotoxin beta receptor (LTBR), and (ii) a second binding domain that specifically binds to extra domain B (EDB) of fibronectin, wherein the multispecific binding molecule activates LTBR upon binding to the EDB.SELECTED DRAWING: None

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Application No. 62 / 946,452, filed on December 11, 2019. The entire contents of the foregoing application are hereby incorporated by reference in their entirety into this specification.

[0002] (Field of the Invention) The present invention relates to anti - LTBR multispecific binding molecules, nucleic acids encoding the binding molecules and expression vectors, recombinant cells containing the vectors, and compositions containing the binding molecules. Also provided are methods of making the binding molecules and methods of using the binding molecules to kill cancer cells.

Background Art

[0003] Cancer immunotherapy has the potential to improve the survival rate of cancer patients by promoting the immune response against tumors. Certain patients experience a deep and long - lasting response to currently available anti - cancer immunotherapies (e.g., anti - PD - 1 / PD - L1 antibodies such as anti - CTLA4 antibody ipilimumab, pembrolizumab, or nivolumab), but the majority of patients do not benefit from such therapeutics (Ribas et al., "Science", Vol. 359: pp. 1350 - 1355 (2018)). For example, patients with so - called "cold" or non - inflammatory tumors, characterized by a lack of immune cell infiltration or the absence of an inflammatory signature, have low benefit from anti - cancer immunotherapy (Chen and Mellman, "Nature", Vol. 541: pp. 321 - 330 (2017)). Thus, there is a need for new anti - cancer immunotherapies with improved efficacy.

[0004] The receptor of the TNF superfamily, lymphotoxin beta receptor (LTBR / TNFRS F3), is one of the many possible targets for 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 lymphokines (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 (200 2 years), Schneider et al., "Immunol. Rev.", Vol. 202: pp. 49- 66 (2004)). LTBR is activated by two different trimeric ligands, LIGHT (TNFSF14) and lymphotoxin α1β2 (LTα1β2). LTα1β2 is specific for LTBR, but LIGHT also binds to and activates HVEM (TNFRSF14), a receptor involved in the regulation of immune cells expressed on immune cells (Pasero et al., "Curr. Opin. Pharmacol.", (2012)). (Dejardin et al., "Immunity", Vol. 17: pp. 525-535 (200 2 years), Schneider et al., "Immunol.Rev.", Vol. 202: pp. 49- 66 (2004)). LTBR is activated by two different trimeric ligands, LIGHT (TNFSF14) and lymphotoxin α1β2 (LTα1β2). LTα1β2 is specific for LTBR, but LIGHT also binds to and activates HVEM (TNFRSF14), a receptor involved in the regulation of immune cells expressed on immune cells (Pasero et al., "Curr. Opin. Pharmacol.", (2012)). Of particular interest from the perspective of cancer immunotherapy is the finding that activation of LTBR by its ligand leads to the 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. (2001); Tang et al., "Cell.Mol.Immunol.", Vol. 14: pp.

[0005] (2001); Tang et al., "Cell.Mol.Immunol.", Vol. 14: pp. (2001); Tang et al., "Cell.Mol.Immunol.", Vol. 14: pp. (2001); Tang et al., "Cell.Mol.Immunol.", Vol. 14: pp. (2001); Tang et al., "Cell.Mol.Immunol.", Vol. 14: pp. Pages 809-18 (2017)). The presence of TLS in the tumor microenvironment typically correlates with immune infiltration and is also associated with a better prognosis, suggesting that TLS contributes to the anti-tumor immune response (Dieu-Nosjean et al., "J. Clin. Oncol.", Vol. 26: pp. 4410-17 (2008); Weinstein and Storkus, "Adv. Cancer Res.", Vol. 128: pp. 197-233 (2015)). Therefore, activation of LTBR may promote TLS formation in the tumor microenvironment, induce an anti-tumor immune response, and have the potential to improve current cancer immunotherapies. The concept of targeting LTBR for the purpose of promoting a protective anti-tumor immune response has been established in several preclinical studies. Some groups target LTBR using its natural ligand LIGHT (TNFSF14). LIGHT binds to LTBR and a second receptor, HVEM (TNFR SF14), 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. 478-85 (2012)). It should be noted that LIGHT-mediated immunobiological effects may depend on either LTBR or HVEM. Yu et al. showed that forced expression of the membrane-bound form of LIGHT in a mouse tumor cell line led to massive infiltration of naive T lymphocytes, which correlated with upregulation of chemokine products and adhesion molecules, resulting in rejection of established tumors at local and distal sites.

[0006]

[0007]

[0008] ​​​​​​​​​​​​​​This has been demonstrated (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 adenoviral delivery of the LIGHT gene to established tumors (Yu et al., "J. Immunol.", Vol. 179: pp. 1960-1968 (2007)). Based on these findings, in an attempt to utilize this mode of action with a more suitable modality for clinical applications, Tang et al. generated a homotrimeric single-chain LIGHT variant with improved stability and human and mouse cross-reactivity, designated 3xhmLIGHT

[0009] (Tang et al., "Cancer Cell", Vol. 29: pp. 285-296 (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, and thereby was able to overcome resistance to checkpoint blockade immunotherapy when combined with anti-PD-L1 antibody in models with low lymphocyte infiltration. Tang et al. reported the tolerance upon intratumoral injection into mice bearing tumors. No significant side effects were observed as no significant changes in body weight or serum cytokines were seen. The authors did not report the tolerance after systemic administration. 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 ).

[0010] ng peptide:VTP) and described it (Johansson-Percival et al., "Nat. Immunol.", Vol. 18 ). Volume: pp. 1207-17 (2017)). In a mouse solid tumor model, VTP-LIG The HT construct homed to tumor blood vessels, promoted vascular normalization, and induced TLS. VT The addition of P-LIGHT enhanced the activity of the combination of anti-CTLA4 and anti-PD-1 antibodies, as well as the activity of anti-tumor vaccination in vivo. After intravenous administration of VTP-LIGHT, body weight loss was observed in the treated mice.

[0011] Gurney et al. reported in vitro and in vivo studies using a bispecific fusion construct consisting of a heterotrimeric single-chain LT fused to a B7-H4-specific tumor-targeting antibody and the α1β2 portion (International Publication No. WO 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. After treatment with the LTα1β2 fusion construct in a mouse tumor model, infiltration of immune cells, induction of cytokine expression, and formation of TLS were observed. The anti-tumor activity of the LTα1β2 antibody fusion in combination with an anti-PD-L1 antibody was superior to that of each of the individual compounds. The efficacy model used by Gurney et al. was an artificial model consisting of engineered cell lines overexpressing B7-H4. The activity in a model with a more representative non-engineered B7-H expression level for B7-H4 levels in human tumors remained unknown. Observations on tolerance in mice were not reported.

[0012] Michaelson et al. constructed a bispecific antibody targeting TRAIL-R2 and LTBR, and the bispecific antibody was achieved by treating with a mixture of the two antibodies. ​​​​​​​​​​​​​​described as having enhanced, synergistic, or broader potential to induce an anti - tumor response (Michaelson et al., "MAbs", Vol. 1: pp. 128 - 41 (2009) . TRAIL - R2 is widely expressed in normal tissues including colon, lung, liver, and brain , a TNF family receptor (Spierings et al., "J. Histochem. Cy tochem.", Vol. 52: pp. 821 - 31 (2004)), but has also been found to co - express with LTBR on the surface of human epithelial cancer cell lines. Bispecific constructs have shown enhanced activity compared to the parental antibodies in in vitro and mouse tumor xenograft models. No observations on tolerance in mice have been reported .

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

[0014] The immune system is tightly regulated to ensure the immune - mediated eradication of pathogens without causing tissue damage or autoimmunity . Systemic immunomodulatory therapies generally result in a delicate balance that is disrupted, and also cause immune - related adverse events such as interstitial pneumonia, colitis, hepatitis, thyroid dysfunction, skin reactions, and eye inflammation , especially in the context of combination therapies where toxicity can be additive or synergistic, posing challenges to the development of new immunotherapies .

[0015] Due to the widespread expression of LTBR in organisms, it induces TLS and creates an activated immune environment ​Agonist LTBR-targeted drugs that can be administered systemically carry a substantial risk of inducing 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.", Vol. 18: pp. 1207-17 (2017)). Thus, as hypothesized in the prior art, therapeutic modalities that specifically activate LTBR in tumors but not in other tissues are needed to reduce the risk of toxicity and to generate well-tolerated drugs that can be used 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 PROBLEM

[0017] Provided herein are multispecific binding molecules, such as bispecific antibodies, that can specifically activate the lymphotoxin beta receptor (LTBR) in tumors. The multispecific binding molecule has a first specificity for LTBR and an It has a second specificity for B (extra-domain B: EDB). EDB is a tumor-associated antigen (TAA) of the extracellular matrix. The multispecific binding molecule activates LTBR in tumors expressing EDB, but activates LTBR to a much lower extent than its ligands LIGHT and LTα1β2, or only slightly activates it, or does not activate it at all in the absence of EDB, thereby reducing the risk of undesirable immune-related events. Unlike the aforementioned LTBR-activating molecules, efficient LTBR activation in the presence of EDB is achieved via EDB binding through the EDB-specific portion of the multispecific binding molecule of the present invention and LTBR binding through the LTBR-specific portion of the multispecific binding molecule of the present invention. When EDB is absent, the multispecific binding molecule does not cause LTBR activation in normal tissues. This is a significant advantage over natural LTBR ligands, such as LIGHT, and molecules described in the prior art, which can activate LTBR independently of TAA and thus, as shown in the examples herein, have far fewer tumors specific for LTBR activation compared to the molecules of the present invention.

[0018] Provided herein is a multispecific binding molecule. The multispecific binding molecule can comprise (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, and the multispecific binding molecule activates LTBR upon binding of EDB. More specifically, the multispecific binding molecule, when the multispecific binding molecule binds to LTBR and EDB simultaneously, binds to these targets.​Activates LTBR via each specific binding domain. Preferably, this is 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. The multispecific binding molecule can be, for example, a bispecific antibody. In certain embodiments, the multispecific binding molecule comprises two antigen-binding domains. In certain embodiments, the multispecific binding molecule comprises three antigen-binding domains . The three antigen-binding domains can comprise, for example, one binding domain that specifically binds to LTBR and, for example, two binding domains that specifically bind to EDB . In certain embodiments, the multispecific binding molecule comprises three antigen-binding domains and, for example, additional binding domains in the form of single-chain variable domains are fused to the N-terminus or C-terminus of, for example, the heavy or light chain of an antibody (e.g., in an IgG format)

[0019] and are composed of an antibody. In the multispecific binding molecule of the present invention that specifically binds to LTBR and TAAs present in the extracellular matrix, the TAAs present in the extracellular matrix are fibronectin . Preferably, the binding domain that binds to the TAA specifically binds to the extra domain B (EDB) of fibronectin. In certain non-limiting embodiments, the binding domain that specifically binds to LTBR is the BHA10 antibody or CBE1

[0020] comprising a heavy chain variable region (VH) and a light chain variable region (VL).

[0021] ​​​​​1 An antibody or a fragment or derivative thereof, such as 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 comprising 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 comprising 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 comprising the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR 3 each comprising 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 comprising the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCD R3 each comprising the amino acid sequences of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively ; or (iv) VH comprises an amino acid sequence having at least 95%, 96%, 9 7%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 9 8%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44, for example VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 43 amino acid sequence, and VL comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 44 comprising an acid sequence, wherein VL has at least 95%, 96% , 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 44 ; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 43 comprising an acid sequence, wherein VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 43, wherein VL has at least 9 5%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 43, wherein VL has at least 9 5%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 99 % identity with the amino acid sequence of SEQ ID NO: 43, wherein VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having 100% identity with the amino acid sequence of SEQ ID NO: 43, wherein VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: ; or (v) VH has at least 95%, 96%, 97 comprising an amino acid sequence having 95%, 98%, 99% identity, or 100% identity, VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98 %, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48, e.g., V H comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96% 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 9 6%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 47 comprising an amino acid sequence having identity, wherein VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; or (vi) SEQ ID NO: 22; or (vii) SEQ ID NO: 23; or (viii) SEQ ID NO: 25. (viii) SEQ ID NO: 25.

[0022] In certain non-limiting embodiments, the second binding domain that specifically binds to EDB is e.g., an L19 antibody or a fragment or derivative thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining region 1 (HCDR1), H CDR2, and HCDR3, and VL comprises light chain complementarity determining region 1 (LCDR1), LC DR2, and LCDR3, and the antibody or fragment thereof comprises any of the following: comprising: (i) HCDR1, HCDR2, and HCDR3 each comprising 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 comprising the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or ; or 76, and SEQ ID NO: 77, respectively; or (ii) VH comprises an amino acid sequence having at least 95%, 96%, 9 7%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 9 8%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46, e.g., VH comprises an amino acid having at least 95% identity to the amino acid sequence of SEQ ID NO: 45 ; or ; or comprising an array, wherein VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 9 6%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46.

[0023] In certain non-limiting embodiments, the multispecific binding molecule comprises: (1) The binding domain that specifically binds to LTBR includes the BHA10 antibody or the CBE11 antibody or a fragment or derivative thereof, such as a single-chain antibody fragment (scFv), which contains a heavy-chain variable region (VH) and a light-chain variable region (VL). 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. The VH and VL include any of the following: (i) HCDR1, HCDR2, and HCDR3 each containing the amino acid sequence of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR 3 each containing the amino acid sequence 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 sequence of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCD R3 each containing the amino acid sequence 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 sequence of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LC DR3 each containing the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97% to the amino acid sequence of SEQ ID NO: 44, comprising an amino acid sequence having 98%, 99% identity, or 100% identity, for example VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 43 and VL comprises an amino acid sequence having at least 95%, 96% 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44 ; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 43 and VL comprises an amino acid sequence having at least 95% 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44 ; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43 and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44 ; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 43 and VL comprises an amino acid sequence having at least 95% 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44 ; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 43 and VL comprises an amino acid sequence having at least 95% 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44 ; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 43 and VL comprises an amino acid sequence having at least 95% 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44 comprising an amino acid sequence having the property; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 9 7%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 9 8%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48, for example VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96% 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 9 At least 95%, 96%, 97%, 98%, 99% identity, or 100% identity VH comprises an amino acid sequence having 100% affinity to the amino acid sequence of SEQ ID NO: 47; and VL has at least one amino acid sequence having at least one amino acid sequence identical to the amino acid sequence of SEQ ID NO: 48. At least 95%, 96%, 97%, 98%, 99% identity, or 100% identity or comprising an amino acid sequence having the following structure: (vi) SEQ ID NO: 22; or (vii) SEQ ID NO: 23; or (viii) SEQ ID NO: 25; and (2) The second binding domain that specifically binds to EDB is, for example, a heavy chain variable region (VH ) and a light chain variable region (VL), or a fragment or derivative thereof. VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. VL contains light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. , the antibody or fragment thereof is SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: HCDR1, HCDR2, and HCDR3 containing the amino acid sequence of No. 74, and the sequence LCDR1, LCDR2, LCDR3, LCDR4, LCDR5, LCDR6, LCDR7, LCDR8, LCDR9, LCDR10, LCDR11, LCDR12, LCDR13, LCDR14, LCDR15, LCDR16, LCDR17, LCDR18, LCDR19, LCDR19, LCDR111, LCDR19, LCDR112, LCDR13, LCDR14, LCDR15, LCDR16 2, and LCDR3.

[0024] In certain non-limiting embodiments, the multispecific binding molecule comprises: (1) The binding domain that specifically binds to LTBR consists of a heavy chain variable region (VH) and a light chain variable region (VL). BHA10 antibody or CBE11 antibody, or a fragment thereof, containing a variable region (VL) or derivatives thereof, including single chain antibody fragments (scFv), in which VH is a heavy chain complementarity VL comprises light chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. The VH and VL are as follows: Contains any of the following: (i) the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively; HCDR1, HCDR2, and HCDR3 containing SEQ ID NO: 63, SEQ ID NO: 64, No. 64, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 65. 3; or (ii) the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively and HCDR1, HCDR2, and HCDR3, each comprising SEQ ID NO: 63, sequence LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 64 and SEQ ID NO: 65 R3; or (iii) the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively. HCDR1, HCDR2, and HCDR3 containing the sequences SEQ ID NO: 69, SEQ ID NO: 70, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 70 and SEQ ID NO: 71 DR3; or (iv) VH has at least 95%, 96%, or Contains amino acid sequences with 97%, 98%, 99% identity, or 100% identity and VL is at least 95%, 96%, 97%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. 98%, 99% or 100% identity, e.g., For example, the VH may be an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 43. and VL has at least 95%, 96%, or more amino acid sequence identical to the amino acid sequence of SEQ ID NO: 44. %, 97%, 98%, 99% identity, or 100% identity comprising; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95% 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 9 7%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; or or (v) VH comprises an amino acid sequence having at least 95%, 96%, 9 7%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; comprising an amino acid sequence having 8%, 99% identity, or 100% identity, for example wherein VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ​ comprising an amino acid sequence having the identity of; or (vi) SEQ ID NO: 22; or (vii) SEQ ID NO: 23; or (viii) SEQ ID NO: 25; and, (2) The second binding domain that specifically binds to EDB includes, by way of example, the heavy chain variable region (VH ) and the light chain variable region (VL), an L19 antibody or a fragment or derivative thereof 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 its fragment has at least 9 5%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 45 and includes a VH having an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 46, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 46 and includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 45, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 46 and includes an amino acid sequence having at least 9 6% identity with the amino acid sequence of SEQ ID NO: 45, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 46 and includes an amino acid sequence having at least 9 7% identity with the amino acid sequence of SEQ ID NO: 45, and VL has the amino acid sequence of SEQ ID NO: 46 at least 95%, 96%, 97%, 98%, 99% identity, or 100 % identity amino acid sequence; VH contains an amino acid sequence having at least 98% identity 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% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence having at least 99% identity 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% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH contains an amino acid sequence having 100% identity 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% identity, or [[ID=!4]]100% identity to the amino acid sequence of SEQ ID NO: 46. In certain non-limiting embodiments, the multispecific molecule comprises: (1) a binding domain that specifically binds to LTBR comprising SEQ ID NO: 22; and (2) a second binding domain that specifically binds to EDB, for example, the L19 antibody or a fragment or derivative thereof 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 a fragment thereof comprises any of the following:

[0025] (i) amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively ​​​​​​HCDR1, HCDR2, and HCDR3 containing SEQ ID NO: 75, SEQ ID NO: 8, No. 76, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 77. 3; or (ii) VH has a sequence similar to that of SEQ ID NO: 45, but is at least 95%, 96%, or Contains amino acid sequences with 97%, 98%, 99% identity, or 100% identity and VL is at least 95%, 96%, 97%, or It includes amino acid sequences with 98%, 99% identity, or 100% identity.

[0026] In certain non-limiting embodiments, the multispecific molecule comprises: (1) a binding domain that specifically binds to LTBR comprising SEQ ID NO: 23; and (2) The second binding domain that specifically binds to EDB is, for example, a heavy chain variable region (VH ) and a light chain variable region (VL), or a fragment or derivative thereof. VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. VL contains light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. , the antibody or fragment thereof comprises any of the following: (i) the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively HCDR1, HCDR2, and HCDR3 containing SEQ ID NO: 75, SEQ ID NO: 8, No. 76, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 77. 3; or (ii) VH has a sequence similar to that of SEQ ID NO: 45, but is at least 95%, 96%, or Contains amino acid sequences with 97%, 98%, 99% identity, or 100% identity See, VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46.

[0027] In certain non-limiting embodiments, the multispecific molecule comprises: and (1) a binding domain that specifically binds to LTBR comprising SEQ ID NO: 25; (2) a second binding domain that specifically binds to EDB is, by way of example, a heavy chain variable region (VH ) and a light chain variable region (VL) comprising an L19 antibody or a fragment or derivative thereof 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 comprising the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, and LCDR1, LCDR2, and LCDR 3 each comprising the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77; or (ii) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity 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% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46.

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

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

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

[0031] Also provided are one or more nucleic acid molecules encoding the multispecific binding molecules disclosed herein. Also provided are one or more vectors comprising one or more nucleic acid molecules disclosed herein. Also provided are isolated host cells comprising one or more isolated vectors disclosed herein. Also provided are one or more vectors comprising one or more nucleic acid molecules disclosed herein. Also provided are isolated host cells comprising one or more isolated vectors disclosed herein. Also provided are isolated host cells comprising one or more isolated vectors disclosed herein. .

[0032] A medicament comprising a multispecific binding molecule disclosed herein and a pharmaceutically acceptable carrier A composition is also provided.

[0033] A method for treating cancer in a subject in need of treatment is also provided. The method comprises administering to the 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. 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. 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. Comprising administering.

[0034] Use of 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 for activating LTBR in tumor tissue. 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. Is also provided.

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

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

[0037] In certain embodiments, a multispecific binding molecule such as a bispecific antibody or an antigen-binding fragment thereof forms two binding domains for EDB, two heavy chains (HC) and In certain embodiments, a multispecific binding molecule such as a bispecific antibody or an antigen-binding fragment thereof forms two binding domains for EDB, two heavy chains (HC) and and two light chains (LC).

[0038] In certain embodiments, the scFv is fused to the carboxy (C)-terminus or amino (N)-terminus of one HC. In certain embodiments, the scFv fused to the HC has a sequence selected from SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 56.

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

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

[0041] Also provided is a nucleic acid encoding the heavy chain, light chain, and / or functional fragment of a multispecific binding molecule as disclosed herein.

[0042] Also provided is a vector comprising a nucleic acid molecule disclosed herein.

[0043] Also provided is a host cell comprising a nucleic acid molecule or vector disclosed herein.

[0044] In preferred embodiments, the multispecific binding molecule, bispecific antibody, nucleic acid, vector, or host cell according to the invention is an isolated multispecific binding molecule, an isolated bispecific ​It is a heterologous antibody, an isolated nucleic acid, an isolated vector, or an isolated host cell.

[0045] Also provided is a pharmaceutical composition comprising a multispecific binding molecule such as a bispecific antibody or an antigen-binding fragment thereof as disclosed herein, and a pharmaceutically acceptable carrier. Also provided is a method of treating cancer in a subject in need of treatment. The method comprises (a) identifying a subject in need of cancer treatment, and (b) administering to the subject in need thereof the multispecific binding molecule of the present invention, for example, in the form of a pharmaceutical composition, wherein administering the pharmaceutical composition to the subject in need thereof treats cancer in the subject.

[0046] Also provided is a method of activating LTBR-expressing cells. The method comprises contacting the LTBR-expressing cells with a multispecific binding molecule, for example, in the form of the pharmaceutical composition of the present invention, wherein contacting the LTBR-expressing cells with the multispecific binding molecule or the pharmaceutical composition results in an increase in the 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 as compared to cells expressing LTBR in an environment where EDB is absent. Also provided is a method of inhibiting the growth or proliferation of cancer cells in a tumor expressing EDB. The method comprises 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, wherein contacting the cancer cells and / or cells in the tumor microenvironment with the pharmaceutical composition inhibits the growth or proliferation of the cancer cells. Also provided is a method of inhibiting the growth or proliferation of cancer cells in a tumor expressing EDB. The method comprises 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, wherein contacting the cancer cells and / or cells in the tumor microenvironment with the pharmaceutical composition inhibits the growth or proliferation of the cancer cells. Also provided is a method of inhibiting the growth or proliferation of cancer cells in a tumor expressing EDB. The method comprises 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, wherein contacting the cancer cells and / or cells in the tumor microenvironment with the pharmaceutical composition inhibits the growth or proliferation of the cancer cells.

[0047] Also provided is a method of activating LTBR-expressing cells. The method comprises contacting the LTBR-expressing cells with a multispecific binding molecule, for example, in the form of the pharmaceutical composition of the present invention, wherein contacting the LTBR-expressing cells with the multispecific binding molecule or the pharmaceutical composition results in an increase in the 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 as compared to cells expressing LTBR in an environment where EDB is absent. Also provided is a method of activating LTBR-expressing cells. The method comprises contacting the LTBR-expressing cells with a multispecific binding molecule, for example, in the form of the pharmaceutical composition of the present invention, wherein contacting the LTBR-expressing cells with the multispecific binding molecule or the pharmaceutical composition results in an increase in the 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 as compared to cells expressing LTBR in an environment where EDB is absent. Also provided is a method of activating LTBR-expressing cells. The method comprises contacting the LTBR-expressing cells with a multispecific binding molecule, for example, in the form of the pharmaceutical composition of the present invention, wherein contacting the LTBR-expressing cells with the multispecific binding molecule or the pharmaceutical composition results in an increase in the 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 as compared to cells expressing LTBR in an environment where EDB is absent. Also provided is a method of activating LTBR-expressing cells. The method comprises contacting the LTBR-expressing cells with a multispecific binding molecule, for example, in the form of the pharmaceutical composition of the present invention, wherein contacting the LTBR-expressing cells with the multispecific binding molecule or the pharmaceutical composition results in an increase in the 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 as compared to cells expressing LTBR in an environment where EDB is absent. -8, MIP-3b, ICAM-1, I-TAC, IP-10, IL-12p70, TN F-a, MIP-3a, and / or SDF-1a expression.

[0048] Also provided is a method of inhibiting the growth or proliferation of cancer cells in a tumor expressing EDB. The method comprises 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, wherein contacting the cancer cells and / or cells in the tumor microenvironment with the pharmaceutical composition inhibits the growth or proliferation of the cancer cells. Also provided is a method of inhibiting the growth or proliferation of cancer cells in a tumor expressing EDB. The method comprises 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, wherein contacting the cancer cells and / or cells in the tumor microenvironment with the pharmaceutical composition inhibits the growth or proliferation of the cancer cells. Also provided is a method of inhibiting the growth or proliferation of cancer cells in a tumor expressing EDB. The method comprises 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, wherein contacting the cancer cells and / or cells in the tumor microenvironment with the pharmaceutical composition inhibits the growth or proliferation of the cancer cells. Also provided is a method of inhibiting the growth or proliferation of cancer cells in a tumor expressing EDB. The method comprises 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, wherein contacting the cancer cells and / or cells in the tumor microenvironment with the pharmaceutical composition inhibits the growth or proliferation of the cancer cells.

[0049] Also provided is a method of manufacturing a pharmaceutical composition disclosed herein. The method includes combining an isolated multispecific binding molecule of the invention, such as a bispecific antibody or an antigen-binding fragment thereof, with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition. Also provided is a method of making a multispecific binding molecule such as a bispecific antibody or an antigen-binding fragment thereof. The method includes culturing a host cell containing a nucleic acid disclosed herein under conditions for producing a multispecific binding molecule such as a bispecific antibody or an antigen-binding fragment thereof and recovering the multispecific binding molecule such as a bispecific antibody or an antigen-binding fragment thereof.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The above summary and the following detailed description of the preferred embodiments of the present application will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present application is not limited to the embodiments shown in the drawings.

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MODE FOR CARRYING OUT THE INVENTION

[0052] In the background art, and throughout this specification, various publications, papers, and patents are cited or described, and each of these references is hereby incorporated by reference in its entirety into this specification. The discussions of documents, operations, materials, devices, articles, etc. included in this specification are for the purpose of providing the context of the present invention. Such discussions do not admit that any or all of these things constitute a part of the prior art with respect to any of the inventions disclosed or claimed.

[0053] Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. If not, the specific terms used in this specification have the meanings set forth herein.

[0054] When used in this specification and the appended claims, the singular forms "a", "an", and "t he" are to be noted as including plural referents unless the context clearly dictates otherwise.

[0055] Unless otherwise specified, any numerical values such as concentrations or concentration ranges described in this specification are to be understood as being modified in all instances by the term "about". Thus, the 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 in this specification, the use of a numerical range includes all possible sub-ranges, including integers and fractional values within that range, and all individual numerical values within that range, unless the context clearly indicates otherwise. within that range. Explicitly include.

[0056] Unless otherwise stated, the term "at least" preceding a series of elements is to be understood as referring to all of the elements of the series. One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. It is contemplated.

[0057] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" "includes," "including," "has," "having," ")," "contains," or "containing," or any other variation thereof, are intended to cover the recited integer or group of integers, but are not intended to exclude other integers or groups of integers not recited, and are intended to be non- exclusive or non-limiting. For example, a composition, mixture, process, method, article, or apparatus that includes a series of elements is not necessarily limited to only those elements, and may include other elements not expressly enumerated or inherently present in such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied if A is true (or present) and B is false (or absent), if A is false (or absent) and B is true (or present), and also if both A and B are true (or present). For example, condition A or B is satisfied when A is true (or present) and B is false (or absent), when A is false (or absent) and B is true (or present), and also when both A and B are true (or present). If both A and B are true (or exist), then it is satisfied by one of .

[0058] As used herein, the conjunction "and / or" between multiple listed elements The term is understood to encompass both individual and combined options, e.g. When two elements are connected by "and / or," the first option is the second option, etc. The second option refers to the second option without the first option. The third option indicates that the first and second elements are applicable together. Any one of these options is included in the meaning and therefore and are understood to satisfy the requirement of the term "and / or" as used herein. The simultaneous applicability of two or more of the alternatives is also included in the meaning, hence the use of "and / It is understood that the requirement of the term "or" is met.

[0059] As used herein, the terms "consists of" or "consist" Variations such as "of" or "consisting of" are used throughout the specification and claims. When used throughout the range, it includes any enumerated integer or group of integers, but does not include additional Indicates that no integer or group of integers is added to the specified method, structure, or composition.

[0060] As used herein, the term "consists essentially of" or "consist essentially of" or "consist essentially of" Variations such as "isting essentially of" and "is not" are used throughout the specification and claims. When used, it encompasses any enumerated integer or group of integers, and optionally includes the specified method, structure, or any enumerated integer or that does not materially alter the basic or novel characteristics of the composition. Indicates inclusion of the group of integers. See MPEP §2111.03.

[0061] As used herein, a "subject" refers to any animal, preferably a mammal, most preferably a mammalian animal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, any mammal. Examples of mammals include, but are not limited to: , cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys , humans, etc., preferably humans.

[0062] The term "about" as used herein when referring to dimensions or characteristics of components of the preferred invention The terms "approximately," "generally," "substantially," and the like, as will be understood by those skilled in the art, are intended to The dimensions / features listed are not strict boundaries or parameters, but are functionally the same or similar. It should also be understood that this does not exclude slight variations from the above. By value, such references involving numerical parameters are understood to be within the scope of art-accepted principles. Mathematical and industrial principles (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, etc.) that are used When using a digit, the least significant figure will include the variant that does not change.

[0063] The terms "identical" or percent "identity" refer to the degree to which two or more nucleic acids or polypeptides peptide sequences (e.g., anti-LTBR bispecific antibodies and polynucleotides encoding same) , anti-LTBR / anti-EDB bispecific antibody and polynucleotide encoding the same, LTB R polypeptide and LTBR / polynucleotide encoding the same, EDB polypeptide and EDB polynucleotide encoding the same), when measured using one of the following sequence comparison algorithms or by visual inspection, refers to two or more sequences or subsequences that are the same or a specific percentage of the same amino acid residues or nucleotides when compared and aligned to maximize identity [[ID=ll]].

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

[0065] [[ID=!6]] Optimal alignment of sequences for comparison is achieved, for example, using 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. Natl. Acad. Sci. USA 85:2444 (1988), or computerized implementations of these algorithms (Wisconsin Genetics s Software Package, Genetics Computer Gro up, Genetics Computer Group , Madison, Wis.), or by visual inspection. up, Madison, Wis.).​ GAP, BES in up, 575 Science Dr., Madison, WI TFIT, FASTA, and TFASTA), or visual inspection (generally, Current Protocols in Molecular Biology, F.M. Ausu bel et al., eds., Current Protocols, a join t venture between Greene Publishing Asso ciates, Inc. and John Wiley & Sons, Inc., (1 995 Supplement)(Ausubel) should be referred to). It can be done.

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

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

[0068] In addition to calculating percent sequence identity, the BLAST algorithm also calculates the percent sequence identity between two sequences. Statistical analysis of similarities between columns is also performed (e.g., Karlin & Altschul, Pro c. Nat'l. Acad. Sci. USA 90:5873-5787(1993) One measure of similarity provided by the BLAST algorithm is the minimum sum probability. The probability of a sequence fragment being amplified (P(N)) between two nucleotide sequences or two amino acid sequences is provides an indication of the probability that a match would occur by chance. For example, nucleic acids may be expressed as a ratio of a test nucleic acid to a reference nucleic acid. The minimum sum probability in the comparison is less than about 0.1, more preferably less than about 0.01, and most preferably A sequence is considered similar to a reference sequence if its nucleotide sequence is less than about 0.001.

[0069] Further indications that two nucleic acid sequences or polypeptides are substantially identical include the following: As described above, the polypeptide encoded by the first nucleic acid is encoded by the second nucleic acid. Therefore, the polypeptide is immunologically cross-reactive with the polypeptide to be synthesized. The peptide is typically substantially identical to the second polypeptide, e.g., the two peptides Another indication that two nucleic acid sequences are substantially identical is that the two The ability of two molecules to hybridize to each other under stringent conditions.

[0070] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid molecule." , also called "nucleotides" or "nucleic acids," and may be unmodified RNA or DNA or modified RNA or DNA. Any polyribonucleotide or polydeoxyribonucleotide, which may be NA or DNA. "Polynucleotide" refers to a nucleotide, but is not limited to these. , a single-stranded and double-stranded DNA, a DNA that is a mixture of single-stranded and double-stranded regions, a single-stranded and double-stranded RNA, and an RNA that is a mixture of single-stranded and double-stranded regions, a single-stranded or more typ ically double-stranded or a mixture of single-stranded and double-stranded regions, including DNA and RNA that may be hybrid molecules. In addition, "polynucleotide" refers to a triple-stranded region containing RNA or D NA 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. Thus, "polynucleotide" typically includes chemically, enzymatically, or metabolically modified forms of polynucleotides found in nature, as well as chemical forms having the characteristics of viral and cellular DNA and RNA. "Polynucleot ide" also includes relatively short nucleic acid strands (often referred to as oligonucleotides). When used herein, the term "vector" refers to a replicon that can replicate or express another nucleic acid segment by functionally inserting it. When used herein, the term "host cell" refers to a cell containing a nucleic acid molecule of the present invention. "Host cell" can be, for example, any type of cell, such as a primary cell, a cell in culture, or a cell derived from a cell line. In one embodiment, "host cell" contains the nucleic acid molecule of the present invention. "Host cell" also includes relatively short nucleic acid strands (often referred to as oligonucleotides).

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

[0072] As used herein, the term "host cell" refers to a cell containing a nucleic acid molecule of the present invention. "Host cell" can be, for example, any type of cell, such as a primary cell, a cell in culture, or a cell derived from a cell line. In one embodiment, "host cell" contains the nucleic acid molecule of the present invention. ​​It is a transfected cell. In another embodiment, a "host cell" is a progeny or potential progeny of such a transfected cell. The progeny of a cell may, for example, have mutations or environmental influences that occur in subsequent generations, or may not have the same identity as the parental cell due to integration of a nucleic acid molecule into the host cell genome.

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

[0074] As used herein, the terms "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. In this specification, the conventional one-letter or three-letter codes for amino acid residues are used. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention. Examples of intervention include, for example, disulfide bond formation, or modification by introduction of non-natural amino acids. Examples of intervention include, for Conformation formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, for example, conjugates with labeling components. Also included in the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), and other modifications known in the art.

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

[0076] As used herein, a "multispecific binding molecule" means a molecule that specifically binds to at least two different molecules. Preferably, the molecule is a protein including, by way of 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 view of the presence of binding specificity for LTBR, is sometimes referred to herein as an "anti-LTBR" binding molecule or antibody.

[0077] As used herein, a "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, and are formed by two or more chains of an antibody, for example, the variable domain of the heavy chain paired with the variable domain of the light chain, or by an scFv molecule or by single chains such as VHHs from llamas, e.g. nanobodies. The target molecule of the present invention may be formed by LTBR or fibronectin. The EDB of fibronectin, especially fibronectin.

[0078] The term "specific binding," as used herein, refers to the ability of an antibody to bind to a given antigen and to bind to other antigens. Typically, antibodies bind with a higher affinity than the original antibody. - 7 M or less, e.g., about 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 More than M Bottom, about 1×10 -11 M or less, approximately 1×10 -12 M or less, approximately 1×10 -13 M or less, or Approximately 1×10 -14 Dissociation constant (K D ) binds to the specified antigen and non-specific antigen For binding to an epitope (e.g., BSA casein), typically its K D At least 10 times lower K D The dissociation constant should be determined using standard procedures. However, antibodies that specifically bind to a given antigen may also bind to other related antigens, e.g. Humans or monkeys, e.g., Macaca fascicularis (cynomolgus monkey, cyn o) or other species such as Pan troglodytes (chimps) These may have cross-reactivity to the same given antigen (homologue).

[0079] The term "tumor-associated antigen" or "TAA" as used herein refers to a tumor-associated antigen (TAA) found on tumor cells. This refers to an antigen present in the extracellular matrix of a normal tumor or in the extracellular matrix of a tumor. not in a form that is qualitatively different from antigens found on cells or in the extracellular matrix of normal tissues but is quantitatively different in several respects, e.g., present in significantly greater amounts, at higher density, at different sites of expression, and / or differentially accessible to the immune system, etc., on tumor cells or in the extracellular matrix of tumors In certain embodiments, a tumor - associated antigen is present on tumor cells or in the tumor extracellular matrix in at least two - fold greater amount, more preferably at least five - fold greater amount, e.g., at least ten - fold greater amount, even more preferably at least one - hundred - fold greater amount, e.g., at least one - thousand - fold greater amount, most preferably at least ten - thousand - fold greater amount than on non - tumor cells or on the extracellular matrix. EDB is present in fibronectin in the extracellular matrix of tumor tissue, while typically undetectable in the fibronectin form present in normal tissues (i.e., the same tissue under normal conditions and not in a tumor environment). The term "extracellular matrix" as used herein means the non - cellular component present in all tissues and organs, in the form of a three - dimensional network of extracellular macromolecules such as collagen, enzymes, and glycoproteins, which provides structural and biochemical support to surrounding cells. Its exact composition varies by tissue, but 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. Two main classes of extracellular matrix molecules are matrix proteoglycans and fibrous proteins (e.g., collagen, elastin, fibronectin, and laminin).

[0080] When used herein, the term "extracellular matrix" refers to the non - cellular component present in all tissues and organs, in the form of a three - dimensional network of extracellular macromolecules such as collagen, enzymes, and glycoproteins, which provides structural and biochemical support to surrounding cells. Its exact composition varies by tissue, but 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. Two main classes of extracellular matrix molecules are matrix proteoglycans and fibrous proteins (e.g., collagen, elastin, fibronectin, and laminin). The term "extracellular matrix" as used herein means the non - cellular component present in all tissues and organs, in the form of a three - dimensional network of extracellular macromolecules such as collagen, enzymes, and glycoproteins, which provides structural and biochemical support to surrounding cells. Its exact composition varies by tissue, but 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. Two main classes of extracellular matrix molecules are matrix proteoglycans and fibrous proteins (e.g., collagen, elastin, fibronectin, and laminin). comprising).

[0081] antibody The present invention generally relates to anti-LTBR multispecific binding molecules, nucleic acids encoding the multispecific binding molecules and expression vectors, recombinant cells containing the vectors, and compositions comprising the multispecific binding molecules. In preferred embodiments, the anti-LTBR multispecific binding molecule is an anti-LTBR multispecific antibody such as an anti-LTBR bispecific antibody or an antigen-binding fragment thereof . In certain embodiments, the anti-LTBR multispecific binding molecule can comprise a binding domain that specifically binds to LTBR in a form different from an antibody or a functional fragment thereof , for example, these can be anti-LTBR Fynomers, anti-LTBR Affimers, anti-LTBR Darrpins, and / or other protein scaffolds that have been screened for candidates that specifically bind to LTBR . In the multispecific binding molecules of the present invention, the binding domain specific for LTBR is not provided by LIGHT or LTα1β2 (the natural ligand of LTBR) or by a functional fragment or derivative thereof such as 3xhmLIGHT . In preferred embodiments, the binding domain specific for LTBR in the multispecific binding molecules of the present invention comprises an antibody against LTBR, preferably an agonist antibody against LTBR, or a functional fragment or derivative thereof such as an scFv. Agonist antibodies against LTBR themselves have been described , non-limiting examples are BHA10 (e.g., WO 2004002431) and CBE 11 (e.g., WO 0230986), or alternatively, immunization of mice . In the multispecific binding molecules of the present invention, the binding domain specific for LTBR is not provided by LIGHT or LTα1β2 (the natural ligand of LTBR) or by a functional fragment or derivative thereof such as 3xhmLIGHT . In preferred embodiments, the binding domain specific for LTBR in the multispecific binding molecules of the present invention comprises an antibody against LTBR, preferably an agonist antibody against LTBR, or a functional fragment or derivative thereof such as an scFv. Agonist antibodies against LTBR themselves have been described , non-limiting examples are BHA10 (e.g., WO / 2004 / 002431) and CBE 11 (e.g., WO 0230986), or alternatively, immunization of mice . Agonist antibodies against LTBR themselves have been described, non-limiting examples are BHA10 (e.g., International Publication No. 2004002431) and CBE 11 (e.g., International Publication No. 0230986), or alternatively, immunization of mice , can be generated according to known methods for antibody production such as phage display to be.

[0082] The Fyn SH3-derived polypeptide or "Fynomer" is well-known in the art and is described, for example, in Grabulovski et al. (2007), "JBC", Vol. 282, No. 3196 - 3204; International Publication No. 2008 / 022759; Bertschinge r et al. (2007), "Protein Eng Des Sel", Vol. 20 (No. 2 ): pp. 57 - 68; and Gebauer and Skerra (2009), "Cu rr Opinion in Chemical Biology", Vol. 13: pp. 24 5 - 255. The term "Fynomer" and the term "Fyn SH3-derived polypeptide" used interchangeably herein refer to a non-immunoglobulin-derived binding polypeptide (e.g., as described in Gebauer and S kerra (2009), "Curr Opinion in Chemical B iology", Vol. 13: pp. 245 - 255) derived from the human Fyn SH3 domain. Fynomer is a small globular polypeptide of about 7 kDa. The SH3 domain of human Fyn kinase binds to different target proteins (International Publication Nos. 2008 / 022759 , 2011 / 023685, 2013 / 135588, 2014 / 1 70063, Grabulovski D. et al. (2007), "J Biol Ch em", Vol. 282, pp. 3196 - 3204, Bertschinger J. et al. (20 07), "Protein Eng Des Sel", Vol. 20, pp. 57 - 68, and et al. (2014 / 1 and is described in detail in the following documents: Grabulovski D. et al. (2007), "J Biol Chem", Vol. 282, pp. 3196 - 3204; Bertschinger J. et al. (20 07), "Protein Eng Des Sel", Vol. 20, pp. 57 - 68; and et al. (2014 / 1 and Schlatter et al. (2012) "mAbs", Vol. 4 (No. 4), pp. 497-50 pages) was successfully used as a scaffold for engineering a protein (Fyn SH3-derived binding protein called Fynomer) that binds with high affinity and specificity.

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

[0084] DARPins (in the case of designed ankyrin repeat proteins) are typically highly specific protein binders and are genetically engineered antibody mimetic proteins derived from natural ankyrin proteins. DARPins consist of at least three repeat motifs and typically their molecular weights are about 14 kDa or 18 kDa for 4- or 5-repeat DARPins, respectively. The DARPin design is described, for example, in Binz et al., 20 03, "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 of certain embodiments of the multispecific binding molecules of the present invention.

[0086] In a preferred embodiment of the present invention, the binding domain that binds to LTBR binds LT during binding​​​​​​​​​ activates BR and is derived from an antibody, preferably an agonist antibody that specifically binds to LTBR to do. In a specific embodiment, the binding domain that binds to LTBR is the single-chain variable do of the antibody main (scFv), and the scFv can be stabilized by any available format, such as those described above and / or by the methods described herein.

[0087] In certain embodiments, the present invention provides an anti-LTBR / anti-EDB bispecific antibody or antigen-binding fragment thereof, a nucleic acid encoding the antibody and an expression vector, a recombinant cell containing the vector, and a composition containing the bispecific antibody. Also provided are methods of making multispecific binding molecules and / or antibodies, and methods of using multispecific binding molecules and / or antibodies for treating diseases including cancer. The multispecific binding 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 for LTBR and EDB, and / or the ability to treat or prevent cancer when administered alone or in combination with other anti-cancer therapies. to do. In a specific embodiment, the binding domain that binds to LTBR is the single-chain variable do of the antibody main (scFv), and the scFv can be stabilized by any available format, such as those described above and / or by the methods described herein. Also provided are methods of making multispecific binding molecules and / or antibodies, and methods of using multispecific binding molecules and / or antibodies for treating diseases including cancer. The multispecific binding 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 for LTBR and EDB, and / or the ability to treat or prevent cancer when administered alone or in combination with other anti-cancer therapies. The multispecific binding 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 for LTBR and EDB, and / or the ability to treat or prevent cancer when administered alone or in combination with other anti-cancer therapies. to do. In a specific embodiment, the binding domain that binds to LTBR is the single-chain variable do of the antibody main (scFv), and the scFv can be stabilized by any available format, such as those described above and / or by the methods described herein.

[0088] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulins, or human, humanized, chimeric, and bispecific antibodies, which are monoclonal or polyclonal, and includes immunoglobulin or antibody molecules containing antigen-binding domains. Generally, an antibody is a protein or peptide chain that exhibits binding specificity for a particular antigen. The structure of an antibody is known. Immunoglobulins are classified into five main classes according to the amino acid sequence of the heavy chain constant domain It can be assigned to a class (i.e., IgA, IgD, IgE, IgG, and IgM). IgA and IgG can be further subclassified as the isotypes IgA1, IgA2, IgG1, IgG 2, IgG3, and IgG4. Thus, the antibodies of the present invention can be of any of the five major classes or corresponding subclasses. The antibodies of the present invention are preferably IgG1, IgG2, IgG3, or IgG4. Antibody light chains of vertebrate species can be assigned to one of two distinct types based on the amino acid sequence of their constant domains, i.e., either κ or λ. Thus, the antibodies of the present invention can contain a κ or λ light chain constant domain. According to certain embodiments, the antibodies of the present invention contain heavy and / or light chain constant regions derived from rat or human antibodies. In addition to the heavy and light constant domains, the antibody contains an antigen-binding region consisting of 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, C DR2, and CDR3). The light chain variable region domain is alternatively referred to as LCDR1, L CDR2, and LCDR3, and the heavy chain variable region domain is alternatively referred to as HCDR1, H CDR2, and HCDR3. As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds to LTBR is substantially free of antibodies that do not bind to LTBR; an isolated bispecific antibody that specifically binds to LTBR and / or EDB is substantially free of bispecific antibodies that do not bind to LTBR and / or EDB). Further, an isolated antibody is substantially free of other cellular materials and / or chemicals

[0089] When used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds to LTBR is substantially free of antibodies that do not bind to LTBR; an isolated bispecific antibody that specifically binds to LTBR and / or EDB is substantially free of bispecific antibodies that do not bind to LTBR and / or EDB). Further, an isolated antibody is substantially free of other cellular materials and / or chemicals antibodies (e.g., an isolated bispecific antibody that specifically binds to LTBR is substantially free of antibodies that do not bind to LTBR; an isolated bispecific antibody that specifically binds to LTBR and / or EDB is substantially free of bispecific antibodies that do not bind to LTBR and / or EDB). Further, an isolated antibody is substantially free of other cellular materials and / or chemical substances ; an isolated bispecific antibody that specifically binds to LTBR and / or EDB is substantially free of bispecific antibodies that do not bind to LTBR and / or EDB). Further, an isolated antibody is substantially free of other cellular materials and / or chemical substances ; an isolated bispecific antibody that specifically binds to LTBR and / or EDB is substantially free of bispecific antibodies that do not bind to LTBR and / or EDB). Further, an isolated antibody is substantially free of other cellular materials and / or chemical substances ; an isolated antibody is substantially free of other cellular materials and / or chemical substances Is not substantially included.

[0090] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies i.e., the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in trace amounts. The monoclonal antibodies of the present invention can be produced by hybridoma methods, phage display technology, single lymphocyte gene cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be generated by hybridomas containing B cells obtained from transgenic non-human animals, such as transgenic mice or rats, and having a genome containing a human heavy chain transgene and a light chain transgene. In certain embodiments, monoclonal antibodies are produced by recombinant host cells that express a nucleic acid sequence encoding the antibody. Such recombinant host cells can be obtained, for example, by transfection of a nucleic acid sequence into a parental cell, such as a CHO cell . The recombinant host cells can be cultured under conditions that contribute to the expression of the antibody within the host cell, and the antibody can be isolated from the host cell, the culture medium, or both. In certain embodiments, the multispecific binding molecules of the present invention comprise an antibody or one or more of its antigen-binding fragments. As used herein, the term "antigen-binding fragment" refers to, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments , disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies .

[0091] In certain embodiments, the multispecific binding molecules of the present invention comprise an antibody or one or more of its antigen-binding fragments. As used herein, the term "antigen-binding fragment" refers to, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments , disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies , (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies ) Single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure, such as an antibody fragment. 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 an Fd fragment of a heavy chain. According to other certain embodiments, the antigen-binding fragment comprises Fab and F(ab’). In some embodiments, the antigen-binding fragments include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include formed from a portion of an antibody containing one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure, such as an antibody fragment. 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 an Fd fragment of a heavy chain. According to other certain embodiments, the antigen-binding fragment comprises Fab and F(ab’). In some embodiments, the antigen-binding fragments include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include 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 an Fd fragment of a heavy chain. According to other certain embodiments, the antigen-binding fragment comprises Fab and F(ab’). In some embodiments, the antigen-binding fragments include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include According to certain embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd fragment of a heavy chain. According to other certain embodiments, the antigen-binding fragment comprises Fab and F(ab’). In some embodiments, the antigen-binding fragments include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include a light chain variable region, a light chain constant region, and an Fd fragment of a heavy chain. According to other certain embodiments, the antigen-binding fragment comprises Fab and F(ab’). In some embodiments, the antigen-binding fragments include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH / 3 domain molecules include According to other certain embodiments, the antigen-binding fragment comprises Fab and F(ab’). In some embodiments, the antigen-binding fragments include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include In some embodiments, the antigen-binding fragments include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include IgG-like molecules having complementary CH3 domains for heterodimer formation, both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or fused to another protein or carrier molecule). In some embodiments, the IgG-like molecules having complementary CH3 domain molecules include IgG-like molecules having complementary CH3 domains for heterodimer formation, recombinant IgG-like bispecific molecules (both sides of this molecule each contain a Fab fragment or a part of a Fab fragment of at least two different antibodies), IgG fusion molecules (a full-length IgG antibody is fused to an extra Fab fragment or a part of a Fab fragment), Fc fusion molecules (a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or a part thereof), Fab fusion molecules (different Fab fragments are fused together), ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are​​​​​​​​ , Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-in-Holes (Genentech), Cros sMAbs (Roche), and electrostatically engineered ones (Amgen), LUZ-Y (G enentech), strand-exchanged and engineered domain bodies (SEEDbody) (EMD Serono), Biclonic (Merus), or DuoBody (G enmab A / S, for example, Labrijn et al., 2013, PNAS 110:514 5 - 5150). In some embodiments, the antigen-binding fragment includes "stapled single-chain Fv" or "scFv" and refers to an scFv that includes one or more disulfide bonds between VH and the linker or between VL and the linker. Typically , an scFv may include one disulfide bond between VH and the linker, one disulfide bond between VL and the linker, or two disulfide bonds between VH and the linker and between VL and the linker. An scFv molecule that includes a disulfide bond between VH and VL is excluded from "scFv".

[0092] As used herein, the term "single-chain antibody" refers to a single-chain antibody conventionally known in the art that includes a heavy-chain variable region and a light-chain variable region connected by a short peptide of, for example, 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 that includes a heavy-chain variable region and a heavy-chain constant region or includes only the heavy-chain variable region.

[0093] In certain embodiments, the multispecific binding molecules of the invention comprise antibodies having one or more mutations in the Fc that suppress binding to Protein A. Such mutations facilitate the production of heterodimers and are described, for example, in WO 2010151792. As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, antigen-binding fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide. As used herein, the term "humanized antibody" means a non-human antibody that retains the antigen-binding properties of the antibody but has been modified to increase sequence homology to a human antibody such that the antigenicity of the antigen in the human body is reduced.

[0094] As used herein, the term "chimeric antibody" refers to an antibody whose amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of an antibody from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of an antibody from another species of mammal (e.g., human) to avoid eliciting an immune response in that species. As used herein, the term "multispecific antibody" refers to an antibody having multiple immunoglobulin variable domains.

[0095]

[0096]

[0097] Refers to an antibody comprising an array, wherein the first immunoglobulin variable domain sequence among a plurality has binding specificity for a first epitope, and the second immunoglobulin variable domain sequence among the plurality has binding specificity for a second epitope. In certain 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 multimeric protein). In certain specific 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, trispecific antibody molecule, or tetravalent antibody molecule. 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 for a first epitope (e.g., an epitope on the LTBR antigen), and a second immunoglobulin variable domain having binding specificity for a second

[0098] epitope (e.g., an epitope on EDB). In one embodiment, the bispecific antibody comprises a first heavy chain variable domain and a first light chain variable domain that form a binding domain having binding specificity for the first epitope, and a second heavy chain variable domain and a second light chain variable domain that form a binding domain having binding specificity for the second epitope. In certain embodiments, the bispecific antibody comprises a first epitope (e.g., an epitope on the LTBR antigen), and a second immunoglobulin variable domain having binding specificity for a second epitope (e.g., an epitope on EDB). In one embodiment, the bispecific antibody comprises a first heavy chain variable domain and a first light chain variable domain that form a binding domain having binding specificity for the first epitope, and a second heavy chain variable domain and a second light chain variable domain that form a binding domain having binding specificity for the second epitope. In certain embodiments, the bispecific antibody comprises a first heavy chain variable domain and a first light chain variable domain that form a binding domain having binding specificity for the first epitope, and a second heavy chain variable domain and a second light chain variable domain that form a binding domain having binding specificity for the second epitope. In certain embodiments, the bispecific antibody comprises a first A half antibody or a fragment thereof having binding specificity for a pitoep, and a second epitope It comprises a half antibody or a fragment thereof having binding specificity for. In certain embodiments, The bispecific antibody is an scFv or a fragment thereof having binding specificity for a first epitope, or a fragment thereof And an scFv or a fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody has binding specificity for a first epitope It comprises an scFv or a fragment thereof and an scFv or a fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody has binding specificity for a first epitope It comprises an scFv or a fragment thereof and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In a preferred embodiment of the present invention The first epitope is located on LTBR and the second epitope is on fibronectin In particular, it is located on its EDB.

[0099] In certain embodiments, the multispecific binding molecule according to the present invention comprises an antibody, for example an IgG having an scFv fused thereto The scFv may, in certain embodiments, have binding specificity for LTBR Both arms (including the variable region) of the antibody may, in certain embodiments, bind to the EDB of fibronectin. The scFv may be fused to the light chain or the heavy chain of the antibody, and may be fused to the N-terminus or C-terminus of the heavy chain or light chain It may also be. 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. For example, 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 It will be apparent to those skilled in the art based on the present disclosure that other forms are also possible, such as 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 the present disclosure that other forms are also possible One arm that specifically binds to LTBR and the other arm that specifically binds to EDB. For example, 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 It will be apparent to those skilled in the art based on the present disclosure that other forms are also possible, such as fusing an scFv that specifically binds to EDB to one of the antibody chains It will be apparent to those skilled in the art based on the present disclosure that other forms are also possible.​

[0100] As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession number P36941. As used herein, the term "LTBR" refers to a polypeptide that is a cell surface receptor for lymphotoxin 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 lineage cells. LTBR can specifically bind to the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can induce apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin 8. Unless otherwise stated, preferably, LTBR is human LTBR. The human LTBR amino acid sequence is provided by UniProt accession 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 pre-mRNA. Extra domain B is a complete fibronectin (FN) type III repeat containing 91 amino acid residues. Generally, EDB is undetectable in normal adult tissues but shows greater expression in fetal and tumor tissues in the extracellular matrix and accumulates around the new vasculature during the angiogenesis process, so EDB is 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 pre-mRNA. Extra domain B is a complete fibronectin (FN) type III repeat containing 91 amino acid residues. Generally, EDB is undetectable in normal adult tissues but shows greater expression in fetal and tumor tissues in the extracellular matrix and accumulates around the new vasculature during the angiogenesis process, so EDB is 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 pre-mRNA. Extra domain B is a complete fibronectin (FN) type III repeat containing 91 amino acid residues. Generally, EDB is undetectable in normal adult tissues but shows greater expression in fetal and tumor tissues in the extracellular matrix and accumulates around the new vasculature during the angiogenesis process, so EDB is 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 pre-mRNA. Extra domain B is a complete fibronectin (FN) type III repeat containing 91 amino acid residues. Generally, EDB is undetectable in normal adult tissues but shows greater expression in fetal and tumor tissues in the extracellular matrix and accumulates around the new vasculature during the angiogenesis process, so EDB is 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 pre-mRNA. Extra domain B is a complete fibronectin (FN) type III repeat containing 91 amino acid residues. Generally, EDB is undetectable in normal adult tissues but shows greater expression in fetal and tumor tissues in the extracellular matrix and accumulates around the new vasculature during the angiogenesis process, so EDB is 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 pre-mRNA. Extra domain B is a complete fibronectin (FN) type III repeat containing 91 amino acid residues. Generally, EDB is undetectable in normal adult tissues but shows greater expression in fetal and tumor tissues in the extracellular matrix and accumulates around the new vasculature during the angiogenesis process, so EDB is Potential markers and targets for angiogenesis. Unless otherwise stated, preferably, ED B is human EDB. Human E DB is provided by UniProt number P02751.

[0102] The term "fibronectin" refers to a polypeptide that is a high-molecular-weight glycoprotein 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 proteoglycan. Fibronectin can exist as a protein dimer consisting of two nearly identical monomers linked by a pair of disulfide bonds. Fibronectin is produced from a single gene, but alternative splicing of fibronectin pre-mRNA molecules creates several isoforms of fibronectin, one of which is EDB fibronectin. Fibronectin can play a role in cell adhesion, growth, migration, and differentiation and can be important for processes such as wound healing and embryogenesis. The amino acid sequence of human fibronectin is provided by UniProt number P02751, which contains exon domain B, as well as NCBI accession numbers NP_001263337 (isoform B), N P_001263338 (isoform c), NP_001263339 (isoform d), NP_001263340 (isoform e), and NP_0012633 41 (isoform f), NP_001293058 (isoform 8), NP_0 is produced from a single gene, but alternative splicing of fibronectin pre-mRNA molecules creates several isoforms of fibronectin, one of which is EDB fibronectin. Fibronectin can play a role in cell adhesion, growth, migration, and differentiation and can be important for processes such as wound healing and embryogenesis. The amino acid sequence of human fibronectin is provided by UniProt number P02751, which contains exon domain B, but alternative splicing of fibronectin pre-mRNA molecules creates several isoforms of fibronectin, one of which is EDB fibronectin. Fibronectin can play a role in cell adhesion, growth, migration, and differentiation and can be important for processes such as wound healing and embryogenesis. The amino acid sequence of human fibronectin is provided by UniProt number P02751, which contains exon domain B, and NCBI accession numbers NP_001263337 (isoform B), NP_001263338 (isoform c), NP_001263339 (isoform d), NP_001263340 (isoform e), and NP_0012633 41 (isoform f), NP_001293058 (isoform 8), NP_0 and NCBI accession numbers NP_001263337 (isoform B), N P_001263338 (isoform c), NP_001263339 (isoform d), NP_001263340 (isoform e), and NP_0012633 41 (isoform f), NP_001293058 (isoform 8), NP_0 01293059 (Isoform 9), NP_001293060 (Isoform 1 0), NP_001293061 (Isoform 11), and NP_002017 (a Isoform 3) are provided by.

[0103] As used herein, an antibody or binding molecule that "specifically binds to LTBR" binds to LT BR, preferably human LTBR, with a KD of 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1×10 M or less and includes an antibody or molecule containing its antigen-binding domain that binds. -10 The term "KD" is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and refers to the dissociation constant expressed as molar concentration (M). The KD value of an antibody can be determined using methods in the art in view of the present disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using the biosensor system of the BIACORE® system, or by using any biolayer interferometry technique such as the Octet RED96 system. In a preferred embodiment, the binding domain specific for 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 or derivative thereof such as scFv. As used herein, an "agonist antibody against LTBR" binds to LTBR and directly or specific binding domain for LTBR is an antibody against LTBR, preferably an agonist antibody against LTBR, or a functional fragment or derivative thereof such as scFv. As used herein, an "agonist antibody against LTBR" binds to LTBR and directly or indirectly activates LTBR function. is in any of the higher-order clustering, for example, fixation to a solid support, use of a cross-linked antibody, etc. An antibody that can induce downstream signal transduction by any of the following. Against LTBR The agonist antibody itself has been described, and non-limiting examples are BHA10 (e.g., International Publication No. 2004002431), CBE11 (e.g., International Publication No. 0230986), R EA412 (commercially available from Miltenyi Biotec), 31G4D8 (commercially available from BioLeg end), and 71319 / MAB629 (commercially available from Novus Biological s), or alternatively, can be generated according to known methods of antibody production such as mouse immunization, phage display, etc.

[0104] As used herein, an antigen-binding domain or antigen-binding fragment that "specifically binds to EDB" has a KD of 1×10 -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1×1 0 -10 M or less and binds to EDB (e.g., in the formation of EDB fibronectin ) refers to an antigen-binding domain or antigen-binding fragment.

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

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

[0107] According to certain aspects of the present invention, multispecific binding molecules are provided herein. Multispecific binding molecules comprise (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, and the multispecific binding molecule activates LTBR upon binding to EDB. In certain embodiments, the multispecific binding molecule activates LTBR in a tumor - specific manner. Activating LTBR in a tumor - specific manner, as used herein, means that upon simultaneous binding of the multispecific binding molecule to LTBR and EDB, both present in the tumor microenvironment either on the surface of cells or in the extracellular matrix, LTBR is activated to cause signal transduction via standard

[0108] and / or non - standard NF - kB pathways. Activation of the NF - kB pathway can lead to the establishment of an inflammatory tumor microenvironment via the secretion of inflammatory chemokines and cytokines and the expression of adhesion molecules on the surface of cells. Simultaneous binding of the multispecific binding molecule results in activation of LTBR in tumors. E DB is either not present in normal tissue, i.e., normal cells, or in the extracellular matrix adjacent to normal tissue. Activation of the NF - kB pathway can lead to the establishment of an inflammatory tumor microenvironment via the secretion of inflammatory chemokines and cytokines and the expression of adhesion molecules on the surface of cells. Simultaneous binding of the multispecific binding molecule results in activation of LTBR in tumors. E DB is either not present in normal tissue, i.e., normal cells, or in the extracellular matrix adjacent to normal tissue. DB is either not present in normal tissue, i.e., normal cells, or in the extracellular ​When not present in the matrix, the multispecific binding molecule can bind only to LTBR on normal tissue and does not result in LTBR activation. This can activate LTBR independent of TAA, and thus, as shown in the examples herein, there are far fewer tumors specific for LTBR activation compared to the molecules of the present invention, which is a significant advantage over prior art described molecules, such as those based on LIGHT antibody fusions. In certain embodiments, the multispecific binding molecule comprises a bispecific antibody comprising two binding domains, e.g., 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, e.g., one binding to LTBR and two bindings to EDB. In certain embodiments, the multispecific binding molecule comprises three binding domains. In certain embodiments, the three binding domains are all 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 are present in a 2:1 stoichiometry. The three antigen-binding domains can comprise, for example, one first binding domain that specifically binds to LTBR on LTBR-expressing cells. The three antigen-binding domains can comprise, for example, two second binding domains that specifically bind to EDB. In certain embodiments, the two second binding domains have the same binding specificity for EDB, e.g., the two second binding domains can be identical. In certain embodiments, the multispecific binding molecule can activate LTBR independent of TAA, and thus, as shown in the examples herein, there are far fewer tumors specific for LTBR activation compared to the molecules of the present invention, which is a significant advantage over prior art described molecules, such as those based on LIGHT antibody fusions. In certain embodiments, the multispecific binding molecule can activate LTBR independent of TAA, and thus, as shown in the examples herein, there are far fewer tumors specific for LTBR activation compared to the molecules of the present invention, which is a significant advantage over prior art described molecules, such as those based on LIGHT antibody fusions. In certain embodiments, the multispecific binding molecule can activate LTBR independent of TAA, and thus, as shown in the examples herein, there are far fewer tumors specific for LTBR activation compared to the molecules of the present invention, which is a significant advantage over prior art described molecules, such as those based on LIGHT antibody fusions. In certain embodiments, the multispecific binding molecule can activate LTBR independent of TAA, and thus, as shown in the examples herein, there are far fewer tumors specific for LTBR activation compared to the molecules of the present invention, which is a significant advantage over prior art described molecules, such as those based on LIGHT antibody fusions.

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

[0110] According to certain aspects, an isolated anti-lymphotoxin beta receptor (LTBR) bispecific antibody or antigen-binding fragment thereof is provided herein. In certain non-limiting embodiments, the binding domain that specifically binds to LTBR comprises a heavy chain variable region (VH) and a light chain variable region (VL), an agonist anti-LTBR antibody or fragment or derivative thereof, such as a single-chain antibody fragment (scFv), 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, and the VH and VL comprise any of the following : (i) HCDR1, HCDR2, and HCDR3 each comprising 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 comprising 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 comprising the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR 3 each comprising the amino acid sequences of SEQ ID NO: 63, SEQ ID NO : 64, and SEQ ID NO: 65, respectively ; 3; or (iii) HCDR1, HCDR2, and HCDR3 each containing the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 each containing the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; or (iv) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44. For example, VH contains an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; [[ID= or 27]] VH contains an amino acid sequence having at least 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; [[ID= or 36]]VH contains an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; ​​​​​​​​​​having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity comprising an amino acid sequence; VH comprises an amino acid sequence having at least 99 % identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 97 %, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98 %, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48, for example VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 9 6%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47, and comprising the amino acid sequence, wherein VL has at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having 100% identity with the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity with the amino acid sequence of SEQ ID NO: 48 ; or (vi) SEQ ID NO: 22; or (vii) SEQ ID NO: 23; or (viii) SEQ ID NO: 25.

[0111] In certain non - limiting embodiments, the second binding domain that specifically binds to EDB comprises, by way of 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 regions 1 (HCDR1), HCDR2, and HCDR3, and VL comprises light - chain complementarity - determining regions 1 (LCDR1), LCDR2, and LCDR3, and the antibody or fragment thereof comprises any of the following: (i) each comprising the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74 for HCDR1, HCDR2, and HCDR3, respectively, and each comprising the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77 for LCDR1, LCDR2, and LCDR3 ; or (ii) VH comprises an amino acid sequence having at least 95%, 96%, 9 7%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46, for example VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 45 and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 45 and VL comprises an amino acid sequence having at least 95%, 9 6%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 45 and VL comprises an amino acid sequence having at least 95 %, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 45 and VL comprises an amino acid sequence having at least 95 %, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 45 and VL comprises an amino acid sequence having at least 95 %, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 45 and VL comprises an amino acid sequence having at least 95 also have 95%, 96%, 97%, 98%, 99% identity, or 100% identity and comprises an amino acid sequence; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46.

[0112] In certain non-limiting embodiments, the multispecific binding molecule comprises: (1) A binding domain that specifically binds to LTBR comprises the heavy chain variable region (VH) and the light chain variable region (VL) of the BHA10 antibody or the CBE11 antibody or a fragment or derivative thereof, such as a single-chain antibody fragment (scFv), wherein VH comprises the heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and VL comprises the 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 comprising the amino acid sequence of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or (ii) HCDR1, HCDR2, and HCDR3 each comprising the amino acid sequence of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or HCDR1, HCDR2, and HCDR3 containing the same, 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; or (ii) (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, 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; or (iv)VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44. For example, VH contains an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96% 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95% 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96% 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95% 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95% 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; comprising an amino acid sequence; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48, for example, VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 9 7%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 9 8%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48, for example, VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; amino acids having 96%, 97%, 98%, 99% identity, or 100% identity and includes; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% identity, or 100% identity and includes; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99% identity, or 100% identity and includes; VH comprises an amino acid sequence having at least 99 % identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 10 0% identity and includes; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity and includes; or (vi) SEQ ID NO: 22; or (vii) SEQ ID NO: 23; or (viii) SEQ ID NO: 25; and (2) The second binding domain that specifically binds to EDB includes, by way of example, the heavy chain variable region (VH ) and the light chain variable region (VL) of the L19 antibody or a fragment or derivative thereof and VH includes the heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 and VL includes the light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 When the antibody or its fragment contains, respectively, HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, and LCDR1, LCDR 2, and LCDR3 having the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively. In certain non-limiting embodiments, the multispecific binding molecule comprises: (1) The binding domain that specifically binds to LTBR is an antibody such as BHA10 antibody or CBE11 antibody or its fragment or derivative, including a single-chain antibody fragment (scFv), which contains a heavy-chain variable region (VH) and a light-chain variable region (VL). VH contains heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, and VL contains light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3. The VH and VL contain any of the following:

[0113] In certain non-limiting embodiments, the multispecific binding molecule comprises: (1) The binding domain that specifically binds to LTBR is an antibody such as BHA10 antibody or CBE11 antibody or its fragment or derivative, including a single-chain antibody fragment (scFv), which contains a heavy-chain variable region (VH) and a light-chain variable region (VL). VH contains heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, and VL contains light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3. The VH and VL contain any of the following: (i) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR 3 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 containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR 3 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 containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCD (i) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR 3 containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or 3; or (ii) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR (ii) HCDR1, HCDR_{2}, and HCDR_{3} comprising the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR (ii) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and LCDR1, LCDR2, and LCDR 3 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 containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCD (iii) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and LCDR1, LCDR2, and LCD 3 containing the amino acid sequences of SEQ ID NO: 69, SEQ LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of column number 70 and sequence number 71 or (iv) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44, such as VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 43, and VL comprises an amino acid sequence having at least 95% , 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH comprises an amino acid sequence having at least 9% identity to the amino acid sequence of SEQ ID NO: 43 comprising an amino acid sequence having 9% identity, wherein VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44 ; VH comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 44 ; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48, e.g., VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48, e.g., VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48, e.g., VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48 ; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48, e.g., comprising an amino acid sequence; VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having at least 99 % identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH comprises an amino acid sequence having 100 % identity to the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; or (vi) SEQ ID NO: 22; or (vii) SEQ ID NO: 23; or (viii) SEQ ID NO: 25; and, (2) The second binding domain that specifically binds to EDB comprises, by way of example, the heavy chain variable region (VH ) and the light chain variable region (VL) of the L19 antibody or a fragment or derivative thereof, wherein VH comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, VL comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or a fragment thereof comprises a VH having at least 9 5%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46 ; and VL comprises light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, and the antibody or a fragment thereof comprises a VH having at least 9 5%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46 ; having identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46 comprising an amino acid sequence; for example, VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 10 0% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises the amino acid sequence of SEQ ID NO: 45 having at least 98% identity, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46; VH comprises the amino acid sequence of SEQ ID NO: 45 having 100% identity, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46.

[0114] In certain non-limiting embodiments, the multispecific molecule comprises: (1) a binding domain that specifically binds to LTBR comprising SEQ ID NO: 22; and, (2) a second binding domain that specifically binds to EDB is, for example, a heavy chain variable region (VH ) and a light chain variable region (VL) comprising the L19 antibody or a fragment or derivative thereof 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 comprising the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, and LCDR1, LCDR2, and LCDR 3 each comprising the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77; or (ii) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46.

[0115] In certain non-limiting embodiments, the multispecific molecule comprises: (1) a binding domain that specifically binds to LTBR comprising SEQ ID NO: 23; and, (2) a second binding domain that specifically binds to EDB is, for example, a heavy chain variable region (VH ) and a light chain variable region (VL) comprising the L19 antibody or a fragment or derivative thereof ​​VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. VL contains light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. , the antibody or fragment thereof comprises any of the following: (i) the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively HCDR1, HCDR2, and HCDR3 containing SEQ ID NO: 75, SEQ ID NO: 8, No. 76, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 77. 3; or (ii) VH has a sequence similar to that of SEQ ID NO: 45, but is at least 95%, 96%, or Contains amino acid sequences with 97%, 98%, 99% identity, or 100% identity and VL is at least 95%, 96%, 97%, or It includes amino acid sequences with 98%, 99% identity, or 100% identity.

[0116] In certain non-limiting embodiments, the multispecific molecule comprises: (1) a binding domain that specifically binds to LTBR comprising SEQ ID NO: 25; and (2) The second binding domain that specifically binds to EDB is, for example, a heavy chain variable region (VH ) and a light chain variable region (VL), or a fragment or derivative thereof. VH includes heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. VL contains light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. , the antibody or fragment thereof comprises any of the following: (i) the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively HCDR1, HCDR2, and HCDR3 containing SEQ ID NO: 75, SEQ ID NO: 8, LCDR1, LCDR2, and LCDR containing the amino acid sequences of SEQ ID NO: 76 and SEQ ID NO: 77 or (ii) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity 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% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46. In certain specific non-limiting embodiments, the multispecific binding molecule comprises any of the following:

[0117] In certain specific non-limiting embodiments, the multispecific binding molecule comprises any of the following: (a) (i) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 that forms a binding domain together with a first light chain comprising the amino acid sequence of SEQ ID NO: 2, and (ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 4 that forms a binding domain together with a second light chain comprising the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule designated COVA14121); or (b) (i) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 9 that forms a binding domain together with a first light chain comprising the amino acid sequence of SEQ ID NO: 10, and (ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 4 that forms a binding domain together with a second light chain comprising the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule designated COVA14122). (b) (i) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 9 that forms a binding domain together with a first light chain comprising the amino acid sequence of SEQ ID NO: 10, and (ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 4 that forms a binding domain together with a second light chain comprising the amino acid sequence of SEQ ID NO: 5 (a multispecific binding molecule designated COVA14122). In certain further non-limiting embodiments, the multispecific binding molecule comprises any of the following:

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

[0119] In some embodiments, the multispecific molecule is (i) a heavy chain portion, wherein the amino a light chain comprising the amino acid sequence of SEQ ID NO: 38, which forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO: 39; cFv heavy chain fusion, and (ii) a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5. and a heavy chain comprising the amino acid sequence of SEQ ID NO:4, which forms the

[0120] In some embodiments, the multispecific (e.g., bispecific) molecule is characterized by the absence of EDB. NF-κB signaling induced under the same conditions is at least 2 times higher than that induced under the same conditions. fold, at least 3-fold, e.g., at least 4-fold greater than that of NF-κB in the presence of EDB. In some cases, the assay involves the detection of NF-κB luciferase receptors. NF-κB luciferase reporter assay is performed It may be carried out using the protocol of Example 2.

[0121] In some embodiments, the multispecific (e.g., bispecific) molecule is characterized by the absence of EDB. The ICAM-1 expression induced under the same conditions was at least two-fold lower. at least 4-fold greater, such as at least 3-fold greater, ICA at the cell surface in the presence of EDB In some cases, the assay is performed using the A375 / WI38A subline 2R A. In vitro LTBR activation assays, such as co-culture cell assays. The WI38A / WI38A subline 2RA co-culture cell assay was performed using the protocol in Example 3. This may also be done.

[0122] According to a particular embodiment, the heavy and light chains are humanized.

[0123] In some embodiments, the bispecific antibodies of the invention are Diabodies, cross-bodies, scFvs, or bispecific antibodies obtained by controlled Fab arm exchange, such as include Fv, Duobody, spFv, or bispecific antibodies.

[0124] In some embodiments, the bispecific antibodies include IgG-like molecules having a complementary CH3 domain that forces heterodimer formation, recombinant IgG-like dual-targeting molecules (where the two sides of this molecule each contain a Fab fragment or a portion of a Fab fragment of at least two different antibodies), IgG fusion molecules (where the full-length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment), Fc fusion molecules (where a single-chain Fv molecule or a stabilized diabody is fused to a heavy-chain constant domain, Fc region, or a portion thereof), Fab fusion molecules (where different Fab fragments are fused together), ScFv and diabody-based and heavy-chain antibodies (e.g., domain antibodies, nanobodies) (where different single-chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, nano bodies) are fused to each other or fused to another protein or carrier molecule). Examples include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche) and electrostatically engineered ones (Amgen), LUZ-Y ( Genentech), strand-exchanged and engineered domain bodies (SEEDbody ), etc.

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

[0126] In some embodiments, examples of recombinant IgG-like bispecific molecules include Dual Tar geting (DT)-Ig (GSK / Domantis), Two-in-one A ntibody (Genentech), Cross-linked Mabs (Kar manos Cancer Center), mAb2 (F-Star), or CovX -body (CovX / Pfizer).

[0127] In some embodiments, examples of IgG fusion molecules include Dual Variable D omain (DVD)-Ig (Abbott), IgG-like Bispecifi c (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ ), and BsAb (Zymogenetics), HERCULES (Biogen I dec), or TvAb (Roche).

[0128] In some embodiments, examples of Fc fusion molecules may include ScFv / Fc Fusions (Ac ademic Institution), SCORPION (Emergent Bi oSolutions / Trubion, Zymogenetics / BMS), Dua l Affinity Retargeting Technology (Fc-DAR T) (MacroGenics), or Dual(ScFv)2-Fab (Nation al Research Center for Antibody Medicine --China).

[0129] In some embodiments, Fab fusion bispecific antibodies include F(ab)2 (Med arex / AMGEN), Dual-Action or Bis-Fab (Genen tech), Dock-and-Lock (DNL) (ImmunoMedics), B ivalent Bispecific (Biotecnol), or Fab-Fv (U CB-Celltech). ScFv-, diabody-based and domain antibodies include Bispecific T Cell Engager (BITE) (M icromet), Tandem Diabody (Tandab) (Affimed) , Dual Affinity Retargeting Technology (DA RT) (MacroGenics), Single-chain Diabody (Ac ademic), TCR-like Antibodies (AIT, Receptor Logics), Human Serum Albumin ScFv Fusion( Merrimack), or COMBODY (Epigen Biotech), dual-labeled nanobody (dual targeting nanobody) (Ablynx), dual targeting heavy chain only domain antibody but are not limited to these.

[0130] The full-length bispecific antibodies of the present invention can be, for example, Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies, in vitro, in a cell-free environment or co-expression use In any of the following, it can be produced by introducing substitutions at the heavy chain CH3 interface in each half - molecule so as to facilitate the formation of a heterodimer of two antibody half - molecules having distinct specificities. The Fab - arm exchange reaction is the result of disulfide - bond isomerization reaction and dissociation - association of the CH3 domain. The heavy - chain disulfide bond in the hinge region of a single - specificity parental antibody is reduced. One of the resulting free cysteines of the parental single - specificity antibody forms an intra - heavy - chain disulfide bond with the cysteine residue of the second parental single - specificity antibody molecule, and at the same time, the CH3 domain of the parental antibody is released and reformed by dissociation - association. The CH3 domain of the Fab - arm can be engineered to favor heterodimer formation over homodimer formation. The resulting product is a bispecific antibody having two Fab - arms or half - molecules that bind to distinct epitopes, namely, an epitope in LTBR and an epitope in the EDB of fibronectin, respectively. It can be achieved. The Fab - arm exchange reaction is the result of the disulfide - bond isomerization reaction and the dissociation - association of the CH3 domain. The heavy - chain disulfide bond in the hinge region of a single - specificity parental antibody is reduced. One of the resulting free cysteines of the parental single - specificity antibody forms an intra - heavy - chain disulfide bond with the cysteine residue of the second parental single - specificity antibody molecule, and at the same time, the CH3 domain of the parental antibody is released and reformed by dissociation - association. The CH3 domain of the parental antibody is released and reformed by dissociation - association. The CH3 domain of the Fab - arm can be engineered to favor heterodimer formation over homodimer formation. The resulting product is a bispecific antibody having two Fab - arms or half - molecules that bind to distinct epitopes, namely, an epitope in LTBR and an epitope in the EDB of fibronectin, respectively. The resulting product is a bispecific antibody having two Fab - arms or half - molecules that bind to distinct epitopes, namely, an epitope in LTBR and an epitope in the EDB of fibronectin, respectively. The resulting product is a bispecific antibody having two Fab - arms or half - molecules that bind to distinct epitopes, namely, an epitope in LTBR and an epitope in the EDB of fibronectin, respectively. It is a bispecific antibody.

[0131] As used herein, "homodimer formation" means the interaction of two heavy chains having the same CH3 amino - acid sequence. As used herein, "homodimer" means an antibody having two heavy chains with the same CH3 amino - acid sequence. As used herein, "homodimer" means an antibody having two heavy chains with the same CH3 amino - acid sequence.

[0132] As used herein, "heterodimer formation" means the interaction of two heavy chains having non - identical CH3 amino - acid sequences. As used herein, "heterodimer" means an antibody having two heavy chains with non - identical CH3 amino - acid sequences. As used herein, "heterodimer" means an antibody having two heavy chains with non - identical CH3 amino - acid sequences.

[0133] The "knob - in - hole" strategy (e.g., International Publication No. WO 2006 / 02893 Using the method described in reference 6, a full-length bispecific antibody can be generated. Briefly stated, selected amino acids that form the interface of the CH3 domain in human IgG are mutated at positions that affect CH3 domain interactions to promote heterodimer formation . Amino acids with small side chains (holes) are introduced into the heavy chain of an antibody that specifically binds to a first antigen , and amino acids with large side chains (knobs) are 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 the preferential interaction between the heavy chain with a "hole" and the heavy chain with a "knob" . Exemplary CH3 substitution pairs that form knobs and holes are T366Y / F405A , T366W / F405W, F405W / Y407A, T394W / Y407T, T39 4S / Y407A, T366W / T394S, F405W / T394S, or T366W / T366S_L368A_Y407V (expressed as the modified position in the first CH3 domain of the first heavy chain / the modified position in the second CH3 domain of the second heavy chain using Kabat numbering ). ). ).

[0134] Other strategies, such as using electrostatic interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface to promote heavy chain heterodimer formation, can be used as described, for example, in U.S. Patent Application Publication No. 2010 / 0015133, U.S. Patent Application Publication No. 2009 / 0182127, U.S. Patent Application Publication No. 2010 / 028637 , or U.S. Patent Application Publication No. 2011 / 0123532 . In other strategies, heterodimer formation is described, for example, in U.S. Patent Application Publication No. 2012 / 01498 . . As described in No. 76 or US Patent Application Publication No. 2013 / 0195849, the following substitutions: L351Y_F405A Y407V / T394W, T366I_K392M_T3 94W / F405A_Y407V, T366L_K392M_T394W / F405A_ Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407 A / T366V K409F Y407A / T366A_K409F, or T350V_ L351Y_F405A Y407V / T350V_T366L_K392L_T394 W (expressed as the modification position in the first CH3 domain of the first heavy chain / the modification position in the second CH3 domain of the second heavy chain) can be promoted. In addition to the above methods, the bispecific antibodies of the present invention can be prepared according to the method described in International Publication No. 2011 / 131746 by introducing asymmetric mutations into the CH3 regions of two single - specificity homodimer antibodies and forming bispecific heterodimer antibodies from the two parental single - specificity homodimer antibodies under reducing conditions that isomerize disulfide bonds, thereby generating in vitro in a cell - free environment.

[0135] In this method, the first single - specificity bivalent antibody and the second single - specificity bivalent antibody are engineered to have specific substitutions in the CH3 domain that promote the stability of the heterodimer, but these antibodies are co - incubated under reducing conditions sufficient for the cysteine in the hinge region to isomerize the disulfide bond, thereby generating bispecific antibodies by Fab - arm exchange. The incubation conditions can optimally be returned to non - reducing conditions. Exemplary reducing conditions that can be used are such that the first single - specificity bivalent antibody and the second single - specificity bivalent antibody are co - incubated under reducing conditions sufficient for the cysteine in the hinge region to isomerize the disulfide bond, thereby generating bispecific antibodies by Fab - arm exchange. In the CH3 domain that promotes the stability of the heterodimer, but these antibodies are co - incubated under reducing conditions sufficient for the cysteine in the hinge region to isomerize the disulfide bond, thereby generating bispecific antibodies by Fab - arm exchange. The incubation conditions can optimally be returned to non - reducing conditions. Exemplary reducing conditions that can be used are The agent is 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol, preferably, it is a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, at a temperature of at least 20°C, in the presence of at least 25 mM of 2-MEA or in the presence of at least 0.5 mM of dithiothreitol, at pH 5-8, for example pH 7.0 or pH 7.4, incubation for at least 90 minutes can be used. In some embodiments described herein, the immune effector properties of a multispecific binding molecule such as a bispecific antibody of the present invention can be modified, preferably silenced, by techniques known to those skilled in the art, for example, by modifying the Fc. For example, Fc effector functions, such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptor, BCR), etc., can be provided and / or controlled by modifying the residues in these active Fc. For example, see the following: 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, for example, the aspartic acid at position 265

[0136] ​ Paraheptanoic acid (D), asparagine (N) at position 297, and proline (P) at position 329 mutation. Here, the numbering is as detailed in International Publication No. 2019 / 068632 to obtain so-called DANAPA variants, for example, each against alanine (A), as indicated by EU indexes such as Kabat.

[0137] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors recognize an antibody bound to a target cell and subsequently cause lysis of the target cell.

[0138] In certain embodiments, the multispecific binding molecule of the invention comprises a chimeric bispecific anti body.

[0139] In certain embodiments, the multispecific binding molecule of the invention comprises a human or humanized bispecific antibody.

[0140] In another general aspect, the invention relates to one or more isolated nucleic acids encoding the multispecific binding molecule of the invention, such as a bispecific anti body, or an antigen-binding fragment thereof. As a non-limiting example, the heavy chain of a bispecific antibody can be encoded by one nucleic acid, and the light chain can be encoded by a second nucleic acid. In another example, the heavy chain and light chain of a bispecific antibody may be encoded on a single nucleic acid molecule. Considering the degeneracy of the genetic code, those skilled in the art will understand that the coding sequence of a protein can be changed (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Therefore ​​​​ and 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 changing the amino acid sequence of the protein. In addition, one or more nucleic acids of the present invention can be isolated nucleic acids. Accordingly, the present invention relates to any nucleic acid molecule or combination of nucleic acid molecules encoding the molecules 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 a plasmid, cosmid, phage vector, or viral vector, etc. can be used. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element for establishing the conventional functions of an expression vector, such as a promoter, ribosome binding element, terminator, enhancer, selection marker, and / or origin of replication. The promoter can be a constitutive, inducible, or reconstitutable promoter. A number of expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to generate antibodies or antigen-binding fragments thereof intracellularly. 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 one or more vectors comprising one or more nucleic acids encoding multispecific binding molecules such as the bispecific antibody or antigen-binding fragment thereof of the present invention. relates to a host cell comprising. In view of the present disclosure, any host cell known to those skilled in the art can be , and can be used for the recombinant expression of a multispecific binding molecule such as the bispecific antibody or its antigen-binding fragment of the present invention. In some embodiments, the host cell is Escherichia coli TG1 or BL21 cells (e.g., for the expression of scFv or Fab antibodies), CHO-DG 44 or CHO-K1 cells, or HEK293 cells (e.g., for the expression of full-length IgG antibodies ). According to certain embodiments, the recombinant expression vector can be stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed, and the host cell is transformed with a protein by conventional methods such as chemical transfection , heat shock, or electroporation.

[0143] In another general aspect, the present invention relates to a method for producing a multispecific binding molecule such as the bispecific antibody or its antigen-binding fragment disclosed herein. The method comprises culturing a cell comprising a nucleic acid encoding a multispecific binding molecule such as a bispecific antibody or its antigen-binding fragment under conditions for producing a multispecific binding molecule such as the bispecific antibody or its antigen-binding fragment disclosed herein, and recovering a multispecific binding molecule such as a bispecific antibody or its antigen-binding fragment from the cell or cell culture (e.g., supernatant). The expressed multispecific binding molecule such as a bispecific antibody or its antigen-binding fragment is collected from the cell and can be purified according to conventional techniques known in the art and as described herein.

[0144] Pharmaceutical composition In another general aspect, the invention relates to a pharmaceutical composition comprising a multispecific binding molecule of the invention (e.g., a bispecific antibody or an antigen-binding fragment thereof) and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" means a product comprising a multispecific binding molecule of the invention together with a pharmaceutically acceptable carrier. The multispecific binding molecules (e.g., bispecific antibodies) of the invention and compositions containing them are also useful in the manufacture of medicaments for the therapeutic uses mentioned herein. As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer solution, stabilizer, solubilizer, oil, lipid, lipid-containing vesicles, microspheres, liposome encapsulation,

[0145] or other materials well known in the art for use in pharmaceutical formulations. It will be understood that the properties of the 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 either the efficacy of the composition according to the invention or the biological activity of the composition according to the invention. According to certain embodiments, any pharmaceutically acceptable carrier suitable for use of an antibody in a pharmaceutical composition may be used herein, in view of the present disclosure. Formulations of pharmaceutically active ingredients with pharmaceutically acceptable carriers are known in the art, for example, as in Remingto n: The Science and Practice of Pharmacy (e.g., the 21st edition (2005) and any subsequent revised editions). Non-limiting examples of additional ingredients include buffers, diluents As will be appreciated, the properties of the 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 either the efficacy of the composition according to the invention or the biological activity of the composition according to the invention. According to certain embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use of an antibody in a pharmaceutical composition may be used herein. Formulations of pharmaceutically active ingredients with pharmaceutically acceptable carriers are known in the art, for example, as in Remingto

[0146] n: The Science and Practice of Pharmacy (e.g., the 21st edition (2005) and any subsequent revised editions). Non-limiting examples of additional ingredients include buffers, diluents For example, the 21st edition (2005) and any subsequent revised editions), are known in the art. Non-limiting examples of additional ingredients include buffers, diluents As will be appreciated, the properties of the 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 either the efficacy of the composition according to the invention or the biological activity of the composition according to the invention. According to certain embodiments, , solvents, tonicity regulators, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can 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. Preferred examples of liquid formulations are aqueous formulations, i.e., formulations containing water. Liquid formulations may include solutions, suspensions, emulsions, micro emulsions, gels, and the like. Aqueous formulations typically contain at least 50% by weight of water, or at least 60% by weight, 70% by weight, 75% by weight, 80% by weight, 8 5% by weight, 90% by weight, or at least 95% by weight of water.

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

[0149] In another embodiment, the pharmaceutical composition is a solid formulation, for example, can be used as such or a lyophilized or spray-dried composition to which a solvent and / or diluent can be added by a physician or a patient before use. 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 can also be in the form of, for example, sachets, dragees, powders, granules, lozenges, or powders for reconstitution .

[0150] The dosage form may be an immediate release, in which case it may contain a water-soluble or water-dispersible carrier, or the dosage form may be a delayed release, sustained release, or controlled release, in which case the stomach It may contain a water-insoluble polymer that controls the dissolution rate of the dosage form in the intestinal tract or subcutaneously. .

[0151] In other embodiments, the pharmaceutical composition can be delivered intranasally, orally, or sublingually.

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

[0153] In certain embodiments, the pharmaceutical composition contains a buffering agent. Non-limiting examples of buffering agents include arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, f umaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, acetic sodium, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinic salt, tartaric acid, tricine, or tris(hydroxymethyl)-aminomethane, and mixtures thereof are included. The buffering agent can be present individually or in total at a concentration of about 0.01 mg / mL to about 50 mg / mL, for example, at a concentration of about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each of these specific buffering agents constitute alternative embodiments of the present invention.

[0154] In certain embodiments, the pharmaceutical composition contains a preservative. Non-limiting examples of preservatives include benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hy droxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlo rphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxy Seven benzoates, imidourea, methyl 4-hydroxybenzoate, phenol, 2-f enoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thimerosal, and mixtures thereof. The preservative is , individually or in total, present at a concentration of about 0.01 mg / mL to about 50 mg / mL, such as about 0.1 mg / mL to about 20 mg / mL. Pharmaceutical compositions containing each one of these specific preservatives constitute alternative embodiments 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), alditols (such as glycerol, 1,2-propanediol propylene glycol, etc.), 1,3-p ropanediol, or 1,3-butanediol, etc.), polyethylene glycol (such as , PEG400), and mixtures thereof. Another example of an isotonic agent is sugar . Non-limiting examples of sugars include, for example, fructose, glucose, mannose, sor bose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch, or carboxymethylcellulose sodium containing monosaccharides, disaccharides, or polysaccharides, or water-soluble glucans. Another example of an isotonic agent is a sugar alcohol, and the term "sugar alcohol" refers to at least one -OH It is defined as a C(4-8) hydrocarbon having a base. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xyl itol, or arabinitol. The isotonic agent may be present individually or in total at a concentration of about 0.01 m g / mL to about 50 mg / mL, for example about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition containing each one of these specific isotonic agents constitutes an alternative embodiment of the present invention.

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

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

[0158] In certain embodiments, the pharmaceutical composition contains a stabilizer, and the stabilizer is carboxy- / hydroxycellulose and their derivatives (such as HPC, HPC-SL, HPC-L, and H PMC, etc.), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, A salt (such as sodium chloride), a sulfur-containing substance (for example, monothioglycerol), or thiogly colic acid. The stabilizer may be present, individually or in total, at a concentration of about 0.01 mg / mL to about 50 mg / mL, for example, about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition containing each one 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) portion and a lipid-soluble (lipophilic) portion. The surfactant may be selected, for example, from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactant may be present, individually or in total, at a concentration of about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition containing each one of these specific surfactants constitutes an alternative embodiment of the present invention.

[0160] In certain embodiments, the pharmaceutical composition comprises one or more protease inhibitors, for example, EDTA, and / or benzamidine hydrochloride (HCl). The protease inhibitor may be present, individually or in total, at a concentration of about 0.1 mg / mL to about 20 mg / mL. A pharmaceutical composition containing each one of these specific protease inhibitors constitutes an alternative embodiment of the present invention.

[0161] In another general aspect, the present invention is a method for producing a pharmaceutical composition comprising a multispecific binding molecule such as a bispecific antibody of the present invention or an antigen-binding fragment thereof, wherein the bispecific antibody or the multispecific binding molecule such as an antigen-binding fragment thereof is combined with a​​ A method for obtaining a pharmaceutical composition, which comprises combining with

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

[0163] The functional activity of a multispecific binding molecule that binds to LTBR and / or EDB (e.g., a bispecific antibody and its antigen-binding fragment) can be characterized by methods known in the art and as described herein. Methods for characterizing a multispecific binding molecule that binds to LTBR and / or EDB include affinity and specificity assays including Biacore, ELISA, and / or OctetRed analysis; binding assays for detecting the binding of a multispecific binding molecule to LTBR on cancer cells and other cells by FACS but are not limited thereto. According to a particular embodiment, methods for characterizing a multispecific binding molecule that binds to LTBR and / or EDB include those described below.

[0164]

[0165] In another general aspect, the invention relates to a method of establishing an inflammation-induced tumor microenvironment. The method comprises contacting LTBR-expressing cells in the tumor microenvironment with the multispecific binding molecule of the invention wherein contacting the LTBR-expressing cells with the multispecific binding molecule results in the secretion of inflammatory chemokines and cytokines and the expression of adhesion molecules on the cell surface.

[0165] ​In another general aspect, the present invention specifically binds to LTBR and EDB of fibronectin A multispecific binding molecule of the present invention (e.g., a bispecific antibody or an antigen-binding fragment thereof) that binds Or a pharmaceutical composition disclosed herein, to a subject in need thereof, related to a method for treating cancer in a subject in need thereof. The cancer is preferably an EDB-expressing cancer Yes. The cancer can be, for example, an LTBR-expressing cancer. The cancer can be, for example, prostate cancer, lung cancer, gastric cancer Esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, 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 It can be selected from the group consisting of liquid tumors. mphocytic leukemia, CLL), chronic myelogenous leukemia (chronic myelogenous leukemia, C ML), multiple myeloma (multiple myeloma, MM), acute myeloid leukemia, AML), and other liquid tumors.

[0166] According to an embodiment of the present invention, the pharmaceutical composition comprises an effective amount of an anti-LTBR multispecific binding molecule ( For example, an anti-LTBR / anti-EDB bispecific antibody or an antigen-binding fragment thereof). 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. Answer.

[0167] According to a particular embodiment, the effective amount is one, two, three, four, or Refers to an amount of treatment sufficient to achieve more: (i) reducing or ameliorating 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 the symptoms associated therewith, (vii) reducing hospitalization of a subject having the disease, disorder or condition being treated, or the symptoms associated therewith, (viii) shortening the length of hospitalization of a subject having the disease, disorder or condition being treated, or the symptoms associated therewith, (ix) increasing the survival rate of a subject having the disease, disorder or condition being treated, or the symptoms associated therewith, (xi) inhibiting or reducing the disease, disorder or condition being treated, or the symptoms associated therewith in the subject being treated, and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another treatment. In some embodiments, an effective amount of the multispecific binding molecule of the invention can be administered at a dosage in the range of 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. The effective amount or dosage will depend on the disease, disorder or condition being treated, the means of administration, the site of targeting, the physiology of the subject, the disease, disorder or condition being treated, or the symptoms associated therewith, (viii) shortening the length of hospitalization of a subject having the disease, disorder or condition being treated, or the symptoms associated therewith, (ix) increasing the survival rate of a subject having the disease, disorder or condition being treated, or the symptoms associated therewith, (xi) inhibiting or reducing the disease, disorder or condition being treated, or the symptoms associated therewith in the subject being treated, and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another treatment. The effective amount or dosage will depend on the disease, disorder or condition being treated, the means of administration, the site of targeting, the physiology of the subject, the disease, disorder or condition being treated, or the symptoms associated therewith, (viii) shortening the length of hospitalization of a subject having the disease, disorder or condition being treated, or the symptoms associated therewith, (ix) increasing the survival rate of a subject having the disease, disorder or condition being treated, or the symptoms associated therewith, (xi) inhibiting or reducing the disease, disorder or condition being treated, or the symptoms associated therewith in the subject being treated, and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another treatment. In some embodiments, an effective amount of the multispecific binding molecule of the invention can be administered at a dosage in the range of 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

[0168] about 5 mg / kg. The effective amount or dosage will depend on the disease, disorder or condition being treated, the means of administration, the site of targeting, the physiology of the subject, the disease, disorder or condition being treated, or the symptoms associated therewith, (viii) shortening the length of hospitalization of a subject having the disease, disorder or condition being treated, or the symptoms associated therewith, (ix) increasing the survival rate of a subject having the disease, disorder or condition being treated, or the symptoms associated therewith, (xi) inhibiting or reducing the disease, disorder or condition being treated, or the symptoms associated therewith in the subject being treated, and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another treatment.

[0169] The effective amount or dosage will depend on the disease, disorder or condition being treated, the means of administration, the site of targeting, the physiology of the subject, The physical state (including, for example, age, weight, health status), whether the subject is human or animal , other drugs to be administered, and whether the treatment is prophylactic or therapeutic, etc. can vary depending on various factors. The therapeutic dosage is optionally incremented to optimize safety and efficacy.

[0170] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intravenous administration, subcutaneous administration, or intramuscular administration. In some embodiments, the compositions disclosed herein can be administered to a 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

[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 not necessarily be recognized in the subject, but may be recognizable in the subject. The terms "treat", "treating", and "treatment" also may refer to regressing a disease, disorder, or condition, preventing its progression, or at least delaying its progression. In certain embodiments, "treat", "treating", and "treatment" refer to one related to a disease, disorder, or condition such as a tumor or more preferably cancer Refers to alleviating the above symptoms, preventing progression or onset, or shortening the duration thereof. In certain embodiments the terms "treating", "treat", and "treatment" refer to preventing recurrence of a disease, disorder, or condition. In certain embodiments the terms "treating", "treat", and "treatment" refer to improving the survival rate of a subject having a disease , disorder, or condition. In certain embodiments the terms "treating ", "treat", and "treatment" refer to disappearance of a disease, disorder, or condition in a subject.

[0172] According to certain embodiments, there is provided a composition for treating cancer. For cancer treatment , the composition can be used in combination with 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, cytokine, oncolytic virus, TLR agonist, STING agonist, other immuno-oncology drugs, anti-angiogenic agent, radiotherapy, antibody-drug conjugate (A DC), targeted therapy, or other anti-cancer agents, and can be used in combination with another treatment not limited thereto .

[0173] As used herein, the term "in combination" when used in connection with the administration of two or more therapeutic agents to a subject refers to the use of two or more therapeutic agents. The use of the term "in combination" does not limit the order in which the therapeutic agents are administered to the subject. For example, a first therapeutic agent (e.g., the composition described herein ) can be administered to the subject before the administration of a second therapeutic agent (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 before 12 weeks), simultaneously, or thereafter (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 later) and can be administered. .

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

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

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

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

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

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

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

[0181] Embodiment 7 is the 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 the 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 an antibody.

[0183] Embodiment 9 is the multispecific binding molecule according to any one of Embodiments 1 to 8, wherein the first binding domain that specifically binds to LTBR includes a heavy-chain variable region (VH) and a light-chain variable region (VL), VH includes heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, VL includes light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL are as follows ((i) to (viii)): HCDR1, HCDR2, and HCDR3 each containing the amino acid sequences of SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, 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; or (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, 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; or ; 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, and LCDR1, LCDR2, and LCDR3 each containing the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: LCDR1, LCDR2, and LCDR containing the amino acid sequences of SEQ ID NO: 64 and SEQ ID NO: 65 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, 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; or (iv) VH contains an amino acid sequence having at least 95%, 96%, 9 7%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 9 8%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44. For example VH contains an amino acid sequence having at least 95% identity to 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 to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 96% identity to 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 to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43 and VL contains an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 43 and VL contains an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 43 and VL contains an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 43 and VL contains an amino acid sequence having at least 9 5%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 44; VH contains an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 43 comprising an amino acid sequence having homogeneity, wherein VL has at least 95%, 96%, 97%, 98%, 99% homogeneity, or 100% homogeneity with the amino acid sequence of SEQ ID NO: 44 ; VH comprises an amino acid sequence having at least 99 % homogeneity with the amino acid sequence of SEQ ID NO: 43, and VL has at least 95%, 96%, 97%, 98%, 99% homogeneity, or 100% homogeneity with the amino acid sequence of SEQ ID NO: 44 ; VH comprises an amino acid sequence having 100% homogeneity with the amino acid sequence of SEQ ID NO: 43, and VL has at least 95%, 96%, 97%, 98%, 99% homogeneity, or 100% homogeneity with the amino acid sequence of SEQ ID NO: 44; or (v) VH comprises an amino acid sequence having at least 95%, 96%, 97 %, 98%, 99% homogeneity, or 100% homogeneity with the amino acid sequence of SEQ ID NO: 47, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98 %, 99% homogeneity, or 100% homogeneity with the amino acid sequence of SEQ ID NO: 48, for example, V H comprises an amino acid sequence having at least 95% homogeneity with the amino acid sequence of SEQ ID NO: 47 and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% homogeneity, or 100% homogeneity with the amino acid sequence of SEQ ID NO: 48 ; VH comprises an amino acid sequence having at least 96% homogeneity with the a mino acid sequence of SEQ ID NO: 47, and VL has at least 95%, 9 6%, 97%, 98%, 99% homogeneity, or 100% homogeneity with the amino acid se comprising columns; VH has at least 97% identity to the amino acid sequence of SEQ ID NO: 47 and comprises an amino acid sequence, and VL has at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH has at least 98% identity to the amino acid sequence of SEQ ID NO: 47 and comprises an amino acid sequence, and VL has at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH has at least 99% identity to the amino acid sequence of SEQ ID NO: 47 and comprises an amino acid sequence, and VL has at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48; VH has 100% identity to the amino acid sequence of SEQ ID NO: 47 and comprises an amino acid sequence, and VL has at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 48: or (vi) SEQ ID NO: 22; or (vii) SEQ ID NO: 23; or (viii) SEQ ID NO: 25 and is a multispecific binding molecule comprising any one or more thereof. (vi) SEQ ID NO: 22; or (vii) SEQ ID NO: 23; or (viii) SEQ ID NO: 25 and is a multispecific binding molecule comprising any one or more thereof.

[0184] Embodiment 10 is the multispecific binding molecule according to any one of Embodiments 1 to 9, wherein a second binding domain that specifically binds to EDB comprises a heavy chain variable region (VH) and a light chain variable region (VL), and VH comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and comprising an HCDR3, wherein the VL is a light chain complementarity determining region 1 (LCDR1), LCDR2, and L comprising a CDR3, wherein the VH and VL are as follows ((i)-(ii)): (i) respectively comprising the amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74 for HCDR1, HCDR2, and HCDR3, and respectively comprising the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77 for LCDR1, LCDR2, and LCDR3 ; or (ii) the VH comprises an amino acid sequence having at least 95%, 96%, 9 7%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 45 and the VL comprises an amino acid sequence having at least 95%, 96%, 97%, 9 8%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46, for example the VH comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 45 and the VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46 ; the VH comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 45 and the VL comprises an amino acid sequence having at least 95%, 9 6%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46 ; the VH comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 45 and the VL comprises an amino acid sequence having at least 95 %, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46 ; the VH comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 45 comprising an amino acid sequence having properties, wherein VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46 and comprises an amino acid sequence; VH has at least 99% identity to the amino acid sequence of SEQ ID NO: 45 and comprises an amino acid sequence, wherein VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46 and comprises an amino acid sequence; VH has 100% identity to the amino acid sequence of SEQ ID NO: 45 and comprises an amino acid sequence, wherein VL has at least 95%, 96%, 97%, 98%, 99% identity, or 100% identity to the amino acid sequence of SEQ ID NO: 46 and comprises an amino acid sequence.

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

[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 comprising the amino acid sequence of SEQ ID NO: 30, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 that forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5; or (d) (i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 31, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 that forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5; or (e) (i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 32, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 that forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5; or (f) (i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 33, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 that forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5; or (g) (i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 34, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 that forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5; or ​​​​​​​​​​​​​​​(h) (i) a heavy chain portion that comprises a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO: 5; (ii) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 35, and 4, which forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5 a heavy chain comprising: (j) (i) a heavy chain portion that forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO: 5; (ii) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 38, 4, which forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5 a heavy chain comprising: (k) (i) a heavy chain portion that forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO: 5; (ii) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 39, 4, which forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5 a heavy chain comprising: (l) (i) a heavy chain portion that forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO: 5; (ii) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 56, and 4, which forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain comprising either

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

[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 containing the one or more nucleic acid molecules according to Embodiment 14.

[0190] Embodiment 16 is an isolated host cell containing 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 for treating cancer in a subject in need of treatment, the method 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 for producing the multispecific binding molecule according to any one of Embodiments 1 to 13, the method comprising the one or more nucleic acid molecules according to Embodiment 14 or the one or more vectors according to Embodiment 15. ​​​​​​​​​​​Expressing one or more vectors in a host cell and collecting a multispecific binding molecule is a method comprising:

[0195] Embodiment 21 is the multispecific molecule according to any one of Embodiments 1 to 10, wherein the NF-κB signaling induced in the presence of EDB is at least 2-fold, at least 3-fold, for example at least 4-fold greater than the NF-κB signaling induced in the absence of EDB, inducing NF-κB signaling in the presence of EDB.

[0196] Embodiment 22 is the multispecific molecule according to any one of Embodiments 1 to 10 or 21, wherein the ICAM-1 expression induced in the presence of EDB is at least 2-fold, at least 3-fold, for example at least 4-fold greater than the ICAM-1 expression on the cell surface induced in the absence of EDB, inducing ICAM-1 expression on the cell surface in the presence of EDB.

Examples

[0197] Example 1: Generation of EDB / LTBR Bispecific Antibody and Control Molecule The bispecific antibody and the control molecule are derived from the target binding sequences shown in Table 1 and are transiently expressed in a CHO suspension culture in a serum-free / animal component-free medium, and purified by protein A affinity chromatography followed by size exclusion chromatography (SEC) on a Superdex 200 10 / 300GL column (GE Healthcare) using an Akta Pure instrument (GE Healthcare). The heavy chain contained the knob-in-hole (KiH) mutation that promotes heterodimer formation (Ridgway et al., "Protein Eng.", Vol. 9 (No. 7): 61 Healthcare). Healthcare) and purified by size exclusion chromatography (SEC) on a Superdex 200 10 / 300GL column (GE Healthcare). The heavy chain contained the knob-in-hole (KiH) mutation that promotes heterodimer formation (Ridgway et al., "Protein Eng.", Vol. 9 (No. 7): 61 column chromatography, followed by size exclusion chromatography (SEC) on a Superdex 200 10 / 300GL column (GE Healthcare). The heavy chain contained the knob-in-hole (KiH) mutation that promotes heterodimer formation (Ridgway et al., "Protein Eng.", Vol. 9 (No. 7): 61 by size exclusion chromatography (SEC). The heavy chain contained the knob-in-hole (KiH) mutation that promotes heterodimer formation (Ridgway et al., "Protein Eng.", Vol. 9 (No. 7): 61 containing the knob-in-hole (KiH) mutation that promotes heterodimer formation (Ridgway et al., "Protein Eng.", Vol. 9 (No. 7): 61 (Ridgway et al., "Protein Eng.", Vol. 9 (No. 7): 61 7-21(1996), Atwell et al., "J. Mol. Biol.", Vol. 270 (No. 1): pp. 26-35 (1997); Merchant et al., "Nat. Biotec hnol.", Vol. 16 (No. 7): pp. 677-81 (1998)). The antibody contains IgG1 sigma Fc with seven Fc mutations - L234A compared to wild-type IgG1 that reduces Fc receptor interaction, L235A , L235A, G237A, P238S, H268A, A330S, and P331S set of (Tam et al., "Antibodies" ( 2017)).

[0198] The symmetric single antibody and bispecific antibody were generated with IgG1 sigma mutations and without KiH mutations .

[0199]

Table 1

[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 loaded onto the column, and elution was recorded by OD280 .

[0201] Table 2 shows an overview of the structural characteristics of the bispecific antibodies and control molecules described in this example. Bold molecules are the molecules according to the invention, and the others are controls in different embodiments.

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

[0203]

Table 2

[0204]

Table 3

[0205]

Table 4

[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 is the heavy chain (HC; SEQ ID NO: 1) and light chain (LC; SEQ ID NO: 2) of the agonist LTBR antibody LTBRmAb1, and the heavy chain (H Generated by co-expression with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) (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 the agonist LTBR antibody LTBRmAb1 with the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV antibody B21M (Figure 1K). iii. COVA14122 was generated by co-expression of the heavy chain (H C; SEQ ID NO: 9) and light chain (LC; SEQ ID NO: 10) of the agonist LTBR antibody LTBRmAb2 with the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the 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 the agonist LTBR antibody LTBRmAb2 with the heavy chain (HC ; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the 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 with 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 trimeric LIGHT engineered for better stability and human and mouse cross-reactivity (Tangr et al., “Cancer Cell,” Volume 29: pp. 285–96 (2016)) by fusion to the N terminus of IgG1 sigma Fc. ​vii. COVA1418 was generated by co-expression with 3xhmLIGHT-Fc (SEQ ID NO: 15) and the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV V antibody B21M (Fig. 1E). 3xhmLIGHT-Fc is a single-chain trimeric LIGHT that was engineered for better stability and cross-reactivity in humans and mice due to its fusion to the N-terminus of IgG1 sigma Fc (SEQ ID NO: 58) (Tangr et al., "Cancer Cell", Vol. 29: pp. 285-96 (2016)).

[0209] Symmetric antibodies (all IgG1 sigma, without KiH mutation): viii. COVA14114 was generated by expressing the anti-RSV B21M antibody heavy chain carrying a C-terminal LTα1β2 fusion (SEQ ID NO: 18) together with the light chain (LC; SEQ ID NO: 8) of the B21M antibody (Fig. 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 (Fig. 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 (Fig. 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 (Fig. 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 (Fig. 1C). xiii. COVA1452 is the heavy chain (HC; SEQ ID NO: ​​​​​​​​​12) and generated by co-expression with a light chain (LC; SEQ ID NO: 5) (Figure 1D).

[0210] Asymmetric antibodies with a 2:1 stoichiometry (all are IgG1 sigma and all are accompanied by KiH mutations) xiv. COVA14116 is generated by co-expression of an EDBmAb1 heavy chain carrying a C-terminal LTα1β2 fusion (SEQ ID NO: 21, including SEQ ID NO: 8 4) 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 is generated by co-expression of an anti-RSV B21M antibody heavy chain carrying a C-terminal LTα1β2 fusion (SEQ ID NO: 19, including SEQ ID NO: 85 with the heavy chain (H C; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV B21M antibody (Figure 1J) . xvi. COVA1484 is generated by co-expression of an anti-RSV B21M antibody heavy chain carrying an N-terminal stapled scFv BHA10 (VH-V L orientation SEQ ID NO: 22) fusion (including SEQ ID NO: 26, SEQ ID NO: 85) with the heavy chain (HC; SEQ ID NO: 7) and light chain (L C; SEQ ID NO: 8) of the anti-RSV B21M antibody (Figure 1P). xvii. COVA1485 is generated by co-expression of an anti-RSV B21M antibody heavy chain carrying an N-terminal stapled scFv BHA10 (VL- VH orientation SEQ ID NO: 23) fusion (including SEQ ID NO: 27, SEQ ID NO: 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 is generated by co-expression of an anti-RS carrying a C-terminal stapled scFv BHA10 (VH ​The V B21M antibody heavy chain, and the heavy chain (HC; SEQ ID NO: 7) and light chain of the anti-RSV B21M antibody were generated by co-expression with the light chain (LC; SEQ ID NO: 8) (Figure 1R). xix. COVA1487 is an anti-RSV carrying a C-terminal stapled scFv BHA10 (VL-V H orientation SEQ ID NO: 23) fusion (including SEQ ID NO: 29, SEQ ID NO: 85). The V B21M antibody heavy chain, and the heavy chain (HC; SEQ ID NO: 7) and light chain (L C; SEQ ID NO: 8) of the anti-RSV B21M antibody were generated by co-expression (Figure 1S). xx. COVA1480 is an anti-EDB antibody carrying an N-terminal stapled scFv BHA10 (VH-VL orientation SEQ ID NO: 22) fusion (including SEQ ID NO: 30, SEQ ID NO: 84). The EDBmAb1 heavy chain, and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 were generated by co-expression (Figure 1T). xxi. COVA1481 is an anti-EDB antibody carrying an N-terminal stapled scFv BHA10 (VL-V H orientation SEQ ID NO: 23) fusion (including SEQ ID NO: 31, SEQ ID NO: 84). The EDBmAb1 heavy chain of the antibody, and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 were generated by co-expression (Figure 1U). xxii. COVA1482 is an anti-EDB antibody carrying a C-terminal stapled scFv BHA10 (VH- VL orientation SEQ ID NO: 22) fusion (including SEQ ID NO: 32, SEQ ID NO: 84). The EDBmAb1 heavy chain of the antibody, and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 were generated by co-expression (Figure 1V). xxiii. COVA1483 is an anti-ED carrying a C-terminal stapled scFv BHA10 (VL -VH orientation SEQ ID NO: 23) fusion (including SEQ ID NO: 33, SEQ ID NO: 84). The heavy chain of the B antibody EDBmAb1 and the heavy chain (HC; SEQ ID NO: 4) of the anti-EDB antibody EDBmAb1 were generated by co-expression with the light chain (LC; SEQ ID NO: 5) (Figure 1W). xxiv. COVA14107 is the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminal stapled scFv BHA10 (VH -VL orientation, VL3 Y36F_S49Y_F87Y SEQ ID NO: 53) fusion (SEQ ID NO: 34 , including SEQ ID NO: 84), and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody E DBmAb1 were generated by co-expression (Figure 1X). xxv. COVA14108 is the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminal stapled scFv BHA10 (VH- VL orientation, VH_CDR1_Y33A SEQ ID NO: 54) fusion (SEQ ID NO: 35, SEQ ID NO: 84 , including SEQ ID NO: 84), and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmA b1 were generated by co-expression (Figure 1Y). xxvi. COVA14133 is the anti-EDB antibody EDBmAb1 heavy chain carrying a C-terminal stapled scFv BHA10 (VH -VL orientation SEQ ID NO: 22) fusion (SEQ ID NO: 38, including SEQ ID NO: 3), and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1 were generated by co-expression (Figure 1Z). xxvii. COVA14136 is the anti-RSV B21M antibody heavy chain carrying a C-terminal stapled scFv BHA10 (V H-VL orientation SEQ ID NO: 22) fusion (SEQ ID NO: 41, including SEQ ID NO: 6), and the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-EDB antibody EDBmAb1 were generated by co-expression (Figure 1A1). ​​​​xxviii. COVA14174 carries a C-terminal disulfide-stabilized scFv BHA1 0 (VH-VL orientation SEQ ID NO: 25) fusion (including SEQ ID NO: 39, SEQ ID NO: 3) It was generated by co-expression of the anti-EDB antibody EDBmAb1 heavy chain and 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 carries a C-terminal disulfide-stabilized scFv BHA10 ( VH-VL orientation SEQ ID NO: 25) fusion (including SEQ ID NO: 40, SEQ ID NO: 6) of the anti-R SV B21M antibody heavy chain and the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV B21M antibody, generated by co-expression (Figure 1A3). xxx. COVA1456 carries a C-terminal disulfide-stabilized scFv BHA10 (VH -VL orientation SEQ ID NO: 25) fusion (including SEQ ID NO: 56, SEQ ID NO: 84) of the anti-ED B antibody EDBmAb1 heavy chain and the heavy chain (HC; SEQ ID NO: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1, generated by co-expression (Figure 1A4). xxxi. COVA1462 carries a C-terminal disulfide-stabilized scFv BHA10 (V H-VL orientation SEQ ID NO: 25) fusion (including SEQ ID NO: 57, SEQ ID NO: 85) of the anti-R SV B21M antibody heavy chain and the heavy chain (HC; SEQ ID NO: 7) and light chain (LC; SEQ ID NO: 8) of the anti-RSV B21M antibody, generated by co-expression (Figure 1A5).

[0211] Mesothelin / LTBR bispecificity: asymmetric antibody, with a 2:1 stoichiometry (IgG1 Sigma, with KiH mutation xxxii. COVA14146 is a C-terminal stapled scFv BHA10 (V H-VL alignment number 22) Fusion (including SEQ ID NO: 80 and SEQ ID NO: 86) - carrying anti-Mesothelin antibody MSLNmAb1 heavy chain and the heavy chain (HC; SEQ ID NO: 81) and light chain (LC; SEQ ID NO: 82) of anti-Mesothelin antibody MSLNmAb1, generated by co-expression (Figure 1A6 and Table 3). ; SEQ ID NO: 81) and light chain (LC; SEQ ID NO: 82) generated by co-expression (Figure 1A 6 and Table 3).

[0212] Results All of the above constructs were successfully expressed and purified. Surprisingly, constructs containing LIGHT and LTα1β2 (COVA1418, COVA1454, COVA141 13, COVA14114, COVA14116, and COVA14117; Table 2) showed a purified yield reduced by up to 10-fold compared to the EDB / LTBR bispecific body containing the staple-processed scFv derived from the agonistic anti-LTBR antibody (e.g., COVA14 82 and COVA14133; see Table 4). Furthermore, constructs containing LIGHT (e.g., COVA145 4) showed a tendency for reduced monomer content as can be seen from the size exclusion chromatograms shown in Figure 2 and Table 4. In summary, these facts (purification yield up to 10-fold higher 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. or LTα1β2-Fc fusion, as shown in Figure 2 and Table 4, showed a tendency for reduced monomer content. In summary, these facts (purification yield up to 10-fold higher 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. or LTα1β2-Fc fusion, indicating that the bispecific constructs of the present invention may have better biophysical properties than constructs containing LIGHT or LTα1β2-Fc fusion. or LTα1β2-Fc fusion.

[0213]

Table 6

[0214] Example 2: EDB-dependent in vitro LTBR activation - NF-κB luciferase reporter assay The EDB / LTBR heterodimer can activate LTBR in an EDB-dependent manner To show that, the activity of the compound 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 the regulation of cell development and immune homeostasis. Activation of NF-κB via the tumor necrosis factor receptor (TNFR) or a TNFR superfamily member (e.g., LTBR) occurs upon engagement with their respective ligands. The A549 lung epithelial cell line naturally 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 a stimulant, the endogenous NF-κB transcription factor binds to the DNA response element and induces transcription of the luciferase gene.

[0215] To demonstrate the 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 overnight incubation, the coated plates were washed with PBS and blocked for 2 hours at 37°C in assay medium (DMEM + 10% heat-inactivated FBS). A 1:5 dilution series of the compound to be tested was prepared in assay medium as a 2-fold concentrated stock (tested​​ The final concentration was in the range of 200 nM to 2.6 pM). The blocking solution was removed by suction and then 50 μL of the diluted compound was added to the pre-blocked plate. 50 μL of A549 cell suspension (concentration of cell suspension = 0.4 Mio cells / ml assay medium was added to each well (20,000 cells / well). The A549 cells were pre-separated from the cell culture flask by using Accutase / EDTA and then transferred to the assay medium. The cells were incubated with the compound at 37 °C / 5% CO2 for 18 - 20 hours in a incubator.

[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 fold of LTBR signaling was calculated as follows: Induction fold = RLU cells / mean R LU 刺激済み cells (including as a control in each plate where non-stimulated cells were tested). LU 非刺激

[0218] A dose-response curve including standard deviation was plotted using GraphPad Prism and, where applicable, a non-linear fit was applied (log(agonist) vs response (variable slope - 3-parameter)). To fit the data, the x-values (concentration of the compound) were transformed using the x = Log(x) function of GraphP ad Prism.

[0219] Results Antibody-LIGHT fusion Tan et al. reported that bispecific molecule anti-EGFR-LIGHT fusion has antitumor activity similar to the study published by Tang et al. ("Cancer Cell", Vol. 29: pp. 285-96 (201 6). Similar to the study published b...

Claims

1. A multispecific binding molecule, comprising: (i) a first binding domain that specifically binds to lymphotoxin beta receptor (LTBR); and (ii) a second binding domain that specifically binds to the extra domain B (EDB) of fibronectin, wherein the multispecific binding molecule activates LTBR upon binding to the EDB. A multispecific binding molecule that activates LTBR upon binding to the EDB.

2. The multispecific binding molecule according to claim 1, wherein the multispecific binding molecule activates LTBR in a tumor-specific manner.

3. The multispecific binding molecule according to claim 1 or 2, wherein the multispecific binding molecule is a bispecific antibody.

4. The multispecific binding molecule according to any one of claims 1 to 3, wherein the multispecific binding molecule comprises two antigen-binding domains.

5. The multispecific binding molecule according to any one of claims 1 to 3, wherein the multispecific binding molecule comprises three antigen-binding domains.

6. The multispecific binding molecule according to claim 5, wherein the three antigen-binding domains comprise one binding domain that specifically binds to LTBR.

7. The multispecific binding molecule according to claim 5 or 6, wherein the three antigen-binding domains comprise two binding domains that specifically bind to EDB.

8. The multispecific binding molecule according to any one of claims 5 to 7, wherein the binding domain that specifically binds to LTBR comprises a single-chain variable domain of an 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-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, and the VH and VL are as follows: (i) HCDR1, HCDR2, and HCDR3 each comprising 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 comprising 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 comprising 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 comprising the amino acid sequences of SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; or ​ ​ ​ ​ ３； ​ (iii) HCDR1, HCDR2, and HCDR3 each containing the amino acid sequence 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 sequence of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively; or (iv) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 43, and VL contains an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 44; or (v) VH contains an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 47, and VL contains an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 48: or (vi) SEQ ID NO: 22; or (vii) SEQ ID NO: 23; or (viii) SEQ ID NO: 25 The multispecific binding molecule according to any one of claims 1 to 8, comprising any one of the above.

10. The second binding domain that specifically binds to EDB comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises a heavy chain complementarity determining region 1 (HCDR1), HCDR 2, and HCDR3, the VL comprises a light chain complementarity determining region 1 (LCDR1), LCD R2, and LCDR3, and the VH and VL are as follows: (i) HCDR1, HCDR2, and HCDR3 each containing the amino acid sequence 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 sequence of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; or (ii) VH contains an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 45, and VL contains an amino acid sequence having at least 95 % identity to the amino acid sequence of SEQ ID NO: 46, The multispecific binding molecule according to any one of claims 1 to 9, comprising any one of the above.

11. The following: (a) (i) A first heavy chain containing the amino acid sequence of SEQ ID NO: 1, which forms a binding domain together 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, which forms a binding domain together with a second light chain containing the amino acid sequence of SEQ ID NO: 5; or ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ (b)(i) A first heavy chain comprising the amino acid sequence of SEQ ID NO: 9, which forms a binding domain together with a first light chain comprising the amino acid sequence of SEQ ID NO: 10, and (ii) an amino acid A second heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a second light chain comprising the amino acid Sequence of SEQ ID NO: 5 Of any one of the above, the multispecific binding molecule according to any one of claims 1 to 10 [

12. ] 。 The following: (a)(i) An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 30, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) an amino acid A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid Sequence of SEQ ID NO: 5 Or (b)(i) An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 31, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) an amino acid A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid Sequence of SEQ ID NO: 5 Or (c)(i) An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 32, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) an amino acid A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid Sequence of SEQ ID NO: 5 Or (d)(i) An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 33, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) an amino acid A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid Sequence of SEQ ID NO: 5 Or (e)(i) An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 34, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) an amino acid A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid Sequence of SEQ ID NO: 5 Or (f)(i) An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 35, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) an amino acid A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid Sequence of SEQ ID NO: 5 Or (g)(i) The heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5 ​ An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 38, which is formed, and (ii) the amino acid sequence A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5; or Or (h) (i) An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 39, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) the amino acid sequence A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5; or Or Or Or An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 56, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) the amino acid sequence A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, A multispecific binding molecule according to any one of claims 1 to 3 or 5 to 10, comprising any one of the above. A multispecific Binding molecule.

13. (i) An scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO: 38, wherein the heavy chain portion forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and (ii) the amino acid sequence of SEQ ID NO: 5 A heavy chain comprising the amino acid sequence of SEQ ID NO: 4, which forms a binding domain together with a light chain comprising the amino acid sequence of SEQ ID NO: 5, and a multispecific binding molecule according to any one of claims 1 to 3 or 5 to 10.

14. One or more nucleic acid molecules encoding the multispecific binding molecule according to any one of claims 1 to 13.

15. One or more vectors comprising the one or more nucleic acid molecules according to claim 14.

16. An isolated host cell comprising the one or more nucleic acid molecules according to claim 14 or the one or more vectors according to claim 15.

17. A pharmaceutical composition comprising the multispecific binding molecule according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

18. A method for treating cancer in a subject in need of treatment, comprising administering to the subject the multispecific binding molecule according to any one of claims 1 to 13, the one or more nucleic acid molecules according to claim 14, the one or more vectors according to claim 15, or the pharmaceutical composition according to claim 17.

19. For activating LTBR in tumor tissue, the multispecific binding molecule according to any one of claims 1 to 13, the one or more nucleic acid molecules according to claim 14, the one described in claim 15 One

18. A method for treating cancer in a subject in need of treatment, comprising administering to the subject the multispecific binding molecule according to any one of claims 1 to 13, the one or more nucleic acid molecules according to claim 14, the one or more vectors according to claim 15, or the pharmaceutical composition according to claim 17.

19. For activating LTBR in tumor tissue, the multispecific binding molecule according to any one of claims 1 to 13, the one or more nucleic acid molecules according to claim 14, the one or more vectors according to claim 15, or the pharmaceutical composition according to claim 17.

19. For activating LTBR in tumor tissue, the multispecific binding molecule according to any one of claims 1 to 13, the one or more nucleic acid molecules according to claim 14, the one or more vectors according to claim 15, or the pharmaceutical composition according to claim 17. One or more vectors according to claim 15, or the pharmaceutical composition according to claim 17. A method comprising administering to the subject. Use of one or more vectors or of the pharmaceutical composition according to claim 17.

20. A method for producing a multispecific binding molecule according to any one of claims 1 to 13, comprising expressing in a host cell one or more nucleic acid molecules according to claim 14 or one or more vectors according to claim 15, and collecting the multispecific binding molecule. 。

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