Multispecific binding molecules containing LTBR and EDB binding domains and their applications

Abstract

This document provides an anti-LTBR multispecific binding molecule, a nucleic acid encoding the anti-LTBR multispecific binding molecule, a vector containing the nucleic acid, a host cell containing the vector, and a pharmaceutical composition containing the anti-LTBR multispecific binding molecule. It also provides a method of treating cancer in a subject of need, comprising administering the pharmaceutical composition disclosed herein.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 946,452, filed December 11, 2019. The entire contents of the above application are incorporated herein by reference. Technical Field

[0003] This 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. Methods for preparing the binding molecules and methods for using the binding molecules to kill cancer cells are also provided. Background Technology

[0004] Immunotherapy for cancer has the potential to improve survival in cancer patients by promoting immune responses to tumors. While some patients experience profound and durable responses to currently available anticancer immunotherapies (such as the anti-CTLA4 antibody ipilimumab, and anti-PD-1 / PD-L1 antibodies such as pembrolizumab or nivolumab), most patients do not benefit from such therapies (Ribas et al., Science 359:1350-1355 (2018)). For example, patients with so-called “cold” or non-inflammatory tumors characterized by a lack of immune cell infiltration or the absence of inflammatory features benefit less from anticancer immunotherapy (Chen and Mellman, Nature 541:321-30 (2017)). Therefore, there is a need for novel anticancer immunotherapies with improved efficacy.

[0005] Lymphotoxin β receptor (LTBR / TNFRSF3), a receptor of the TNF superfamily, is one of many potential targets for anticancer 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 17:525-535 (2002), Schneider et al., Immunol. Rev. 202:49-66 (2004)). LTBR is activated by two different trimeric ligands (LIGHT (TNFSF14) and lymphotoxin α1β2 (LTα1β2)). However, LTα1β2 is specific to LTBR, ​​and LIGHT also binds to and activates HVEM (TNFRSF14), which is a receptor expressed on immune cells and involved in the regulation of immune cells (Pasero et al., Curr. Opin. Pharmacol. 12:478-85 (2012)).

[0006] From the perspective of cancer immunotherapy, it is of particular interest to discover that LTBR, ​​through activation of its ligands, leads to the ectopic formation of tertiary lymphoid structures (TLS) (Schrama et al., Immunity 14:111-121 (2001); Tang et al., Cell.Mol.Immunol.14:809-18 (2017)). The presence of TLS in the tumor microenvironment is generally associated with immune infiltration and also with better prognosis, suggesting that TLS is involved in antitumor immune responses (Dieu-Nosjean et al., J.Clin.Oncol.26:4410-17 (2008); Weinstein and Storkus, Adv.Cancer Res.128:197-233 (2015)). Therefore, LTBR activation has the potential to promote TLS formation in the tumor microenvironment and induce antitumor immune responses, as well as improve current cancer immunotherapies.

[0007] Several preclinical studies have established the concept of targeting LTBR for treatment, with the aim of promoting protective anti-tumor immune responses.

[0008] Several groups have used its natural ligand LIGHT (TNFSF14) to target LTBR. LIGHT binds to both LTBR and its second receptor HVEM (TNFRSF14), which is expressed on immune cells such as B cells, T cells, NK cells, monocytes, and dendritic cells (DCs) (Pasero et al., Curr. Opin. Pharmacol. 12:478-85 (2012)). Therefore, it should be noted that LIGHT-mediated immunobiological effects may depend on either LTBR or HVEM.

[0009] Yu et al. demonstrated that forced expression of the membrane-bound form of LIGHT in mouse tumor cell lines led to a large infiltration of primitive T lymphocytes, which was associated with the upregulation of chemokine production and adhesion molecules, resulting in rejection of established tumors at both local and distal sites (Yu et al., Nat. Immunol. 5:141-9 (2004)). Similar findings were obtained in the scenario of forced expression of the membrane-bound LIGHT by delivering the LIGHT gene adenovirus into established tumors (Yu et al., J. Immunol. 179:1960-8 (2007)).

[0010] Building on these findings, and in an attempt to utilize this mode of action in a form more suitable for clinical applications, Tang et al. generated a homotrimeric single-chain LIGHT variant with improved stability and cross-reactivity in humans and mice, termed 3xhmLIGHT (Tang et al., Cancer Cell 29:285-96 (2016)). When fused with an EGFR-specific tumor-targeting antibody, 3xhmLIGHT induces antitumor immunity in mouse and human tumor models by increasing lymphocyte infiltration, thereby overcoming resistance to checkpoint blockade immunotherapy when combined with an anti-PD-L1 antibody in models with low lymphocyte infiltration. Tang et al. reported tolerability after intratumoral injection in tumor-bearing mice. No significant side effects were observed, as no significant changes in body weight or serum cytokines were observed. The authors did not report tolerability after systemic administration.

[0011] 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. 18:1207-17 (2017)). In a mouse solid tumor model, the VTP-LIGHT construct homed to tumor vessels, promoted vascular normalization, and induced TLS. The addition of VTP-LIGHT enhanced the combination of anti-CTLA4 and anti-PD-1 antibodies and the activity of in vivo anti-tumor vaccines. Weight loss was observed in treated mice following intravenous administration of VTP-LIGHT.

[0012] Gurney et al. reported in vitro and in vivo studies of a bispecific fusion construct consisting of a heterotrimeric single-chain LTα1β2 moiety fused to a B7-H4-specific tumor-targeting antibody (WO2018 / 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. Treatment with the LTα1β2 fusion construct in a mouse tumor model resulted in immune cell infiltration, induced cytokine expression, and TLS formation. The combination of the LTα1β2 antibody fusion construct and the anti-PD-L1 antibody exhibited superior antitumor activity compared to each compound alone. The efficacy model used by Gurney et al. was an artificial model composed of engineered cell lines overexpressing B7-H4. The activity of models with unengineered B7-H4 expression levels that are more representative of B7-H4 levels in human tumors remains unclear. Observations on mouse tolerability were not reported.

[0013] Michaelson et al. described the construction of bispecific antibodies targeting TRAIL-R2 and LTBR to explore the possibility that bispecific antibodies could trigger enhanced, synergistic, or broader antitumor responses compared to those achieved by treatment with a mixture of the two antibodies (Michaelson et al., MAbs 1:128-41 (2009)). TRAIL-R2 is a TNF family receptor widely expressed in normal tissues, including the colon, lung, liver, and brain (Spierings et al., J. Histochem. Cytochem. 52:821-31 (2004)), but it has also been found to be co-expressed with LTBR on the surface of human epithelial cancer cell lines. In vitro and in mouse tumor xenograft models, the bispecific constructs showed enhanced activity relative to the parental antibody. Observations on mouse tolerability were not reported.

[0014] These studies indicate the potential of targeting LTBR for tumor immunotherapy and suggest that activating LTBR signaling could enhance immune cell infiltration, induce TLS in the tumor environment, and potentially help overcome resistance to checkpoint inhibitory therapies.

[0015] The immune system is tightly regulated to ensure the eradication of immune-mediated pathogens without causing tissue damage or autoimmunity. Systemic immunomodulatory therapies are often observed to disrupt this delicate balance, leading to immune-related adverse events such as pneumonia, colitis, hepatitis, thyroid dysfunction, skin reactions, and eye inflammation. This represents a challenge in developing novel immunotherapies, specifically in cases where the toxicity can be additive or synergistic in combination therapies.

[0016] Due to the widespread expression of LTBR in organisms, agonistic LTBR-targeting drugs that can induce TLS and generate activating immune environments carry a significant risk of causing systemic immune-related adverse events. Interestingly, Johansson-Percival et al. reported weight loss in mice after systemic administration of the LTBR-activating compound VTP-LIGHT (Johansson-Percival et al., Nat. Immunol. 18:1207-17 (2017)). Therefore, as hypothesized in the prior art, there is a need for therapeutic forms that specifically activate LTBR in tumors rather than in other tissues to reduce the risk of toxicity and generate well-tolerated drugs that can be used for combination therapies (Allen et al., Oncotarget 8:99207-8 (2017); Tang et al., CellMol. Immunol. 14:809-18 (2017)).

[0017] Although several groups have studied LTBR as a therapeutic target using various LTBR-targeting fractions, no therapeutic modality capable of specifically activating LTBR in tumors has been described to date. Summary of the Invention

[0018] This article provides multispecific binding molecules, such as bispecific antibodies, that specifically activate the lymphotoxin β receptor (LTBR) in tumors. The multispecific binding molecule has primary specificity for the LTBR and secondary specificity for the extracellular matrix extracellular domain B (EDB). EDB is a tumor-associated antigen (TAA) of the extracellular matrix. The multispecific binding molecule activates the LTBR in tumors expressing EDB, but in the absence of EDB, it does not activate or only moderately activates the LTBR, ​​to a degree far less than its ligands LIGHT and LTα1β2, thereby reducing the risk of immune-related adverse events. Unlike previously described LTBR-activating molecules, effective LTBR activation is achieved in the presence of EDB through EDB-specific binding and LTBR binding through the LTBR-specific binding portion of the multispecific binding molecule of this invention. In the absence of EDB, the multispecific binding molecule will not lead to LTBR activation in normal tissues. This is a significant advantage over molecules based on natural LTBR ligands described in the prior art, such as LIGHT antibody fusions, which can activate LTBR independently of TAA and therefore have much lower tumor specificity for LTBR activation compared to the molecules of the present invention, as illustrated in the examples herein.

[0019] This document provides a multispecific binding molecule. A multispecific binding molecule may comprise (i) a first binding domain that specifically binds to a lymphotoxin β receptor (LTBR), and (ii) a second binding domain that specifically binds to EDB, wherein the multispecific binding molecule activates the LTBR upon binding to EDB. More specifically, when the multispecific binding molecule binds to both LTBR and EDB simultaneously, it activates the LTBR via its respective specific binding domains to these targets. Preferably, this occurs in a tumor environment in which both LTBR-expressing cells and EDB-expressing cells are present, resulting in specific activation of the LTBR in tumor tissue. In some embodiments, the multispecific binding molecule activates the LTBR in a tumor-specific manner. The multispecific binding molecule may be, for example, a bispecific antibody. In some embodiments, the multispecific binding molecule comprises two antigen-binding domains. In some embodiments, the multispecific binding molecule comprises three antigen-binding domains. The three antigen-binding domains may, for example, include one binding domain that specifically binds to LTBR. The three antigen-binding domains may, for example, include two binding domains that specifically bind to EDB.

[0020] In some embodiments, the multispecific binding molecule comprises three antigen-binding domains and is composed of an antibody (e.g., in the form of IgG), with an additional binding domain (e.g., in the form of a single-chain variable domain) fused to the antibody, for example, fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.

[0021] For the multispecific binding molecules of the present invention that specifically bind to LTBR and TAA present in the extracellular matrix, the TAA present in the extracellular matrix is ​​fibronectin. Preferably, the binding domain that binds to TAA specifically binds to the additional domain B (EDB) of fibronectin.

[0022] In some non-limiting embodiments, the binding domain that specifically binds to LTBR comprises a BHA10 antibody or a CBE11 antibody or a fragment or derivative thereof, such as a single-chain antibody fragment (scFv), which includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH includes heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL includes light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the VH and the VL include any of the following:

[0023] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0024] (ii) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0025] (iii) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, respectively; or

[0026] (iv) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:43, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96 ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:43 has at least 97% identity with the amino acid sequence of SEQ ID NO:44, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44. The amino acid sequence of NO:44 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0027] (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity 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%, or 100% identity with the amino acid sequence of SEQ ID NO:48, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:47, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:47, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:47 has at least 97% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48. The amino acid sequence of NO:48 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0028] (vi)SEQ ID NO:22; or

[0029] (vii)SEQ ID NO:23; or

[0030] (viii)SEQ ID NO:25.

[0031] In some non-limiting embodiments, the second binding domain that specifically binds to EDB comprises an L19 antibody or a fragment or derivative thereof, for example, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises any of the following:

[0032] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively; or

[0033] (ii) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:45, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:45 has at least 97% identity with the amino acid sequence of SEQ ID NO:46, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46. The amino acid sequence of NO:46 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

[0034] In some non-limiting embodiments, the multispecific binding molecule comprises:

[0035] (1) A binding domain that specifically binds to LTBR, ​​comprising a BHA10 antibody or a CBE11 antibody or a fragment or derivative thereof, such as a single-chain antibody fragment (scFv), comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the VH and the VL comprise any of the following:

[0036] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0037] (ii) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0038] (iii) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, respectively; or

[0039] (iv) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:43, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96 ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:43 has at least 97% identity with the amino acid sequence of SEQ ID NO:44, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44. The amino acid sequence of NO:44 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0040] (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity 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%, or 100% identity with the amino acid sequence of SEQ ID NO:48, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:47, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:47, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:47 has at least 97% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48. The amino acid sequence of NO:48 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0041] (vi)SEQ ID NO:22; or

[0042] (vii)SEQ ID NO:23; or

[0043] (viii)SEQ ID NO:25; and

[0044] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2 and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2 and LCDR3, wherein the antibody or fragment thereof comprises HCDR1, HCDR2 and HCDR3, which respectively comprise the amino acid sequences of SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74; and LCDR1, LCDR2 and LCDR3, which respectively comprise the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77.

[0045] In some non-limiting embodiments, the multispecific binding molecule comprises:

[0046] (1) A binding domain that specifically binds to LTBR, ​​comprising a BHA10 antibody or a CBE11 antibody or a fragment or derivative thereof, such as a single-chain antibody fragment (scFv), comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the VH and the VL comprise any of the following:

[0047] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0048] (ii) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0049] (iii) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, respectively; or

[0050] (iv) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:43, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96 ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:43 has at least 97% identity with the amino acid sequence of SEQ ID NO:44, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44. The amino acid sequence of NO:44 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0051] (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity 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%, or 100% identity with the amino acid sequence of SEQ ID NO:48, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:47, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:47, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:47 has at least 97% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48. The amino acid sequence of NO:48 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0052] (vi)SEQ ID NO:22; or

[0053] (vii)SEQ ID NO:23; or

[0054] (viii)SEQ ID NO:25; and

[0055] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises VH, which comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:45; and VL, which comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:45; VH comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:45. The amino acid sequence of NO:46 has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence; VH contains an amino acid sequence with at least 96% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 97% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:45. The amino acid sequence of NO:46 has at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:46.

[0056] In some non-limiting embodiments, the multispecific molecule comprises:

[0057] (1) A binding domain that specifically binds to the LTBR containing SEQ ID NO:22; and

[0058] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises any of the following:

[0059] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively; or

[0060] (ii) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46. In some non-limiting embodiments, the multispecific molecule comprises:

[0061] (1) A binding domain that specifically binds to the LTBR containing SEQ ID NO:23; and

[0062] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises any of the following:

[0063] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively; or

[0064] (ii) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:46.

[0065] In some non-limiting embodiments, the multispecific molecule comprises: (1) and (2)

[0066] (1) A binding domain that specifically binds to the LTBR containing SEQ ID NO:25;

[0067] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises any of the following:

[0068] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively; or

[0069] (ii) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:46.

[0070] In some non-limiting embodiments, the multispecific binding molecule comprises any of the following:

[0071] (a)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO:1, which forms a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO:2, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO:4, which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14121]; or

[0072] (b)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO:9, which forms a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO:10, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO:4, which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14122].

[0073] In some other non-limiting embodiments, the multispecific binding molecule comprises any of the following:

[0074] (c)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:30, wherein the heavy chain portion (containing SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1480]; or

[0075] (d)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:31, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1481]; or

[0076] (e)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:32, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1482]; or

[0077] (f)(i) a scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:33, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1483]; or

[0078] (g)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:34, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14107]; or

[0079] (h)(i) a scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:35, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14108]; or

[0080] (j)(i) a scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:38, wherein the heavy chain portion (containing SEQ ID NO:3) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14133]; or

[0081] (k)(i) a scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:39, wherein the heavy chain portion (containing SEQ ID NO:3) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14174]; or

[0082] (l)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:56, wherein the heavy chain portion of the scFv heavy chain fusion (comprising SEQ ID NO:84) forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1456].

[0083] In some embodiments, the multispecific molecule comprises (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO:38, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain with a light chain containing the amino acid sequence of SEQ ID NO:5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO:4, wherein the heavy chain forms a binding domain with a light chain containing the amino acid sequence of SEQ ID NO:5.

[0084] Also provided are one or more nucleic acid molecules encoding multispecific binding molecules as disclosed herein. Also provided are one or more vectors comprising one or more nucleic acid molecules as disclosed herein. Also provided are isolated host cells comprising one or more vectors as disclosed herein.

[0085] Pharmaceutical compositions comprising a multispecific binding molecule as disclosed herein and a pharmaceutically acceptable carrier are also provided.

[0086] Methods for treating cancer in subjects in need are also provided. These methods include administering to the subject a multispecific binding molecule as disclosed herein, one or more nucleic acid molecules as disclosed herein, one or more carriers as disclosed herein, or a pharmaceutical composition as disclosed herein.

[0087] Also provided are the uses of multispecific binding molecules, one or more nucleic acid molecules, one or more carriers, or pharmaceutical compositions as disclosed herein for activating LTBR in tumor tissue.

[0088] Methods for preparing multispecific binding molecules as disclosed herein are also provided, comprising expressing one or more nucleic acid molecules as disclosed herein or one or more vectors as disclosed herein in a host cell, and harvesting the multispecific binding molecules.

[0089] For the multispecific binding molecule of the present invention, the binding domain of the first antigen binds to the LTBR present on cells in the tumor (e.g., tumor cells, fibroblasts, monocytes, etc.). The binding domain of the second antigen binds to EDB, which is a tumor-associated antigen (TAA) present in the extracellular matrix of the tumor.

[0090] In some embodiments, multispecific binding molecules (such as bispecific antibodies or their antigen-binding fragments) comprise two heavy chains (HC) and two light chains (LC) to form two binding domains for EDB.

[0091] In some embodiments, the scFv is fused to the carboxyl (C) terminus or amino (N) terminus of an HC. In some embodiments, the scFv fused to the HC comprises an amino acid sequence selected from the following: 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.

[0092] Also provided are isolated nucleic acids encoding scFv fused with the HC of an isolated anti-LTBR bispecific antibody or its antigen-binding fragment as disclosed herein. Also provided are isolated nucleic acids encoding the HC and LC of an anti-LTBR bispecific antibody or its antigen-binding fragment as disclosed herein.

[0093] In some implementations, the heavy chain, light chain, and / or functional fragments thereof, such as antigen-specific binding domains, are human or humanized.

[0094] It also provides nucleic acids that encode the heavy chain, light chain, and / or functional fragments of multispecific binding molecules as disclosed herein.

[0095] A vector containing nucleic acids as disclosed herein is also provided.

[0096] It also provides a host cell containing nucleic acids or vectors as disclosed herein.

[0097] In a preferred embodiment, the multispecific binding molecule, bispecific antibody, nucleic acid, vector, or host cell according to the present invention are respectively isolated multispecific binding molecules, isolated bispecific antibodies, isolated nucleic acids, isolated vectors, or isolated host cells.

[0098] Pharmaceutical compositions are also provided that comprise multispecific binding molecules as disclosed herein, such as bispecific antibodies, or antigen-binding fragments thereof, and pharmaceutically acceptable carriers.

[0099] A method for treating cancer in a subject in need is also provided. The method includes (a) identifying a subject in need of cancer treatment; and (b) administering the multispecific binding molecule of the invention (e.g., in the form of a pharmaceutical composition) to the subject in need, wherein the pharmaceutical composition is administered to the subject in need to treat the subject's cancer.

[0100] A method for activating cells expressing LTBR is also provided. This method involves contacting cells expressing LTBR with a multispecific binding molecule of the present invention (e.g., in the form of a pharmaceutical composition), wherein contacting cells expressing LTBR with the multispecific binding molecule or pharmaceutical composition results in increased expression of RANTES, IL-6, IL-8, MIP-3b, ICAM-1, I-TAC, IP-10, IL-12p70, TNF-α, MIP-3a, and / or SDF-1a compared to cells expressing LTBR in the absence of EDB.

[0101] A method for inhibiting the growth or proliferation of cancer cells expressing EDB in a tumor is also provided. This method involves contacting cancer cells and / or cells in the tumor microenvironment with the multispecific binding molecule of the present invention (e.g., in the form of a pharmaceutical composition), wherein contacting the cancer cells and / or cells in the tumor microenvironment with the pharmaceutical composition inhibits the growth or proliferation of cancer cells.

[0102] Methods for preparing pharmaceutical compositions as disclosed herein are also provided. These methods involve combining the isolated multispecific binding molecules of the present invention (e.g., bispecific antibodies or antigen-binding fragments thereof) with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0103] A method for preparing multispecific binding molecules (such as bispecific antibodies or antigen-binding fragments thereof) is also provided. This method includes culturing host cells containing nucleic acids as disclosed herein under conditions suitable for preparing multispecific binding molecules (such as bispecific antibodies or antigen-binding fragments thereof), and recovering the multispecific binding molecules (such as bispecific antibodies or antigen-binding fragments thereof). Attached Figure Description

[0104] A better understanding of the foregoing invention and the following detailed description of preferred embodiments of this patent application can be achieved by reading the accompanying drawings. However, it should be understood that this patent application is not limited to the precise embodiments shown in the drawings.

[0105] Figures 1A to 1A6 A schematic diagram of the anti-LTBR bispecific antibody and control molecule is shown. Figures 1A to 1D A control monoclonal antibody with a silent Fc mutant IgG1 is shown. Figures 1E to 1F A 1:1 mortar and pestle (KiH) heterodimer comprising a targeting arm (B21M or EDBmAb1) and a human LIGHT fused to the Fc is shown. A set of mutations were introduced into the Fc fused to the human LIGHT to eliminate binding to protein A and facilitate the purification of the heterodimer. Figures 1G to 1J The human LTα1β2 antibody fusion is shown. Figures 1K to 1O The 1:1 KiH heterodimer was shown. Figures 1P to 1S The 2:1 heterodimer, an isotype control antibody, was shown fused with a bound scFv derived from LTBRmAb1. Figures 1T to 1W A 2:1 heterodimer, EDBmAb1, is shown fused with a scFv derived from LTBRmAb1. Figures 1X to 1Y A 2:1 heterodimer, fused with a binding of EDBmAb1 and a lower affinity variant derived from LTBRmAb1, is shown. Figures 1Z to 1A1 A 2:1 heterodimer, EDBmAb1 or B21M, was shown fused with a scFv derived from LTBRmAb1, but without protein A mutations in the Fc region. Figures 1A2 to 1A5 A 2:1 heterodimer, EDBmAb1 or B21M, is shown fused with a disulfide-stabilized scFv derived from LTBRmAb1. Figure 1A6 A 2:1 heterodimer, MSLNmAb1, is shown fused with a scFv derived from LTBRmAb1.

[0106] Figures 2A to 2G Size exclusion chromatograms (SEC) of the following substances are shown: Figure 2A COVA1418, which consists of the heavy and light chains of 3xhmLIGHT-Fc and anti-RSV antibody B21M; Figure 2B COVA1454 is composed of the heavy and light chains of 3xhmLIGHT-Fc and the anti-EDB antibody EDBmAb1. Figure 2CCOVA14133 is composed of the heavy chain of the anti-EDB antibody EDBmAb1 carrying a C-terminal stapled scFv BHA10 (VH-VL orientation) fusion body, and the heavy and light chains of the anti-EDB antibody EDBmAb1. Figure 2D COVA14113 is composed of a heavy chain of EDBmAb1 carrying the C-terminal LTα1β2 fusion complex and a light chain of the anti-EDB antibody EDBmAb1. Figure 2E COVA14114 is composed of a heavy chain of anti-RSV B21M antibody carrying a C-terminal LTα1β2 fusion complex and a light chain of anti-RSV B21M antibody. Figure 2F COVA14116 is composed of the heavy chain of EDBmAb1 carrying the C-terminal LTα1β2 fusion complex and the heavy and light chains of the anti-EDB antibody EDBmAb1. Figure 2G COVA14117 is composed of a heavy chain of anti-RSV B21M antibody carrying a C-terminal LTα1β2 fusion complex, and heavy and light chains of anti-RSV B21M antibody.

[0107] Figures 3A to 3D The figure shown illustrates the results of the A549 NF-kB reporter gene assay. Figure 3A Compared with COVA1418 and recombinant human LIGHT, COVA1454 induces tumor-associated antigen (TAA)-dependent activation of LTBR. Figure 3B Compared with COVA1418 and recombinant human LIGHT, COVA1454 enables TAA-independent activation of LTBR. Figure 3C Compared with recombinant human LIGHT and recombinant human LTα1β2, TAA-dependent activation of LTBR via COVA14113 and COVA14116 was observed. Figure 3D Compared with recombinant human LIGHT and recombinant human LTα1β2, TAA-independent activation of LTBR was achieved via COVA14113 and COVA14116.

[0108] Figures 4A to 4D The figure shown illustrates the results of A549 NF-kB reporter gene assays using a 1:1 heterodimer composed of EDBmAb1 and LTBRmAb1 or LTBRmAb2. Figure 4A Compared with COVA14120, COVA14124, COVA1413, COVA1440 and recombinant human LIGHT, COVA14121 induces tumor-associated antigen (TAA)-dependent activation of LTBR. Figure 4BCompared with COVA14120, COVA14124, COVA1413, COVA1440 and recombinant human LIGHT, COVA14121 provides TAA-independent activation of LTBR. Figure 4C Compared with COVA14123, COVA14124, COVA1402, COVA1440 and recombinant human LIGHT, COVA14122 showed TAA-dependent activation of LTBR. Figure 4D Compared with COVA14123, COVA14124, COVA1402, COVA1440 and recombinant human LIGHT, TAA-independent activation of LTBR was achieved via COVA14122.

[0109] Figures 5A to 5E The figure shown illustrates the results of the A549 NF-kB reporter gene assay using a 2:1 bispecific antibody. Figure 5A In the presence of fibronectin containing EDB, LTBR was effectively activated by COVA1456 (2:1EDBmAb1×LTBR mAB1). No LTBR activation was observed in the presence of the isotype control molecule COVA1462 (2:1B21M×LTBR mAb1). Figure 5B In the absence of fibronectin containing EDB, LTBR activation via COVA1456 or its isotype control molecule COVA1462 was not measured. Figure 5C Comparison of TAA-dependent LTBR activation results by COVA1456 and COVA1482, their corresponding control molecules COVA1462 and COVA1486, and recombinant human LIGHT; Figure 5D Comparison of TAA-dependent LTBR activation results using COVA1482, bispecific antibodies COVA14107 and COVA14108 containing lower affinity variants of LTBRmAb1, and COVA1486; Figure 5E Comparison of TAA-dependent LTBR activation results using COVA1482 and COVA14133 (constructions without protein A mutation) and their corresponding control molecules COVA1486 and COVA14136; Figure 5FIn the presence of EDB-containing fibronectin, LTBR was effectively activated by COVA14133 (2:1EDBmAb1×LTBR mAB1) and COVA14116 (2:1EDBmAb1×LTα1β2). No LTBR activation was observed in the isotype control molecule COVA14136 (2:1B21M×LTBR mAb1). TAA-independent activation of LTBR was achieved by COVA14117 (2:1B21M×LTα1β2). Figure 5G In the absence of EDB-containing fibronectin, LTBR activation via COVA14133 or its isotype control molecule COVA14136 was not measured. TAA-independent activation of the LTBR was achieved via COVA14116 and COVA14117.

[0110] Figure 6 The results of flow cytometry staining of ICAM-1 on A375 cells after co-culture experiments are shown. COVA1482 and its control molecule COVA1486 are compared with recombinant human LIGHT.

[0111] Figures 7A to 7J The figure shows the cytokine measurements in the supernatant of cocultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133, compared to COVA14136 and COVA1440. Measurements were performed using the MSD platform. Figure 7A : human RANTES concentration; Figure 7B Human IL-6 concentration; Figure 7C : The concentration of human IL-8; Figure 7D : The concentration of human MIP-3b. Figures 7E to 7J The figures shown also include 2:1 antibody x LTα1β2 fusions COVA14116 and COVA14117, as well as EDBmAb1 COVA1452. Figure 7E : human IP-10 concentration; Figure 7F : human SDF-1a concentration; Figure 7G : human IL-12p70 concentration; Figure 7H Human I-TAC concentration; Figure 7I : human MIP-3a concentration; Figure 7J : The concentration of human TNFα.

[0112] Figures 8A to 8B The study demonstrates LTBR activation via MSLN / LTBR bispecific antibody in A549 NF-kB reporter gene / CHOK1MSLN or A549 NF-kB reporter gene / H226 co-cultured cells assays. Figure 8ALTBR activation was measured in A549 NF-κB reporter gene / H226 co-culture. COVA14146 (2:1 MSLNmAb1×LTBRmAb1) was compared with LIGHT and the isotype control 2:1 construct COVA1486; Figure 8B The concentration of RANTES secreted upon activation of the LTBR in an A549NF-kB reporter gene / H226 co-culture assay was measured. COVA14146 (2:1 MSLNmAb1 × LTBRmAb1) was compared with LIGHT and the isotype control 2:1 construct COVA1486.

[0113] Figures 9A to 9B A schematic diagram of a possible LTBR activation mechanism is shown. Figure 9A In the presence of EDB (tumor-associated antigen (TAA)) in the extracellular matrix, bispecific antibodies can cluster LTBRs onto the cell surface via binding to EDB. Activation of LTBRs leads to the secretion of chemically induced cytokines and chemokines. Figure 9B In the absence of EDB in the extracellular matrix, LTBR clustering does not occur. As a result, LTBR activation may not occur.

[0114] Figure 10 The migration of PBMCs toward cytokines induced by LTBR activation is shown. Supernatants from co-cultures treated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 (compared to COVA14136 and COVA1440) and the anti-EDB / LTα1β2 fusion COVA14116 (compared to COVA14117) (shown in Figure 7) were used as inducers for PBMCs in the transwell migration assay. The number of PBMCs migrating toward the co-culture supernatant was counted and is shown in the figure. PBMC migration toward the supernatant from co-cultures stimulated with COVA14133, COVA14116, and COVA14117 (with slightly less migration) was induced in a dose-dependent manner. Supernatants from co-cultures incubated with the non-targeted control molecule COVA14136 did not induce PBMC migration.

[0115] Figures 11A to 11BThe adhesion and migration of monocytes to HUVEC monolayers stimulated with the anti-EDB / anti-LTBR bispecific antibody COVA14133 are shown (compared to its control COVA14136). In an imaging-based assay consisting of continuous flow of monocytes on a HUVEC monolayer grown in the presence of EDB and stimulated with 50 nM COVA14133 or COVA14136, the number of adherent (“A”) and migrating (B) monocytes over time was counted. Student's t-test analysis was performed for COVA14133 versus COVA14136, and the results are labeled as follows: *P<0.05, **P<0.01, ***P<0.005. Detailed Implementation

[0116] The background and description throughout this specification reference or describe various publications, articles, and patents; the full text of each of these references is incorporated herein by reference. Discussions of documents, actions, materials, devices, articles, etc., included in this specification are intended to provide context for the invention. Such discussions are not intended to acknowledge that any or all of these matters constitute prior art with respect to any disclosed or claimed invention.

[0117] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings set forth in this specification.

[0118] It should be noted that, unless the context clearly indicates otherwise, the singular forms “a” and “the” as used herein and in the appended claims include plural references.

[0119] Unless otherwise stated, any numerical value, such as concentrations or concentration ranges described herein, should be understood to be modified by the term "about" in all cases. Therefore, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context clearly indicates otherwise, as used herein, numerical ranges explicitly include all possible subranges, all individual numerical values ​​within that range, including both integers and fractions within such ranges.

[0120] Unless otherwise stated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or be able to determine, various equivalents of specific embodiments of the invention described herein using only conventional experiments. Such equivalents are intended to be covered by this invention.

[0121] As used herein, the terms “comprising,” “including,” “having,” or “containing,” or any other variation thereof, should be understood to mean including the specified integers or groups of integers, but not excluding any other integers or groups of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that includes a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent in such compositions, mixtures, processes, methods, articles, or apparatus. Furthermore, unless expressly indicated otherwise, “or” means inclusive or not exclusive. For example, condition A or B is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0122] As used herein, the connecting term "and / or" between multiple listed elements is understood to include both individual options and combined options. For example, in the case where two elements are connected by "and / or", the first option means that the first element applies even without the second element. The second option means that the second element applies even without the first element. The third option means that it is appropriate to use the first and second elements together. Any of these options is understood to fall within the meaning and therefore satisfies the requirement of the term "and / or" as used herein. The concurrent applicability of more than one option is also understood to fall within the meaning and therefore satisfies the requirement of the term "and / or".

[0123] As used herein, the term “composed of” as used throughout the specification and claims means that any of the listed integers or groups of integers may be included, but additional integers or groups of integers may not be added to the specified method, structure or composition.

[0124] As used herein, the term "consistent essentially of" as used throughout the specification and claims means to include any enumerated integers or groups of integers, and optionally includes any enumerated integers or groups of integers that do not substantially alter the basic or novel characteristics of the specified method, structure, or composition. See MPEP §2111.03.

[0125] As used herein, “subject” means any animal, preferably a mammal, and most preferably a human. As used herein, the term “mammal” covers 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.

[0126] It should also be understood that when referring to the dimensions or characteristics of components of the preferred invention, the terms “about,” “approximately,” “generally,” “substantially,” and similar terms used herein indicate that the described dimensions / characteristics are not strict boundaries or parameters, and do not exclude minor variations that are functionally identical or similar, as understood by one of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.) without altering the least significant figures.

[0127] In the context of two or more nucleic acid or polypeptide sequences (e.g., an anti-LTBR bispecific antibody and the polynucleotide encoding them, an anti-LTBR / anti-EDB bispecific antibody and the polynucleotide encoding them, an LTBR polypeptide and the LTBR polynucleotide encoding them, an EDB polypeptide and the EDB polynucleotide encoding them), the term "identical" or percentage "identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of the same amino acid residues or nucleotides when compared and aligned to obtain the maximum correspondence, as measured by one of the following sequence comparison algorithms or by visual inspection.

[0128] For sequence alignment, a reference sequence is typically used, and the test sequence is compared to it. When using a sequence alignment algorithm, the test and reference sequences are input into the computer, the coordinates of the subsequences are specified (if necessary), and the program parameters of the sequence alignment algorithm are specified. The sequence alignment algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified program parameters.

[0129] The optimal alignment of sequences for comparison can be performed, for example, by Smith & Waterman’s local homology algorithm, *Advanced Applied Mathematics*, Vol. 2: 482 (1981), by Needleman & Wunsch’s homology alignment algorithm, *Journal of Molecular Biology*, Vol. 48: 443 (1970), or by searching Pearson & Lipman’s similarity method, *Proceedings of the National Academy of Sciences*, Vol. 85: 2444 (1988). 85:2444 (1988)), implemented by computerization or by visual inspection (GAP, BESTFIT, FASTA and TFASTA, Wisconsin Genetics Package, Computational Genetics Group, 575 Science Dr., Madison, WI, Wisconsin, WI).

[0130] Examples of algorithms suitable for determining sequence identity percentages and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., (1990) J.Mol.Biol.215:403-410 and Altschul et al., (1997) Nucleic Acids Res.25:3389-3402, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) in the query sequence by recognizing short words of length W that match or satisfy a positive threshold score T when compared to words of the same length in a database sequence. T is called the neighboring word score threshold (Altschul et al., ibid.). These initial neighboring word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence, as long as the accumulated alignment score can be increased.

[0131] For nucleotide sequences, cumulative scores are calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate cumulative scores. Word hits in each direction cease when the cumulative alignment score decreases by an amount X from its maximum realized value; the cumulative score becomes zero or lower due to the accumulation of one or more negative score residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and a comparison of two strands. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0132] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One similarity measure provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in a comparison of the test nucleic acid with a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0133] Another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid cross-reacts with the polypeptide encoded by the second nucleic acid, as described below. Therefore, the polypeptide is usually substantially identical to the second polypeptide, for example, where the two peptides differ only by conserved substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions.

[0134] As used herein, the term "polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," means any polynucleotide or polydeoxynucleotide that may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA consisting of a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, "polynucleotide" refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with a backbone modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and rare bases such as inosine. DNA and RNA can be modified in various ways; therefore, "polynucleotides" include chemically modified, enzymatically modified, or metabolically modified forms of polynucleotides that are normally found naturally, as well as chemical forms of DNA and RNA specific to viruses and cells. "Polynucleotides" also include relatively short nucleic acid chains, often referred to as oligonucleotides.

[0135] As used in this article, the term "vector" is a replicon in which another nucleic acid segment can be operatively inserted to induce replication or expression of that segment.

[0136] As used herein, the term "host cell" refers to a cell containing the nucleic acid molecules of the present invention. A "host cell" can be any type of cell, such as a primary cell, a cultured cell, or a cell derived from a cell line. In one embodiment, a "host cell" is a cell transfected with the nucleic acid molecules of the present invention. In another embodiment, a "host cell" is a progeny or potential progeny of such transfected cells. The progeny of a cell may or may not be identical to the parent cell, for example, due to mutations or environmental influences that may occur in the progeny, or due to the integration of the nucleic acid molecules into the host cell genome.

[0137] As used herein, the term "expression" refers to the biosynthesis of a gene product. This term encompasses transcription from a gene to RNA. It also encompasses translation from RNA to one or more polypeptides, and all naturally occurring post-transcriptional and post-translational modifications. Expressed multispecific binding molecules, such as bispecific antibodies, may be located in the cytoplasm of the host cell, in an extracellular environment such as a growth medium for cell cultures, or anchored to the cell membrane. Preferably, the multispecific binding molecule is secreted from the producing host cell into the culture medium.

[0138] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule composed of amino acids and can be recognized by those skilled in the art as a protein. Conventional single-letter or three-letter codes for amino acid residues are used herein. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length. This polymer can be linear or branched, may contain modified amino acids, and may be intercalated with non-amino acids. The term also covers amino acid polymers that have been naturally modified or modified by intervention; natural or interventional modifications include, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art.

[0139] The peptide sequences described herein are written according to usual convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomers of amino acids are known, they are the L-forms of the amino acids represented unless otherwise explicitly stated.

[0140] 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, such as comprising 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 is sometimes referred to herein as an "anti-LTBR" binding molecule or antibody in view of the presence of binding specificity against LTBR.

[0141] As used herein, "binding domain" refers to the functional portion of a binding molecule, such as that derived from an antibody, that imparts specific binding to a target molecule. Examples of binding domains are variable regions of antibodies that impart specific binding to a target molecule, and can be formed from more than one chain of the antibody, for example, by pairing a variable domain of the heavy chain with a variable domain of the light chain, or by a single chain (such as in scFv molecules), or by, for example, a single domain (such as VHH from llamas, e.g., nanobodies), etc. The target molecule of this invention is LTBR or fibronectin, specifically the EDB of fibronectin.

[0142] As used herein, the term "specific binding" refers to the binding of an antibody to a predetermined antigen with a greater affinity than to other antigens. Typically, antibodies bind with a dissociation constant (Ki). D Binding to the predetermined antigen: approximately 1 × 10 -7 M or smaller, for example, about 1×10 -8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10M or smaller, approximately 1×10 -11 M or smaller, approximately 1×10 -12 M or smaller, approximately 1×10 -13 M or smaller, or about 1×10 -14 M or smaller, usually K D Compared to its binding to non-specific antigens or epitopes (such as BSA, casein), K D The dissociation constant can be measured using standard procedures. However, antibodies that specifically bind to a predetermined antigen may be cross-reactive to other related antigens, for example, to the same predetermined antigen from other species (homologous) (such as humans or monkeys, such as cynomolgus (cyno) or chimpanzee (chimp)).

[0143] As used herein, the term "tumor-associated antigen" or "TAA" refers to an antigen present on tumor cells or in the extracellular matrix of a tumor that is not qualitatively different from antigens found on normal cells or in the extracellular matrix of normal tissues, but is quantitatively different in some respects, such as being present on tumor cells or in the extracellular matrix of a tumor in significantly greater amounts, at higher densities, at different expression sites, and / or having different access to the immune system. In some embodiments, tumor-associated antigens are present on tumor cells or in the extracellular matrix at at least two times higher, more preferably at least five times higher, such as at least 10 times higher, even more preferably at least 100 times higher, such as at least 1000 times higher, and most preferably at least 10,000 times higher, than on non-tumor cells or in the extracellular matrix. EDB is present in fibronectin in the extracellular matrix of tumor tissues; however, it is generally not detectable in the form of fibronectin present in normal tissues (i.e., the same tissue under normal conditions and not in a tumor environment).

[0144] As used herein, the term "extracellular matrix" refers to the noncellular components present in all tissues and organs as a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, which provide structural and biochemical support to surrounding cells. Its exact composition varies from tissue to tissue, but it is generally composed of proteoglycans, water, minerals, and fibrous proteins. Proteoglycans consist of a protein core surrounded by long chains of amyloid molecules called glycosaminoglycans. Two main types of extracellular matrix molecules constitute the matrix: proteoglycans and fibrous proteins, including, for example, collagen, elastin, fibronectin, and laminin.

[0145] Antibody

[0146] This invention generally relates to anti-LTBR multispecific binding molecules, nucleic acids and expression vectors encoding multispecific binding molecules, recombinant cells containing the vectors, and compositions comprising multispecific binding molecules. In a preferred embodiment, the anti-LTBR multispecific binding molecule is an anti-LTBR multispecific antibody, such as an anti-LTBR bispecific antibody or its antigen-binding fragment. In some embodiments, the anti-LTBR multispecific binding molecule may comprise a binding domain that specifically binds to LTBR, ​​which is in a form different from the antibody or its functional fragment, for example, it may comprise an anti-LTBR finomer, an anti-LTBR affimier, an anti-LTBR darpin, and / or other protein scaffolds screened for candidates for specific binding to LTBR. In the multispecific binding molecules of this invention, the LTBR-specific binding domain is not provided by LIGHT or LTα1β2 (the natural ligand of LTBR), nor by its functional fragments or derivatives, such as 3xhmLIGHT. In a preferred embodiment, the LTBR-specific binding domain in the multispecific binding molecule of this invention comprises an antibody against LTBR, ​​preferably an agonist antibody against LTBR, ​​or its functional fragments or derivatives, such as scFv. Such agonistic antibodies against LTBR have been described, and non-limiting examples include BHA10 (e.g., WO2004002431) and CBE11 (e.g., WO0230986), or alternatively generated according to known methods for antibody generation such as mouse immunization, phage display, etc.

[0147] Fyn SH3-derived peptides, or “fynomers,” are well known in the art and have been described, for example, in Grabulovski et al., (2007) JBC, 282, pp. 3196-3204; WO 2008 / 022759; Bertschinger et al., (2007) Protein Eng Des Sel 20(2):57-68; and Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255. The term “Fyn SH3-derived peptide” is used interchangeably with the term “fynomer” herein, referring to a non-immunoglobulin-derived binding peptide (e.g., a so-called scaffold, as described in Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255) derived from the human Fyn SH3 domain. Fynomers are small, globular peptides of approximately 7 kDa. The SH3 domain of human Fyn kinase has been successfully used as a scaffold to engineer proteins (called Fynomer, a Fyn SH3-derived binding protein) that bind to different target proteins with high affinity and specificity (WO 2008 / 022759, WO 2011 / 023685, WO 2013 / 135588, WO 2014 / 170063; Grabulovski D. et al., (2007) J Biol Chem 282, pp. 3196-3204; Bertschinger J. et al., (2007) Protein Eng Des Sel, 20, pp. 57-68; and Schlatter et al., (2012) mAbs, 4(4), pp. 497-450).

[0148] Affimer molecules are small proteins (12kDa to 14kDa) 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 various applications (see, for example, Tiede et al., eLife 2017, DOI:10.7554 / eLife.24903).

[0149] DARPin (used for designing repeating ankyrin proteins) are genetically engineered antibody-mimicking proteins that typically exhibit high protein binding specificity and are derived from natural ankyrin proteins. They consist of at least three repeating motifs, and for four- or five-repeating DARPin, their molecular weight is typically about 14 kDa or 18 kDa, respectively. DARPin design is described, for example, in Binz et al., 2003, J. Mol. Biol. 332:489-503.

[0150] Other forms of protein binding (such as protein scaffolds) are known in the art and can also be used to provide one or more binding domains for certain embodiments of the multispecific binding molecules of the present invention.

[0151] In a preferred embodiment of the invention, the binding domain that binds to the LTBR activates the LTBR upon binding and is derived from an antibody that specifically binds to the LTBR, ​​preferably an agonist antibody. In a specific embodiment, the binding domain that binds to the LTBR is a single-chain variable domain (scFv) of the antibody, which can be in any available form and, for example, stabilized by the methods described previously and / or herein.

[0152] In some embodiments, the present invention relates to anti-LTBR / anti-EDB bispecific antibodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies and expression vectors, recombinant cells containing the vectors, and compositions comprising bispecific antibodies. Methods for preparing multispecific binding molecules and / or antibodies, and methods for treating diseases (including cancer) using multispecific binding molecules and / or antibodies are also provided. The multispecific binding molecules and / or antibodies disclosed herein have one or more desired functional properties, including but not limited to one or more of the following: 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 anticancer therapies.

[0153] As used herein, the term "antibody" is used broadly and includes immunoglobulins or antibody molecules, including human antibodies, humanized antibodies, complex antibodies, and chimeric antibodies, as well as monoclonal or polyclonal antigen-binding domains. Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen. Antibody structures are well known. Immunoglobulins can be designated into five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) based on the amino acid sequence of their heavy chain constant domain. IgA and IgG are further subclassed into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibodies of the present invention can be any of these five major classes or their corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. Based on the amino acid sequence of their constant domain, the light chains of vertebrate antibodies can be designated into two completely different types, namely κ and λ. Therefore, the antibodies of the present invention can contain either a κ or λ light chain constant domain. According to a specific embodiment, the antibody of the present invention comprises a heavy chain and / or light chain constant region derived from a rat or human antibody. In addition to the heavy chain and light chain constant domains, the antibody also comprises an antigen-binding region consisting of a light chain variable region and a heavy chain variable region, each variable region comprising three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0154] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated bispecific antibody that specifically binds to the LTBR is substantially free of bispecific antibodies that do not bind to the LTBR; an isolated bispecific antibody that specifically binds to the LTBR and / or EDB is substantially free of bispecific antibodies that do not bind to the LTBR and / or EDB). Additionally, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0155] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies constituting the group are identical except for the possibility of naturally occurring mutations present in small amounts. The monoclonal antibodies of this invention can be prepared by hybridoma methods, phage display technology, single-lymphocyte gene cloning technology, or by recombinant DNA methods. For example, monoclonal antibodies can be produced from hybridomas comprising B cells derived from transgenic nonhuman animals such as transgenic mice or rats, the transgenic nonhuman animals having a genome containing human heavy chain transgenes and light chain transgenes. In some embodiments, monoclonal antibodies are produced from recombinant host cells expressing nucleic acid sequences encoding antibodies. Such recombinant host cells can be obtained, for example, by transfecting the nucleic acid sequences into parental cells (e.g., CHO cells). The recombinant host cells can be cultured under conditions conducive to antibody expression in the host cells, and the antibodies can be isolated from the host cells, the culture medium, or both.

[0156] In some embodiments, the multispecific binding molecule of the present invention comprises an antibody or one or more antigen-binding fragments thereof. As used herein, the term “antigen-binding fragment” refers to an antibody fragment such as, biantibody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibody (ds biantibody), single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv dimer (bivalent biantibody), multispecific antibody formed from a portion of an antibody comprising one or more CDRs, camelified single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds an antigen but does not contain a complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen as the parent antibody or the antigen bound to the parent antibody fragment. According to a specific embodiment, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the heavy chain. According to other specific embodiments, the antigen-binding fragment comprises Fab and F(ab'). In some embodiments, the antigen-binding fragment includes an IgG-like molecule having a complementary CH3 domain to force heterodimerization; a recombinant IgG-like dual-targeting molecule wherein each side of the molecule contains a portion or fragment of a Fab fragment of at least two different antibodies; an IgG fusion molecule wherein a full-length IgG antibody is fused with an additional Fab fragment or a portion of a Fab fragment; an Fc fusion molecule wherein a single-chain Fv molecule or a stable bispecific antibody is fused with a heavy chain constant domain, an Fc region, or a portion thereof; a Fab fusion molecule wherein different Fab fragments are fused together; and a heavy chain antibody (e.g., a domain antibody, nanobody) based on ScFv and a bispecific antibody, wherein different single-chain Fv molecules or different bispecific antibodies or different heavy chain antibodies (e.g., domain antibodies, nanobody) are fused to each other or to another protein or carrier molecule. In some implementations, IgG-like molecules with complementary CH3 domains include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-in-Holes (Genentech), CrossMAbs (Roche), electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain Body (SEEDbody) (EMD Serono), Biclonic (Merus), or DuoBody (Genmab A / S, see, for example, Labrijn et al., 2013, PNAS 110:5145-5150).In some implementations, the antigen-binding fragment includes a “bound single-chain Fv” or “spFv”, which refers to an scFv containing one or more disulfide bonds between VH and the linker or between VL and the linker. Typically, an spFv may contain one disulfide bond between VH and the linker, one disulfide bond between VL and the linker, or two disulfide bonds between VH and the linker and between VL and the linker. scFv molecules containing a disulfide bond between VH and VL are excluded from the term “scFv”.

[0157] As used herein, the term "single-chain antibody" refers to conventional single-chain antibodies in the art that comprise a heavy chain variable region and a light chain variable region linked by, for example, a short peptide of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to conventional single-domain antibodies in the art that comprise a heavy chain variable region and a heavy chain constant region or comprise only a heavy chain variable region.

[0158] In some embodiments, the multispecific binding molecule of the present invention includes an antibody having one or more mutations in the Fc that eliminate binding to protein A. Such mutations facilitate the purification of heterodimers and have been described, for example, in WO2010151792.

[0159] As used herein, the term "human antibody" refers to an antibody produced by a human being or an antibody having an amino acid sequence corresponding to a human-produced antibody prepared using any technique known in the art. This definition of human antibody includes full-length or complete antibodies, their antigen-binding fragments, and / or antibodies containing at least one human heavy-chain polypeptide and / or light-chain polypeptide.

[0160] As used in this article, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology with human antibodies, so that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced.

[0161] As used herein, the term "chimeric antibody" refers to an antibody whose amino acid sequence of an 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 antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions correspond to sequences in antibodies derived from another mammalian species (e.g., human) in order to avoid eliciting an immune response in that species.

[0162] As used herein, the term "multispecific antibody" refers to an antibody comprising multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the multiple immunoglobulin variable domain sequences has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the multiple immunoglobulin variable domain sequences has binding specificity for a second epitope. In one embodiment, the first and second epitopes do not overlap or substantially do not overlap. In one embodiment, the first and second epitopes are located on different antigens, such as different proteins (or different subunits of multimeric proteins). In some embodiments, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain, or even more immunoglobulin variable domains. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0163] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes, preferably no more than two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain having binding specificity to a first epitope (e.g., an epitope on an LTBR antigen) and a second immunoglobulin variable domain having binding specificity to a second epitope (e.g., an epitope on an EDB). In one embodiment, the bispecific antibody comprises a first heavy chain variable domain and a first light chain variable domain forming a binding domain having binding specificity to the first epitope, and a second heavy chain variable domain and a second light chain variable domain forming a binding domain having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises a hapten or a fragment thereof having binding specificity to the first epitope and a hapten or a fragment thereof having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises an scFv or a fragment thereof having binding specificity to the first epitope and an scFv or a fragment thereof having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises an scFv or a fragment thereof having binding specificity to a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a second epitope. In a preferred embodiment of the invention, the first epitope is located on the LTBR, ​​and the second epitope is located on fibronectin (specifically its EDB).

[0164] In some embodiments, the multispecific binding molecule according to the invention comprises an antibody, such as IgG, wherein the scFv is fused to the antibody. In some embodiments, the scFv may have binding specificity to LTBR. In some embodiments, both arms of the antibody (including the variable region) may 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. In some embodiments, the scFv is fused to the N-terminus of the heavy chain. In other embodiments, the scFv is fused to the C-terminus of the heavy chain. It will be apparent to those skilled in the art based on this disclosure that other forms are also possible, such as bispecific antibodies comprising one arm specifically binding to LTBR and another arm specifically binding to EDB, supplemented by fusing the EDB-specifically binding scFv to one chain of the antibody, etc.

[0165] As used herein, the term "LTBR" refers to a polypeptide that acts as a cell surface receptor for lymphotoxin, participating in apoptosis and cytokine release, and is a member of the tumor necrosis factor receptor superfamily. LTBR may also be referred to as "tumor necrosis factor receptor superfamily member 3 (TNFRSF3)". LTBR is expressed on the surface of many cell types, including epithelial and myeloid cells. LTBR specifically binds to the membrane form of lymphotoxin (a complex of lymphotoxin-α and lymphotoxin-β). Activation of LTBR can trigger apoptosis via TRAF3 and TRAF5 and can lead to the release of interleukin-8. Unless otherwise specified, LTBR is preferably the human LTBR. The amino acid sequence of human LTBR is provided by UniProt No. P36941.

[0166] The term "EDB" or "Extra Domain B" refers to a domain of fibronectin that can 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 sequence containing 91 amino acid residues. Generally, EDB is undetectable in normal adult tissues, but it exhibits higher expression in the extracellular matrix in fetal and tumor tissues and accumulates around the neovascular system during angiogenesis, making EDB a potential biomarker and target of angiogenesis. Unless otherwise specified, EDB is preferably human EDB. Human EDB containing the fibronectin isotype amino acid sequence is provided by UniProt No. P02751.

[0167] The term "fibronectin" refers to a polypeptide of high molecular weight glycoprotein that forms part of the extracellular matrix. Fibronectin can bind to transmembrane receptor proteins, known as integrins. It can also bind to other extracellular matrix proteins, such as collagen, fibrin, and heparan sulfate proteoglycans. Fibronectin can exist as a protein dimer, composed of two nearly identical monomers linked by a disulfide bond. Fibronectin is produced by a single gene, but alternative splicing of the fibronectin pre-mRNA molecule results in several isoforms of fibronectin, one of which is EDB fibronectin. Fibronectin plays a role in cell adhesion, growth, migration, and differentiation, and may be important for processes such as wound healing and embryonic development. The amino acid sequence of human fibronectin is provided by UniProt No. P02751, which contains an additional domain B, and NCBI accession numbers NP_001263337 (isotype B), NP_001263338 (isotype c), NP_001263339 (isotype d), NP_001263340 (isotype e), and NP_001263341 (isotype f), NP_001293058 (isotype 8), NP_001293059 (isotype 9), NP_001293060 (isotype 10), NP_001293061 (isotype 11), and NP_002017 (isotype 3).

[0168] As used in this article, "antibodies or binding molecules that specifically bind to LTBR" refer to those with a concentration of 1 × 10⁻⁶. -7 M or smaller, preferably 1×10 -8 M or smaller, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller, or 1×10 -10 An antibody or molecule containing an antigen-binding domain that has a KD of M or less with an LTBR, ​​preferably a human LTBR. The term "KD" refers to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). According to this disclosure, the KD value of an antibody can be determined using methods in the art. For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system (e.g.,...). The system can be used to determine this, or by using biolayer interferometry techniques such as the Octet RED96 system. In a preferred embodiment, the LTBR-specific binding domain in the multispecific binding molecule of the present invention comprises an antibody against LTBR, ​​preferably an agonist antibody against LTBR, ​​or a functional fragment or derivative thereof, such as scFv. As used herein, an "agonist antibody against LTBR" is an antibody that binds to LTBR and is capable of inducing downstream signaling directly or in higher-order clustering, for example, by immobilization to a solid support, by using a cross-linked antibody, etc. Such agonistic antibodies against LTBR have been described, and non-limiting examples include BHA10 (e.g., WO2004002431), CBE11 (e.g., WO0230986), REA412 (available from Miltenyi Biotec), 31G4D8 (available from BioLegend), and 71319 / MAB629 (available from Novus Biologicals), or alternatively, antibodies may be generated according to known methods for antibody generation such as mouse immunization, phage display, etc.

[0169] As used in this article, the antigen-binding domain or antigen-binding fragment that "specifically binds to EDB" refers to a domain with a binding capacity of 1 × 10⁻⁶. -7 M or smaller, preferably 1×10 -8 M or smaller, more preferably 5×10 -9 M or smaller, 1×10 -9 M or smaller, 5×10 -10 M or smaller, or 1×10 -10 M or smaller KD binds to the antigen-binding domain or antigen-binding fragment of EDB (preferably in the form of EDB fibronectin).

[0170] In a preferred embodiment, the EDB-specific binding domain in the multispecific binding molecule of the present invention comprises an antibody against EDB, or a functional fragment or derivative thereof, such as scFv. Such antibodies against EDB have been described, and non-limiting examples include L19 (e.g., WO9745544) and other antibodies that bind to ED-B or adjacent domains (e.g., Carnemolla et al., Int. J. Cancer: 68, 397-405 (1996)), or alternatively, antibodies may be generated according to known methods for antibody generation, such as mouse immunization, phage display, etc.

[0171] The smaller the KD value of an antibody, the higher its affinity for binding to the target antigen.

[0172] According to a specific aspect of the invention, a multispecific binding molecule is provided herein. The multispecific binding molecule comprises (i) a first binding domain that specifically binds to a lymphotoxin β receptor (LTBR), and (ii) a second binding domain that specifically binds to EDB, wherein the multispecific binding molecule activates the LTBR upon binding to EDB.

[0173] In some implementations, multispecific binding molecules activate the LTBR in a tumor-specific manner. As used herein, tumor-specific activation of the LTBR means that when a multispecific binding molecule simultaneously binds to both the LTBR and EDB (both present in the tumor microenvironment on the cell surface or in the extracellular matrix), the LTBR is activated to trigger signal transduction via the canonical and / or non-canonical NF-κB pathways. Activation of the NF-κB pathway can lead to the establishment of a pro-inflammatory tumor microenvironment by secreting pro-inflammatory chemokines and cytokines and expressing adhesion molecules on the cell surface. Simultaneous binding of the multispecific binding molecule results in LTBR activation in the tumor. If EDB is not present in normal tissue, i.e., normal cells, or not present in the extracellular matrix adjacent to normal tissue, the multispecific binding molecule can only bind to the LTBR in normal tissue, which will not lead to LTBR activation in normal tissue. This is a significant advantage over molecules based on natural LTBR ligands described in the prior art, such as LIGHT antibody fusions, which can activate LTBR independently of TAA and therefore have much lower tumor specificity for LTBR activation compared to the molecules of the present invention, as illustrated in the examples herein.

[0174] In some embodiments, a multispecific binding molecule comprises two binding domains, such as a bispecific antibody comprising two antigen-binding domains: one antigen-binding domain binding to LTBR and another antigen-binding domain binding to EDB. In a preferred embodiment, a multispecific binding molecule comprises more than two antigen-binding domains, such as one antigen-binding domain binding to LTBR and two antigen-binding domains binding to EDB. In some embodiments, a multispecific binding molecule comprises three binding domains. In some embodiments, all three binding domains are different and bind to three different antigens. In some preferred embodiments, the three antigen-binding domains include one binding domain binding to a first antigen and two binding domains binding to a second antigen. In this embodiment, the three antigen-binding domains are present in a 2:1 stoichiometric ratio. The three antigen-binding domains may, for example, include a first binding domain that specifically binds to LTBR on cells expressing LTBR. The three antigen-binding domains may, for example, include two second binding domains that specifically bind to EDB. In some embodiments, the two second binding domains have the same binding specificity to EDB; for example, the two second binding domains may be identical. This document demonstrates that the multispecific binding molecules of the present invention, having more than one binding domain specific to EDB, possess additional advantageous properties compared to those having only one binding domain specific to EDB. In some embodiments, LTBR is activated upon binding to EDB (which is part of fibronectin present in the extracellular matrix of tumor tissue).

[0175] Depending on the specific aspect, this document provides isolated anti-lymphotoxin β receptor (LTBR) bispecific antibodies or antigen-binding fragments thereof. In some non-limiting embodiments, the binding domain that specifically binds to LTBR comprises an agonistic anti-LTBR antibody or a fragment or derivative thereof, such as a single-chain antibody fragment (scFv) comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the VH and the VL comprise any of the following:

[0176] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0177] (ii) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0178] (iii) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, respectively; or

[0179] (iv) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:43, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96 ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:43 has at least 97% identity with the amino acid sequence of SEQ ID NO:44, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44. The amino acid sequence of NO:44 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0180] (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity 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%, or 100% identity with the amino acid sequence of SEQ ID NO:48, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:47, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:47, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:47 has at least 97% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48. The amino acid sequence of NO:48 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0181] (vi)SEQ ID NO:22; or

[0182] (vii)SEQ ID NO:23; or

[0183] (viii)SEQ ID NO:25.

[0184] In some non-limiting embodiments, the second binding domain that specifically binds to EDB comprises an antibody that binds to EDB, or a fragment or derivative of such an antibody, for example comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises any of the following:

[0185] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively; or

[0186] (ii) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:45, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:45 has at least 97% identity with the amino acid sequence of SEQ ID NO:46, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46. The amino acid sequence of NO:46 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

[0187] In some non-limiting embodiments, the multispecific binding molecule comprises:

[0188] (1) A binding domain that specifically binds to LTBR, ​​comprising a BHA10 antibody or a CBE11 antibody or a fragment or derivative thereof, such as a single-chain antibody fragment (scFv), comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the VH and the VL comprise any of the following:

[0189] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0190] (ii) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0191] (iii) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, respectively; or

[0192] (iv) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:43, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96 ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:43 has at least 97% identity with the amino acid sequence of SEQ ID NO:44, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44. The amino acid sequence of NO:44 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0193] (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity 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%, or 100% identity with the amino acid sequence of SEQ ID NO:48, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:47, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:47, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:47 has at least 97% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48. The amino acid sequence of NO:48 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0194] (vi)SEQ ID NO:22; or

[0195] (vii)SEQ ID NO:23; or

[0196] (viii)SEQ ID NO:25; and

[0197] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2 and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2 and LCDR3, wherein the antibody or fragment thereof comprises HCDR1, HCDR2 and HCDR3, which respectively comprise the amino acid sequences of SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74; and LCDR1, LCDR2 and LCDR3, which respectively comprise the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77.

[0198] In some non-limiting embodiments, the multispecific binding molecule comprises:

[0199] (1) A binding domain that specifically binds to LTBR, ​​comprising a BHA10 antibody or a CBE11 antibody or a fragment or derivative thereof, such as a single-chain antibody fragment (scFv), comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the VH and the VL comprise any of the following:

[0200] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0201] (ii) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0202] (iii) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, respectively; or

[0203] (iv) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:43, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96 ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:43 has at least 97% identity with the amino acid sequence of SEQ ID NO:44, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44. The amino acid sequence of NO:44 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0204] (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity 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%, or 100% identity with the amino acid sequence of SEQ ID NO:48, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:47, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:47, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:47 has at least 97% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48. The amino acid sequence of NO:48 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0205] (vi)SEQ ID NO:22; or

[0206] (vii)SEQ ID NO:23; or

[0207] (viii)SEQ ID NO:25; and

[0208] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises VH, which comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:45; and VL, which comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; such as wherein VH comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:45. The amino acid sequence of NO:46 has at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence; VH contains an amino acid sequence with at least 96% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 97% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:45. The amino acid sequence of NO:46 has at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:46.

[0209] In some non-limiting embodiments, the multispecific molecule comprises:

[0210] (1) A binding domain that specifically binds to the LTBR containing SEQ ID NO:22; and

[0211] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises any of the following:

[0212] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively; or

[0213] (ii) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:46.

[0214] In some non-limiting embodiments, the multispecific molecule comprises:

[0215] (1) A binding domain that specifically binds to the LTBR containing SEQ ID NO:23; and

[0216] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises any of the following:

[0217] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively; or

[0218] (ii) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:46.

[0219] In some non-limiting embodiments, the multispecific molecule comprises:

[0220] (1) A binding domain that specifically binds to LTBR containing SEQ ID NO:25; and

[0221] (2) A second binding domain that specifically binds to EDB, comprising an L19 antibody or a fragment or derivative thereof, such as comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, wherein the antibody or fragment thereof comprises any of the following:

[0222] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively; or

[0223] (ii) VH contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO:46.

[0224] In some non-limiting embodiments, the multispecific binding molecule comprises any of the following:

[0225] (a)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO:1, which forms a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO:2, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO:4, which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14121]; or

[0226] (b)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO:9, which forms a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO:10, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO:4, which forms a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14122].

[0227] In some other non-limiting embodiments, the multispecific binding molecule comprises any of the following:

[0228] (c)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:30, wherein the heavy chain portion (containing SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1480]; or

[0229] (d)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:31, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1481]; or

[0230] (e)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:32, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1482]; or

[0231] (f)(i) a scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:33, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1483]; or

[0232] (g)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:34, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14107]; or

[0233] (h)(i) a scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:35, wherein the heavy chain portion (comprising SEQ ID NO:84) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14108]; or

[0234] (j)(i) a scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:38, wherein the heavy chain portion (containing SEQ ID NO:3) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14133]; or

[0235] (k)(i) a scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:39, wherein the heavy chain portion (containing SEQ ID NO:3) of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA14174]; or

[0236] (l)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:56, wherein the heavy chain portion of the scFv heavy chain fusion (comprising SEQ ID NO:84) forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5 [the multispecific binding molecule is referred to as COVA1456].

[0237] In some embodiments, the multispecific molecule comprises (i) an scFv heavy chain fusion containing the amino acid sequence of SEQ ID NO:38, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain with a light chain containing the amino acid sequence of SEQ ID NO:5, and (ii) a heavy chain containing the amino acid sequence of SEQ ID NO:4, wherein the heavy chain forms a binding domain with a light chain containing the amino acid sequence of SEQ ID NO:5.

[0238] In some embodiments, a multispecific (e.g., bispecific) molecule induces NF-κB signaling in the presence of EDB, which is at least 2, such as at least 3, such as at least 4, times greater than NF-κB signaling induced in the absence of EDB (under the same conditions). Sometimes, the assay is an NF-κB luciferase reporter gene assay. The NF-κB luciferase reporter gene assay can be performed using the protocol of Example 2.

[0239] In some embodiments, a multispecific (e.g., bispecific) molecule induces ICAM-1 expression on the cell surface in the presence of EDB, such ICAM-1 expression being at least 2-fold, such as at least 3-fold, or for example at least 4-fold, higher than ICAM-1 expression induced under the same conditions in the absence of EDB. Sometimes, the assay is an in vitro LTBR activation assay, such as an A375 / WI38A subline 2RA co-culture cell assay. The A375 / WI38A subline 2RA co-culture cell assay can be performed using the protocol of Example 3.

[0240] Depending on the specific aspects, heavy and light chains are humanized.

[0241] In some embodiments, the bispecific antibodies of the present invention include bispecific antibodies, crossbody antibodies, scFv, Duobody, spFv, or bispecific antibodies obtained via controlled Fab arm exchange, as described in the present invention.

[0242] In some embodiments, bispecific antibodies include IgG-like molecules having complementary CH3 domains to force heterodimerization; recombinant IgG-like dual-targeting molecules, wherein each flanking element contains a portion or fragment of Fab fragments of at least two different antibodies; IgG fusion molecules, wherein a full-length IgG antibody is fused with an additional Fab fragment or a portion of a Fab fragment; Fc fusion molecules, wherein a single-chain Fv molecule or a stable bispecific antibody is fused with a heavy chain constant domain, Fc region, or a portion thereof; Fab fusion molecules, wherein different Fab fragments are fused together; and heavy chain antibodies (e.g., domain antibodies, nanobodies) based on ScFv and bispecific antibodies, wherein different single-chain Fv molecules or different bispecific antibodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

[0243] In some implementations, IgG-like molecules with complementary CH3 domains include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche), electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain Body (SEEDbody) (EMD Serono), Biclonic (Merus), or DuoBody (Genmab A / S).

[0244] In some implementations, recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig (GSK / Domantis), dual-antibody (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), or CovX (CovX / Pfizer).

[0245] In some implementations, IgG fusion molecules include dual variable domain (DVD)-Ig (Abbott), IgG-like bispecific antibodies (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec), or TvAb (Roche).

[0246] In some implementations, the Fc fusion molecule may include ScFv / Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), dual-affinity retargeting technology (Fc-DART) (MacroGenics), or bis(ScFv)2-Fab (National Research Center for Antibody Medicine--China).

[0247] In some implementations, Fab fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), dual-action or bi-Fab (Genentech), docking-and-lock (DNL) (ImmunoMedics), bivalent bispecific antibodies (Biotecnol), or Fab-Fv (UCB-Celltech). ScFv-based antibodies, bispecific antibodies, and domain-specific antibodies include, but are not limited to, bispecific T-cell adaptors (BiTE) (Micromet), tandem bispecific antibodies (Tandab) (Affimed), dual-affinity retargeting technology (DART) (MacroGenics), single-chain bispecific antibodies (Academic), TCR-like antibodies (AIT, Receptor Logics), human serum albumin ScFv fusions (Merrimack) or COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), and antibodies that target only the heavy chain domain.

[0248] The full-length bispecific antibody of the present invention can be generated, for example, by Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies, in the following manner: a substitution is introduced at the heavy chain CH3 junction in each half-molecule to facilitate the formation of heterodimers of two antibody half-molecules with different specificities in an in vitro cell-free environment or using co-expression. The Fab arm exchange reaction is the result of disulfide bond isomerization and CH3 domain dissociation-association. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with the cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed through dissociation-association. The CH3 domain of the Fab arm can be engineered to facilitate heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each of which binds a different epitope, namely an epitope on the LTBR and an epitope on the EDB of fibronectin.

[0249] As used herein, “homodimerization” refers to the interaction between two heavy chains having the same CH3 amino acid sequence. As used herein, “homodimer” refers to an antibody having two heavy chains containing the same CH3 amino acid sequence.

[0250] As used herein, "heterodimerization" refers to the interaction between two heavy chains with different CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains containing different CH3 amino acid sequences.

[0251] The "mortar and pestle structure" strategy (see, for example, PCT publication WO2006 / 028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids that form the CH3 domain boundaries in human IgG can be mutated at positions that influence CH3 domain interactions, thereby promoting heterodimer formation. Amino acids with small side chains (mortars) are introduced into the heavy chain of an antibody that specifically binds to the first antigen, and amino acids with large side chains (mortars) are introduced into the heavy chain of an antibody that specifically binds to the second antigen. After co-expression of the two antibodies, heterodimers are formed due to the preferential interaction between the "mortar" and "mortar" heavy chains. Exemplary CH3 substitution pairs forming the pestle and mortar (represented as modification positions in the first CH3 domain of the first heavy chain / modification positions in the second CH3 domain of the second heavy chain, using Kabat numbering) are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, or T366W / T366S_L368A_Y407V.

[0252] Other strategies may also be used, such as promoting heavy chain heterodimerization by treating positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface using electrostatic interactions, as described, for example, in U.S. Patent Publication US2010 / 0015133; U.S. Patent Publication US2009 / 0182127; U.S. Patent Publication US2010 / 028637; or U.S. Patent Publication US2011 / 0123532. In other strategies, heterodimerization can be promoted by the following substitutions (represented 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): L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W, for example, as described in U.S. Patent Publication US2012 / 0149876 or U.S. Patent Publication US2013 / 0195849.

[0253] In addition to the methods described above, the bispecific antibody of the present invention can also be generated in an in vitro cell-free environment by introducing an asymmetric mutation into the CH3 region of two monospecific homodimer antibodies, and forming a bispecific heterodimer antibody from the two parental monospecific homodimer antibodies under reducing conditions, thereby allowing disulfide isomerization according to the method described in International Patent Publication WO2011 / 131746. In this method, the first and second monospecific bivalent antibodies are engineered to have certain substitutions at the CH3 domain that promote the stability of the heterodimer; these antibodies are incubated together under reducing conditions sufficient to cause disulfide isomerization of cysteine ​​in the hinge region; thereby generating a bispecific antibody through Fab arm exchange. The incubation conditions can optionally be restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably those selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation for at least 90 minutes at a temperature of at least 20°C can be used in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5 to 8, for example at pH 7.0 or 7.4.

[0254] In some embodiments described herein, the immune effector properties of the multispecific binding molecules of the present invention, such as bispecific antibodies, can be modified, for example, via Fc modification using techniques known to those skilled in the art, preferably silenced. For example, Fc effector functions such as Clq 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 residues in the Fc that contribute to these activities, see, for example, the N297 mutation (Nose et al., PNAS (1983)); the LALA mutation (Xu et al., Cell Immunol. 200(1):16-26)(2000)); and the DANA mutation (Wilson et al., Cancer Cell). 19(1):101-113(2011)); or, for example, mutations in aspartic acid (D) at position 265, asparagine (N) at position 297 and proline (P) at position 329, wherein the numbers are indicated by EU indexes as in Kabat, for example, each mutated to alanine (A) to obtain the so-called DANAPA mutant, as detailed in WO2019 / 068632.

[0255] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (such as natural killer (NK) cells, neutrophils, and macrophages) recognize binding antibodies on target cells and subsequently cause the target cells to lyse.

[0256] In some embodiments, the multispecific binding molecule of the present invention comprises a chimeric bispecific antibody.

[0257] In some embodiments, the multispecific binding molecule of the present invention comprises a human or humanized bispecific antibody.

[0258] In another general aspect, the present invention relates to one or more nucleic acids encoding the multispecific binding molecules of the present invention (e.g., bispecific antibodies or antigen-binding fragments thereof). As a non-limiting example, the heavy chain of a bispecific antibody may be encoded by one nucleic acid, and the light chain may be encoded by a second nucleic acid. In another example, the heavy and light chains of a bispecific antibody may be encoded on a single nucleic acid molecule. Those skilled in the art will understand that, due to the degeneracy of the genetic code, the coding sequence of a protein can be altered (e.g., substitution, deletion, insertion, etc.) without changing the amino acid sequence of the protein. Therefore, those skilled in the art will understand that the nucleic acid sequence encoding the monoclonal antibody and / or bispecific antibody of the present invention can be changed without changing the amino acid sequence of the protein. Furthermore, the one or more nucleic acids of the present invention may also be isolated nucleic acids. Therefore, the present invention relates to any nucleic acid molecule or combination of nucleic acid molecules encoding the molecules of the present invention.

[0259] In another general aspect, the present invention relates to one or more vectors comprising one or more nucleic acids of the present invention. According to this disclosure, any vector known to those skilled in the art, such as plasmids, sticky-terminal plasmids, phage vectors, or viral vectors, may be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements that establish the conventional function of the expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and / or origin of replication. The promoter may be a constitutive, inducible, or repressive promoter. A variety 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 in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present invention. Such techniques are well known to those skilled in the art according to this disclosure.

[0260] In another general aspect, the present invention relates to a host cell comprising one or more vectors containing one or more nucleic acids encoding a multispecific binding molecule of the present invention (such as a bispecific antibody or an antigen-binding fragment thereof). According to this disclosure, any host cell known to those skilled in the art can be used for recombinant expression of the multispecific binding molecule of the present invention (such as a bispecific antibody or an antigen-binding fragment thereof). In some embodiments, the host cell is *E. coli* TG1 or BL21 cells (for expressing, for example, scFv or Fab antibodies), CHO-DG44 or CHO-K1 cells, or HEK293 cells (for expressing, for example, full-length IgG antibodies). According to specific embodiments, the recombinant expression vector is transformed into the host cell by conventional methods such as chemical transfection, heat shock, or electroporation, wherein the recombinant expression vector can be stably integrated into the host cell genome, thereby enabling efficient expression of the recombinant nucleic acid.

[0261] In another general aspect, the present invention relates to a method for preparing multispecific binding molecules (such as bispecific antibodies or antigen-binding fragments thereof) disclosed herein. The method includes culturing cells containing nucleic acids encoding multispecific binding molecules (such as bispecific antibodies or antigen-binding fragments thereof) under conditions for preparing multispecific binding molecules (such as bispecific antibodies or antigen-binding fragments thereof) disclosed herein, and recovering the multispecific binding molecules (such as bispecific antibodies or antigen-binding fragments thereof) from the cells or cell culture (e.g., from a supernatant). The expressed multispecific binding molecules (such as bispecific antibodies or antigen-binding fragments thereof) can be harvested from cells and purified according to conventional techniques known in the art and as described herein.

[0262] Pharmaceutical Composition

[0263] In another general aspect, the present invention relates to a pharmaceutical composition comprising a multispecific binding molecule of the present 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 the multispecific binding molecule of the present invention together with a pharmaceutically acceptable carrier. The multispecific binding molecules of the present invention (e.g., bispecific antibodies) and compositions comprising them can also be used to manufacture medicaments for therapeutic applications mentioned herein.

[0264] As used herein, the term "carrier" means any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of a carrier, excipient, or diluent will depend on the route of administration for the specific application. As used herein, the term "pharmaceuticalally acceptable carrier" means a nontoxic material that does not interfere with the efficacy of the composition according to the invention or the biological activity of the composition according to the invention. In view of this disclosure, any pharmaceutically acceptable carrier suitable for antibody pharmaceutical compositions, according to specific embodiments, may be used herein.

[0265] Formulations of the active pharmaceutical ingredient and a pharmaceutically acceptable carrier are known in the art, for example, as described in Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tension modifiers, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used to formulate the pharmaceutical compositions of the present invention.

[0266] In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation containing water. Liquid formulations may comprise solutions, suspensions, emulsions, microemulsions, gels, etc. Aqueous formulations typically contain at least 50% w / w water, or at least 60% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, or at least 95% w / w water.

[0267] In one embodiment, the pharmaceutical composition may be formulated as an injectable agent that can be administered, for example, via an injection device (e.g., a syringe or infusion pump). The injection may be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

[0268] In another embodiment, the pharmaceutical composition is a solid dosage form, such as a freeze-dried or spray-dried composition, which can be used as is or by adding solvents and / or diluents by a physician or patient prior to use. Solid dosage forms may include tablets, such as compressed and / or coated tablets, and capsules (e.g., hard gelatin capsules or soft gelatin capsules). The pharmaceutical composition may also be in the form of, for example, small capsules for reconstitution, sugar-coated pills, powders, granules, tablets, or powders.

[0269] Dosage forms can be immediate-release, in which case they may contain water-soluble or water-dispersible carriers, or they may be delayed-release, sustained-release, or modified-release, in which case they may contain water-insoluble polymers in the gastrointestinal tract or subcutaneously to adjust the dissolution rate of the dosage form.

[0270] In other embodiments, the pharmaceutical composition may be delivered intranasally, intrabuccally, or sublingually.

[0271] The pH of the aqueous formulation can be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.

[0272] In some embodiments, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, dihydroxyethylglycine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, diglycine peptide, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tris(hydroxymethyl)methylglycine or tris(hydroxymethyl)-aminomethane, and mixtures thereof. Buffers may be present alone or in aggregates at concentrations from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific buffers constitute alternative embodiments of the invention.

[0273] In some embodiments, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include: benzyl chloride, benzoic acid, benzyl alcohol, bromonitropropylene glycol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorophenylglycerol ether, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidureus, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thimerosal, and mixtures thereof. The preservative may be present alone or in aggregates 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. Pharmaceutical compositions comprising each of these specific preservatives constitute alternative embodiments of the invention.

[0274] In some embodiments, the pharmaceutical composition comprises 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), uronic acids (such as glycerol, 1,2-propanediol / propylene glycol, 1,3-propanediol, or 1,3-butanediol), polyethylene glycol (e.g., PEG400), and mixtures thereof. Another example of an isotonic agent includes sugars. Non-limiting examples of sugars may include monosaccharides, disaccharides, or polysaccharides, or water-soluble dextran, including, for example, fructose, glucose, mannose, sorbitol, xylose, maltose, lactose, sucrose, trehalose, dextran, amylopectin, dextrin, cyclodextrin, α- and β-HPCD, soluble starch, hydroxyethyl starch, or sodium carboxymethyl cellulose. Another example of an isotonic agent is a sugar alcohol, wherein the term "sugar alcohol" is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, hexahexol, xylitol, or arabinitol. Isotonic agents may be present alone or in aggregates at concentrations of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific isotonic agents constitute alternative embodiments of the invention.

[0275] In some embodiments, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agent may be present alone or in aggregates at concentrations of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute alternative embodiments of the invention.

[0276] In some embodiments, the pharmaceutical composition comprises 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.

[0277] In some embodiments, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy / hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances (such as monothioglycerol), or thioglycolic acid. The stabilizer may be present alone or in aggregates 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. Pharmaceutical compositions comprising each of these specific stabilizers constitute alternative embodiments of the invention.

[0278] In some embodiments, the pharmaceutical composition comprises one or more surfactants. The term "surfactant" refers to any molecule or ion consisting of a water-soluble portion (hydrophilic) and a lipophilic portion (lipophilic). For example, surfactants may be selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. Surfactants may be present alone or in aggregates at concentrations from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific surfactants constitute alternative embodiments of the invention.

[0279] In some embodiments, the pharmaceutical composition comprises one or more protease inhibitors, such as, for example, EDTA and / or benzoamide hydrochloride (HCl). The protease inhibitors may be present alone or in aggregates at concentrations of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments of the invention.

[0280] In another general aspect, the present invention relates to a method for preparing a pharmaceutical composition comprising a multispecific binding molecule of the present invention (such as a bispecific antibody or an antigen-binding fragment thereof), the method comprising combining the multispecific binding molecule (such as a bispecific antibody or an antigen-binding fragment thereof) with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0281] How to use

[0282] In another general aspect, the present invention relates to a method for targeting LTBRs on cells present in tumors (e.g., tumor cells, fibroblasts, monocytes, etc.), the method comprising exposing the cells present in the tumor to the multispecific binding molecules or pharmaceutical compositions of the present invention.

[0283] The functional activity of multispecific binding molecules (e.g., bispecific antibodies and their antigen-binding fragments) that bind to LTBR and / or EDB can be characterized by methods known in the art and as described herein. Methods for characterizing multispecific binding molecules that bind to LTBR and / or EDB include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, and / or OctetRed assays; and binding assays to detect the binding of multispecific binding molecules to LTBR on cancer cells and other cell types via FACS. Depending on the specific embodiment, methods for characterizing multispecific binding molecules that bind to LTBR and / or EDB include those described below.

[0284] In another general aspect, the present invention relates to a method for establishing a pro-inflammatory tumor microenvironment. The method includes contacting cells expressing LTBR in the tumor microenvironment with the multispecific binding molecules of the present invention, wherein contacting LTBR-expressing cells with the multispecific binding molecules results in the secretion of pro-inflammatory chemokines and cytokines, as well as the expression of adhesion molecules on the cell surface.

[0285] In another general aspect, the present invention relates to a method of treating a subject with cancer, the method comprising administering to the subject a multispecific binding molecule of the present invention (e.g., a bispecific antibody or an antigen-binding fragment thereof) that specifically binds to LTBR and fibronectin-dependent EDB, or a pharmaceutical composition disclosed herein. The cancer is preferably a cancer expressing EDB. The cancer may be, for example, a cancer expressing LTBR. The cancer may be selected, for example, from the group consisting of prostate cancer, lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, cystourethral 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 lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0286] According to embodiments of the present invention, the pharmaceutical composition comprises an effective amount of an anti-LTBR multispecific binding molecule (e.g., an anti-LTBR / anti-EDB bispecific antibody or 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.

[0287] According to the specific implementation plan, an effective dose refers to a therapeutic dose sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the treated disease, disorder, or condition, or related symptoms; (ii) reducing the duration of the treated disease, disorder, or condition, or related symptoms; (iii) preventing the development of the treated disease, disorder, or condition, or related symptoms; (iv) causing the remission of the treated disease, disorder, or condition, or related symptoms; (v) preventing the development or onset of the treated disease, disorder, or condition, or related symptoms; (vi) (vii) To prevent the recurrence of the treated disease, disorder, or condition or its associated symptoms; (viii) To reduce hospitalization of subjects suffering from the treated disease, disorder, or condition or its associated symptoms; (ix) To improve the survival of subjects suffering from the treated disease, disorder, or condition or its associated symptoms; (xi) To suppress or reduce the treated disease, disorder, or condition or its associated symptoms in subjects; and / or (xii) To enhance or improve the preventive or therapeutic effects of another therapy.

[0288] In some embodiments, the effective amount of the multispecific binding molecule of the present invention can be administered at a dose ranging from about 0.1 mg / kg to about 25 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 15 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 5 mg / kg.

[0289] Effective doses or amounts can vary depending on various factors, such as the disease, disorder, or condition being treated, the route of administration, the target site, the subject's physiological state (including, for example, age, weight, and health), whether the subject is a human or an animal, other medications administered, and whether the treatment is preventative or therapeutic. Therapeutic doses may optionally be titrated to optimize safety and efficacy.

[0290] According to specific embodiments, the compositions described herein are formulated to be administered to a subject via the intended route. For example, the compositions described herein may be formulated to be administered intravenously, subcutaneously, or intramuscularly. In some embodiments, the compositions disclosed herein may be administered to a subject via multiple routes, such as local, oral, or parenteral. Parenteral delivery methods include intra-arterial (directly to tissue), intramedullary, intrathecal, intracardiac, intraperitoneal, or intranasal administration.

[0291] As used herein, the terms “treat,” “treating,” and “treatment” are intended to refer to an improvement or reversal of at least one measurable physical parameter associated with cancer, which is not necessarily identifiable in the subject but is identifiable in the subject. The terms “treat” and “treatment” may also refer to remission, prevention of development, or at least delay in the development of a disease, disorder, or condition. In a particular embodiment, “treat” and “treatment” refer to the reduction, prevention of development or onset, or shortening of the duration of one or more symptoms associated with a disease, disorder, or condition (such as a tumor or more preferably cancer). In a particular embodiment, “treat” and “treatment” refer to the prevention of recurrence of a disease, disorder, or condition. In a particular embodiment, “treat” and “treatment” refer to an improvement in the survival of a subject suffering from a disease, disorder, or condition. In a particular embodiment, “treat” and “treatment” refer to the elimination of a disease, disorder, or condition in a subject.

[0292] According to a specific implementation plan, a composition for treating cancer is provided. For cancer treatment, the composition may be used in combination with another treatment, including but not limited to chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-CTLA-4 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, PD-1 / PD-L1 therapy, indoleamine-pyrrole 2,3-dioxygenase (IDO), anti-OX40 antibody, anti-GITR antibody, anti-CD40 antibody, anti-CD38 antibody, cytokines, oncolytic viruses, TLR agonists, STING agonists, other immuno-oncology drugs, anti-angiogenic agents, radiotherapy, antibody-drug conjugates (ADCs), targeted therapy, or other anticancer drugs.

[0293] As used herein, in the context of administering two or more therapies to a subject, the term “combination” means the use of more than one therapy. The use of the term “combination” does not restrict the order in which the therapies are administered to the subject. For example, a first therapy (e.g., the composition described herein) may be administered before, concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to the administration of a second therapy to the subject.

[0294] Implementation Plan

[0295] The present invention provides the following non-limiting embodiments.

[0296] Implementation scheme 1 is a multispecific binding molecule, said multispecific binding molecule comprising:

[0297] (i) a first binding domain that specifically binds to the lymphotoxin β receptor (LTBR), and

[0298] (ii) A second binding domain that specifically binds to the additional domain B (EDB) of fibronectin.

[0299] The multispecific binding molecule activates the LTBR when it binds to the EDB.

[0300] Implementation scheme 2 is a multispecific binding molecule according to implementation scheme 1, wherein the multispecific binding molecule activates LTBR in a tumor-specific manner.

[0301] Implementation scheme 3 is a multispecific binding molecule according to implementation scheme 1 or 2, wherein the multispecific binding molecule is a bispecific antibody.

[0302] Implementation scheme 4 is a multispecific binding molecule according to any one of implementation schemes 1 to 3, wherein the multispecific binding molecule comprises two antigen-binding domains.

[0303] Implementation scheme 5 is a multispecific binding molecule according to any one of implementation schemes 1 to 3, wherein the multispecific binding molecule comprises three antigen-binding domains.

[0304] Implementation scheme 6 is a multispecific binding molecule according to implementation scheme 5, wherein the three antigen-binding domains include a binding domain that specifically binds to LTBR.

[0305] Implementation scheme 7 is a multispecific binding molecule according to implementation scheme 5 or 6, wherein the three antigen-binding domains include two binding domains that specifically bind EDB.

[0306] Implementation scheme 8 is a multispecific binding molecule according to any one of implementation schemes 5 to 7, wherein the binding domain that specifically binds to LTBR includes a single-chain variable domain of the antibody.

[0307] Implementation scheme 9 is a multispecific binding molecule according to any one of embodiments 1 to 8, wherein the first binding domain specifically binding 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, wherein the VH and the VL comprise any one or more of the following [(i) to (viii)]:

[0308] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0309] (ii) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, respectively; or

[0310] (iii) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, respectively; or

[0311] (iv) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:43, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96 ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH comprising an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:43 has at least 97% identity with the amino acid sequence of SEQ ID NO:44, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:43, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:44. The amino acid sequence of NO:44 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0312] (v) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity 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%, or 100% identity with the amino acid sequence of SEQ ID NO:48, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:47, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:47, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:47 has at least 97% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:47, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48. The amino acid sequence of NO:48 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity; or

[0313] (vi)SEQ ID NO:22; or

[0314] (vii)SEQ ID NO:23; or

[0315] (viii)SEQ ID NO:25.

[0316] Implementation scheme 10 is a multispecific binding molecule according to any one of embodiments 1 to 9, wherein the second binding domain specifically binding to EDB 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, wherein the VH and the VL comprise any one or more of the following [(i) to (ii)]:

[0317] (i) HCDR1, HCDR2, and HCDR3 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 comprising the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively; or

[0318] (ii) VH comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46, such as VH comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:45, and VL comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO:45, and VL comprises ...5%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH comprises an amino acid sequence having at least 96%, 96%, 97%, 98%, 9 The amino acid sequence of NO:45 has at least 97% identity with the amino acid sequence of SEQ ID NO:46, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 98% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with at least 99% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46; VH contains an amino acid sequence with 100% identity with the amino acid sequence of SEQ ID NO:45, and VL contains an amino acid sequence with at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46. The amino acid sequence of NO:46 has at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

[0319] Implementation scheme 11 is a multispecific binding molecule according to any one of embodiments 1 to 10, said multispecific binding molecule comprising any one or more of [(a) to (l)]:

[0320] (a)(i) a first heavy chain comprising the amino acid sequence of SEQ ID NO:1, wherein the first heavy chain forms a binding domain 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, wherein the second heavy chain forms a binding domain with a second light chain comprising the amino acid sequence of SEQ ID NO:5; or

[0321] (b)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO:9, the first heavy chain forming a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO:10, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO:4, the second heavy chain forming a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO:5.

[0322] Embodiment 12 is a multispecific binding molecule according to any one of embodiments 1 to 10, said multispecific binding molecule comprising any one of the following:

[0323] (c)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:30, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or

[0324] (d)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:31, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or

[0325] (e)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:32, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or

[0326] (f)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:33, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or

[0327] (g)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:34, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or

[0328] (h)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:35, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or

[0329] (j)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:38, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or

[0330] (k)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:39, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or

[0331] (l)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:56, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5.

[0332] Embodiment 13 is a multispecific binding molecule according to any one of Embodiments 1 to 10, the multispecific binding molecule comprising (i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:38, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5.

[0333] Implementation scheme 14 is one or more nucleic acid molecules that encode a multispecific binding molecule according to any one of implementation schemes 1 to 13.

[0334] Implementation scheme 15 is one or more vectors, said one or more vectors comprising one or more nucleic acid molecules according to implementation scheme 14.

[0335] Implementation scheme 16 is an isolated host cell that comprises one or more vectors as described in implementation scheme 15.

[0336] Embodiment 17 is a pharmaceutical composition comprising a multispecific binding molecule according to any one of Embodiments 1 to 13 and a pharmaceutically acceptable carrier.

[0337] Implementation Scheme 18 is a method for treating cancer in a subject in need, the method comprising administering to the subject a multispecific binding molecule according to any one of Implementation Schemes 1 to 14, one or more nucleic acid molecules according to Implementation Scheme 15, one or more carriers according to Implementation Scheme 16, or a pharmaceutical composition according to Implementation Scheme 17.

[0338] Embodiment 19 is the use of the multispecific binding molecule according to any one of Embodiments 1 to 14, one or more nucleic acid molecules according to Embodiment 15, one or more carriers according to Embodiment 16, or the pharmaceutical composition according to Embodiment 17 for activating LTBR in tumor tissue.

[0339] Embodiment 20 is a method for preparing a multispecific binding molecule according to any one of Embodiments 1 to 13, the method comprising expressing one or more nucleic acid molecules according to Embodiment 14 or one or more vectors according to Embodiment 15 in a host cell, and harvesting the multispecific binding molecule.

[0340] Implementation scheme 21 is a multispecific molecule according to any one of embodiments 1-10, said multispecific molecule inducing NF-κB signaling in the presence of EDB, said NF-κB signaling being at least 2 times, such as at least 3 times, for example at least 4 times, the NF-κB signaling induced in the absence of EDB.

[0341] Embodiment 22 is a multispecific molecule according to any one of embodiments 1-10 or 21, wherein the multispecific molecule induces ICAM-1 expression on the surface of cells in the presence of EDB, wherein the ICAM-1 expression is at least 2 times, such as at least 3 times, for example at least 4 times, the ICAM-1 expression induced in the absence of EDB.

[0342] Example

[0343] Example 1: Generation of EDB / LTBR bispecific antibody and control molecule

[0344] Bispecific antibodies and control molecules derived from the target-binding sequences shown in Table 1 were transiently expressed in CHO suspension cultures in serum-free / animal-component-free medium, and using... Purification was performed on a Superdex 200 10 / 300GL column (GE Healthcare) using Pure Instruments (GE Healthcare) by protein A affinity chromatography followed by preparative size exclusion chromatography (SEC). The heavy chain contains a mortar structure (KiH) mutation to promote heterodimerization (Ridgway et al., Protein Eng. 9(7):617-21 (1996); Atwell et al., J. Mol. Biol. 270(1):26-35 (1997); Merchant et al., Nat. Biotechnol. 16(7):677-81 (1998)). The antibody contains IgG1σFc, which contains a set of seven Fc mutations (L234A, L235A, G237A, P238S, H268A, A330S, and P331S) to reduce Fc receptor interactions when compared with wild-type IgG1 (Tam et al., Antibodies (2017)).

[0345] Symmetrical monospecific and bispecific antibodies with IgG1σ mutation but without KiH mutation are generated.

[0346] Table 1: Target binding sequences for the constructs used in Example 1 .

[0347]

[0348] *: The EDBmAb1 (WO9745544) used here is an anti-ED-B antibody that has been tested in clinical practice. Other antibodies that bind to ED-B or adjacent domains have been previously described (Carnemolla et al., Int. J. Cancer: 68, 397-405 (1996)).

[0349] Protein concentration and purification yield were determined by absorbance measurements at 280 nm (OD280). A Bio SEC-5 column (Agilent, 5 μm particle size) was used. Analytical SEC was performed on a Thermo Vanquish HPLC system. 10 μl of purified protein was loaded onto the column and eluted by OD280.

[0350] Table 2 provides an overview of the structural properties of the bispecific antibody and control molecules described in this embodiment. Molecules in bold are those according to the invention, while other molecules are controls in different aspects.

[0351] Table 3 shows the structural characteristics of another comparative bispecific antibody that targets LTBR and mesothelin (a tumor-associated antigen not present in the extracellular matrix), as described in Comparative Example 4.

[0352]

[0353]

[0354]

[0355]

[0356] The following describes how the different builds are generated.

[0357] Asymmetric antibodies with a 1:1 stoichiometry (all IgG1σ; all with the club-and-mortar structure (KiH) mutation). :

[0358] i.COVA14121 is generated through the co-expression of the following: 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:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1L ).

[0359] ii. COVA14120 is generated through the co-expression of the following: 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 ).

[0360] iii. COVA14122 is generated through the co-expression of the following: 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: 4) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1. Figure 1M ).

[0361] iv. COVA14123 is generated through the co-expression of the following: 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 ).

[0362] v.COVA14124 is generated through the co-expression of the following: the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1 and the heavy chain (HC; SEQ ID NO:6) and light chain (LC; SEQ ID NO:8) of the anti-RSV antibody B21M. Figure 10 ).

[0363] vi. COVA1454 is generated through the co-expression of 3xhmLIGHT-Fc (SEQ ID NO:15) with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1F 3xhmLIGHT-Fc is a single-chain trimer LIGHT (Tang et al., CancerCell 29:285-96 (2016)) engineered to achieve better stability and cross-reactivity with humans and mice, which is fused to the N-terminus of IgG1σFc.

[0364] vii. COVA1418 is generated through the co-expression of 3xhmLIGHT-Fc (SEQ ID NO:15) with the heavy chain (HC; SEQ ID NO:7) and light chain (LC; SEQ ID NO:8) of anti-RSV antibody B21M. Figure 1E 3xhmLIGHT-Fc is a single-chain trimer LIGHT (Tang et al., CancerCell 29:285-96 (2016)) engineered to achieve better stability and cross-reactivity with humans and mice, which is fused to the N-terminus of IgG1σFc (SEQ ID NO:58).

[0365] Symmetrical antibodies (all IgG1σ, no KiH mutation):

[0366] viii.COVA14114 is generated by the following expression: a heavy chain (SEQ ID NO:18) of anti-RSV B21M antibody carrying the C-terminal LTα1β2 fusion variant and a light chain (LC; SEQ ID NO:8) of anti-RSV B21M antibody. Figure 1G ).

[0367] ix.COVA14113 is generated through the following expression: the heavy chain of EDBmAb1 carrying the C-terminal LTα1β2 fusion variant (SEQ ID NO:20) and the light chain of the anti-EDB antibody EDBmAb1 (LC; SEQ ID NO:5). Figure 1H ).

[0368] x.COVA1413 is generated through the co-expression of the heavy chain (HC; SEQ ID NO: 11) and light chain (LC; SEQ ID NO: 2) of the agonist LTBR antibody LTBRmAb1. Figure 1B ).

[0369] xi.COVA1402 is generated through the co-expression of the heavy chain (HC; SEQ ID NO:13) and light chain (LC; SEQ ID NO:10) of the agonist LTBR antibody LTBRmAb2. Figure 1A ).

[0370] xii.COVA1440 is generated through the co-expression of the heavy chain (HC; SEQ ID NO:14) and light chain (LC; SEQ ID NO:8) of the anti-RSV antibody B21M. Figure 1C ).

[0371] xiii. COVA1452 is generated through the co-expression of the heavy chain (HC; SEQ ID NO: 12) and light chain (LC; SEQ ID NO: 5) of the anti-EDB antibody EDBmAb1. Figure 1D).

[0372] Asymmetric antibodies with a 2:1 stoichiometry (all IgG1σ, all with KiH mutation).

[0373] xiv.COVA14116 is generated through the co-expression of the following: the heavy chain (SEQ ID NO:21, containing SEQ ID NO:84) of EDBmAb1 carrying the C-terminal LTα1β2 fusion variant, and the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1I ).

[0374] xv.COVA14117 is generated through co-expression of the following: a heavy chain (SEQ ID NO:19, containing SEQ ID NO:85) of anti-RSV B21M antibody carrying the C-terminal LTα1β2 fusion variant, along with a heavy chain (HC; SEQ ID NO:7) and a light chain (LC; SEQ ID NO:8) of anti-RSV B21M antibody. Figure 1J ).

[0375] xvi.COVA1484 is generated through the co-expression of the following: an anti-RSV B21M antibody heavy chain (SEQ ID NO:26, containing SEQ ID NO:85) carrying an N-terminal stapled scFv BHA10 (VH-VL orientation SEQ ID NO:22) fusion, and an anti-RSV B21M antibody heavy chain (HC; SEQ ID NO:7) and light chain (LC; SEQ ID NO:8). Figure 1P ).

[0376] xvii.COVA1485 is generated through the co-expression of the following: an anti-RSV B21M antibody heavy chain (SEQ ID NO:27, containing SEQ ID NO:85) carrying an N-terminal stapled scFv BHA10 (VL-VH orientation SEQ ID NO:23) fusion, and an anti-RSV B21M antibody heavy chain (HC; SEQ ID NO:7) and light chain (LC; SEQ ID NO:8). Figure 1Q ).

[0377] xviii.COVA1486 is generated by co-expression of the following: an anti-RSV B21M antibody heavy chain (SEQ ID NO:28, containing SEQ ID NO:85) carrying a C-terminal stapled scFv BHA10 (VH-VL orientation SEQ ID NO:22) fusion, and an anti-RSV B21M antibody heavy chain (HC; SEQ ID NO:7) and light chain (LC; SEQ ID NO:8). Figure 1R ).

[0378] xix.COVA1487 is generated through the co-expression of the following: a heavy chain (SEQ ID NO:29, containing SEQ ID NO:85) of an anti-RSV B21M antibody carrying a C-terminal stapled scFv BHA10 (VL-VH orientation SEQ ID NO:23) fusion, and a heavy chain (HC; SEQ ID NO:7) and a light chain (LC; SEQ ID NO:8) of an anti-RSV B21M antibody. Figure 1S ).

[0379] xx.COVA1480 is generated through the co-expression of the following: the heavy chain (SEQ ID NO:30, containing SEQ ID NO:84) of the anti-EDB antibody EDBmAb1 carrying an N-terminal stapled scFv BHA10 (VH-VL orientation SEQ ID NO:22) fusion, along with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1T ).

[0380] xxi.COVA1481 was generated through the co-expression of the following: the heavy chain (SEQ ID NO:31, containing SEQ ID NO:84) of the anti-EDB antibody EDBmAb1 carrying an N-terminal stapled scFv BHA10 (VL-VH orientation SEQ ID NO:23) fusion, along with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1U ).

[0381] xxii.COVA1482 is generated through the co-expression of the following: the heavy chain (SEQ ID NO:32, containing SEQ ID NO:84) of the anti-EDB antibody EDBmAb1 carrying a C-terminal stapled scFv BHA10 (VH-VL orientation SEQ ID NO:22) fusion, along with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1V ).

[0382] xxiii.COVA1483 is generated through the co-expression of the following: the heavy chain (SEQ ID NO:33, containing SEQ ID NO:84) of the anti-EDB antibody EDBmAb1 carrying a C-terminal stapled scFv BHA10 (VL-VH orientation SEQ ID NO:23) fusion, along with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1W ).

[0383] xxiv.COVA14107 was generated through the co-expression of the following: the heavy chain (SEQ ID NO:34, containing SEQ ID NO:84) of the anti-EDB antibody EDBmAb1 carrying a C-terminal stapled scFv BHA10 (VH-VL orientation, VL3 Y36F_S49Y_F87Y SEQ ID NO:53) fusion with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1X ).

[0384] xxv.COVA14108 is generated through the co-expression of the following: the heavy chain (SEQ ID NO:35, containing SEQ ID NO:84) of the anti-EDB antibody EDBmAb1 carrying a C-terminal stapled scFv BHA10 (VH-VL orientation, VH_CDR1_Y33A SEQ ID NO:54) fusion with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1Y ).

[0385] xxvi.COVA14133 is generated through the co-expression of the following: the heavy chain (SEQ ID NO:38, containing SEQ ID NO:3) of the anti-EDB antibody EDBmAb1 carrying a C-terminal stapled scFv BHA10 (VH-VL orientation SEQ ID NO:22) fusion, along with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1Z ).

[0386] xxvii.COVA14136 was generated through the co-expression of the following: an anti-RSV B21M antibody heavy chain (SEQ ID NO:41, containing SEQ ID NO:6) carrying a C-terminal stapled scFv BHA10 (VH-VL orientation SEQ ID NO:22) fusion, and an anti-EDB antibody EDBmAb1 heavy chain (HC; SEQ ID NO:7) and light chain (LC; SEQ ID NO:8). Figure 1A1 ).

[0387] xxviii.COVA14174 is generated through the co-expression of the following: the heavy chain (SEQ ID NO:39, containing SEQ ID NO:3) of the anti-EDB antibody EDBmAb1 carrying a C-terminal disulfide bond-stabilized scFv BHA10 (VH-VL orientation SEQ ID NO:25) fusion, along with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1A2 ).

[0388] xxix.COVA14175 is generated through the co-expression of the following: a heavy chain (SEQ ID NO:40, containing SEQ ID NO:6) of an anti-RSV B21M antibody carrying a C-terminal disulfide bond-stabilized scFv BHA10 (VH-VL orientation SEQ ID NO:25) fusion variant, along with the heavy chain (HC; SEQ ID NO:7) and light chain (LC; SEQ ID NO:8) of the anti-RSV B21M antibody. Figure 1A3 ).

[0389] xxx.COVA1456 is generated through the co-expression of the following: the heavy chain (SEQ ID NO:56, containing SEQ ID NO:84) of the anti-EDB antibody EDBmAb1 carrying a C-terminal disulfide bond-stabilized scFv BHA10 (VH-VL orientation SEQ ID NO:25) fusion variant, along with the heavy chain (HC; SEQ ID NO:4) and light chain (LC; SEQ ID NO:5) of the anti-EDB antibody EDBmAb1. Figure 1A4 ).

[0390] xxxi.COVA1462 is generated through the co-expression of the following: a heavy chain (SEQ ID NO:57, containing SEQ ID NO:85) of an anti-RSV B21M antibody carrying a C-terminal disulfide bond-stabilized scFv BHA10 (VH-VL orientation SEQ ID NO:25) fusion variant, along with the heavy chain (HC; SEQ ID NO:7) and light chain (LC; SEQ ID NO:8) of the anti-RSV B21M antibody. Figure 1A5 ).

[0391] Mesothelin / LTBR bispecific: an asymmetric antibody with a 2:1 stoichiometry (IgG1σ, with KiH mutation).

[0392] xxxii.COVA14146 is generated through the co-expression of the following: the heavy chain (SEQ ID NO:80, containing SEQ ID NO:86) of the anti-mesothelin antibody MSLNmAb1 carrying a C-terminal stapled scFv BHA10 (VH-VL orientation SEQ ID NO:22) fusion variant, and the heavy chain (HC; SEQ ID NO:81) and light chain (LC; SEQ ID NO:82) of the anti-mesothelin antibody MSLNmAb1. Figure 1A6 (and Table 3).

[0393] result

[0394] All the constructs described above can be expressed and purified; however, surprisingly, constructs containing LIGHT and LTα1β2 (COVA1418, COVA1454, COVA14113, COVA14114, COVA14116, and COVA14117; Table 2) showed purification yields up to 10-fold lower than those containing EDB / LTBR bispecific antibodies (e.g., COVA1482 and COVA14133; see Table 4) derived from bound scFvs of agonistic anti-LTBR antibodies. Furthermore, constructs containing LIGHT (e.g., COVA1454) showed a trend toward lower monomer content, as can be seen in the size exclusion chromatograms shown in Figures 2 and Table 4. In summary, these facts (up to 10-fold higher purification yields and higher monomer content) indicate that the bispecific constructs of the present invention may have better biophysical properties than constructs containing either LIGHT or LTα1β2-Fc fusions.

[0395] Table 4: Yield and purity of selected EBD / LTBR bispecific antibodies

[0396]

[0397] Example 2: EDB-dependent in vitro LTBR activation – NF-κB luciferase reporter gene assay

[0398] To demonstrate that the EDB / LTBR bispecific antibody can activate LTBR in an EDB-dependent manner, the activity of the compound was tested in an A549 cell NF-κB luciferase reporter gene assay with or without EDB-containing fibronectin (EDB + fibronectin). NF-κB signaling plays a crucial role in regulating cell development and immune homeostasis. Activation of NF-κB via tumor necrosis factor receptor (TNFR) or TNFR superfamily members (e.g., LTBR) occurs upon binding to their respective ligands. The A549 lung epithelial cell line naturally expresses LTBR, ​​and the NF-κB luciferase reporter gene construct is stably integrated into the genome of the A549 lung epithelial cell line. Following activation by stimulants, endogenous NF-κB transcription factors bind to DNA response elements to induce transcription of the luciferase gene.

[0399] To demonstrate the EDB-dependent activation of LTBR, ​​highly binding 96-well μClear flat plates (Greiner; Monroe, NC) were coated overnight with 150 ng / well of recombinant human EDB+ fibronectin domain 7-B-8-9 (EDB+; SEQ ID NO:51) or 150 ng / well of recombinant human EDB- fibronectin domain 7-8-9 (EDB-; SEQ ID NO:52).

[0400] After overnight incubation, the coated plates were washed with PBS and blocked at 37°C for 2 hours with assay medium (DMEM + 10% heat-inactivated FBS). A 1:5 dilution series of the test compound was prepared in assay medium as a 2-fold stock solution (final test concentration range: 200 nM to 2.6 pM). After removing the blocking solution by aspiration, 50 μl of the diluted compound was added to the pre-blocked plate. 50 μl of A549 cell suspension (cell suspension concentration = 0.4 Mio cells / ml assay medium) was added to each well (20,000 cells / well). A549 cells were previously isolated from cell culture flasks using Accutase / EDTA and then transferred to assay medium. The cells were incubated with the compound at 37°C / 5% CO2 for 18–20 hours.

[0401] After 18 hours of incubation, use Bio-Glo TM Luciferase activity was detected using a luciferase assay system (Promega; Madison, WI). Luminescence was measured using a Tecan M1000 Pro instrument with an integration time of 500 ms. The induction factor of LTBR signal transduction was calculated from the obtained relative light units (RLU) as follows: Induction factor = RLU 受刺激的细胞 / Average RLU 未受刺激的细胞(Unstimulated cells were included as controls in each test plate).

[0402] Dose-response curves were plotted using GraphPad Prism, including standard deviation, and a nonlinear fit (log(agonist) versus response (variable slope - three parameters)) was applied, if applicable. To fit the data, the x values ​​(compound concentrations) were transformed using GraphPadPrism's X = Log(x) function.

[0403] result

[0404] Antibody-LIGHT fusion

[0405] Similar to the work published by Tang et al. (Tang et al., Cancer Cell 29:285-96 (2016), which described the antitumor activity of a bispecific molecule against the EGFR-LIGHT fusion complex), COVA1454, a bispecific molecule consisting of an EDB-binding arm and a LIGHT trimer-Fc fusion complex, was designed, expressed, and tested in A549 cell NF-κB luciferase reporter gene assays in the presence or absence of EDB-containing fibronectin. Figure 1F (and Table 2). The activity of COVA1454 was compared with that of soluble recombinant human LIGHT (catalog number 664-LI-025 / CF; R&D Systems; Minneapolis, MN) and non-targeted LIGHT (COVA1418); Figure 1E Compare with Table 2). Figure 3A The results showed that, in the presence of fibronectin containing EDB, COVA1454 activated LTBR only slightly more than non-targeted LIGHT (COVA1418) or soluble recombinant human LIGHT. Interestingly, Figure 3B The results show that in the absence of EDB-containing fibronectin, COVA1454, COVA1418, and soluble recombinant LIGHT activate LTBR in contrast to the presence of EDB. Figure 3A The expression of LTBR in normal tissues is similar and of the same degree. These findings, along with the widespread expression of LTBR in normal tissues (Lukashev et al., Cancer Res., 66(19):9617-24(2006)), indicate that the antibody LIGHT fusion is not suitable for achieving tumor-specific activation of LTBR. In fact, activation of LTBR in normal tissues may lead to undesirable tumor exotoxicity.

[0406] Antibody-LTα1β2 fusion

[0407] Subsequently, an LTα1β2 antibody fusion was generated ( Figures 1G to 1J (and Table 2), which contains one or two LTα1β2 moieties fused with the anti-EDB antibody EDBmAb1, and is tested in a reporter gene assay.

[0408] These constructs are designed in a manner similar to that of Gurney et al., who reported in vitro and in vivo studies of a bispecific fusion construct consisting of a heterotrimeric single-chain LTα1β2 moiety fused with a B7-H4-specific tumor-targeting antibody (WO2018 / 119118). Importantly, unlike LIGHT used in previous sections, the LTα1β2 fusion construct is a specific agonist of LTBR and does not activate HVEM.

[0409] Figure 3C and Figure 3D The results obtained using COVA14113, a fusion of two LTα1β2 antibodies with EDBmAb1, are shown. Figure 1H (and Table 2); COVA14116, which is a fusion of LTα1β2 and EDBmAb1 antibody (and Table 2); Figure 1I (and Table 2), comparing it with the following: COVA14114, used as a non-targeting LTα1β2 control, which is a fusion of two LTα1β2 antibodies with the isotype control antibody B21M (and Table 2), and comparing it with the following: Figure 1G (and Table 2); soluble recombinant human LIGHT; and soluble LTα1β2 (recombinant human lymphotoxin α1β2; catalog number 8884-LY / CF; R&D Systems). In the presence of EDB ( Figure 3C Both COVA14113 and COVA14116 achieved more potent activation of LTBR than the soluble natural ligands LIGHT and LTα1β2; however, the non-targeting LTα1β2 control COVA14114 showed comparable activation levels to COVA14113 and COVA14116. In the absence of EDB ( Figure 3D The activities of COVA14113 and COVA14114 (carrying two LTα1β2 moieties) remained unchanged, while the activity of COVA14116 (carrying one LTα1β2 moiety) decreased to slightly below the level of activation achieved by soluble LTα1β2. These data demonstrate that tumor antigen-dependent activation of LTBR is very difficult to achieve with this type of antibody-LTα1β2 construct. In fact, due to the widespread expression of LTBR in normal tissues, it is difficult to achieve LTBR activation in the absence of EDB-containing fibronectin (…). Figure 3D The activation level achieved may be problematic (Lukashev et al., Cancer Res., 66(19):9617-24(2006).

[0410] Bispecific antibodies based on agonist anti-LTBR antibodies

[0411] To achieve tumor antigen-dependent activation of LTBR, ​​we developed a bispecific antibody (a 1:1 heterodimer; the Fc region includes a KiH mutation to promote proper pairing), consisting of the anti-EDB antibody EDBmAb1 and the anti-LTBR agonist antibodies LTBRmAb1 and LTBRmAb2. Figures 1K to 1M The aim is to activate the LTBR (tumor antigen is the EDB of fibronectin, which is a tumor antigen present in the extracellular matrix) only upon binding to a tumor antigen. Corresponding control antibodies were also generated, consisting of isotype control antibody B21M paired with LTBRmAb1 and LTBRmAb2. Figures 1N to 1O ).

[0412] Figure 4A and Figure 4C COVA14121 (1:1 heterodimers EDBmAb1 and LTBRmAb1) are shown; Figure 1L (and Table 2) and COVA14122 (1:1 heterodimers EDBmAb1 and LTBRmAb2; Figure 1M (and Table 2) can activate LTBR in an EDB-dependent manner. Contrary to the molecules previously described, and in the embodiments described above, as... Figure 4B and Figure 4D As shown, in the absence of EDB, COVA14121 and COVA14122 exhibited only minimal LTBR activation. This residual activity may be due to residual impurities in the purified material. Table 5 shows a comparison of the maximum induction folds (in the presence or absence of ED-B-containing fibronectin) obtained with heterodimers COVA14121 and COVA14122 or with LIGHT antibody fusions (COVA1454) or LTα1β2 antibody fusions (COVA14113 and COVA14116). This comparison clearly demonstrates that using agonist antibodies makes the LTBR bispecific molecules more specific. In fact, for COVA14121 and COVA14122, the ratio between the maximum induction fold achieved in the presence of ED-B and the maximum induction fold achieved in the absence of ED-B is in the range of 4.4 to 5.4, but for the ligand-antibody fusion, it is in the range of 1.1 to 1.6, thus demonstrating that these ligand-antibody fusions do not achieve specific TAA-dependent LTBR activation compared to the bispecific antibodies of the present invention.

[0413] Table 5: Maximum induction factor of NF-kB signal transduction with or without ED-B

[0414]

[0415] In summary, these results demonstrate that it is possible to activate the LTBR in a tumor-dependent manner using bispecific antibodies based on agonistic LTBR antibodies, exhibiting minimal to no activation in the absence of tumor antigens. In such bispecific molecules, the LTBR-binding antibody activates the LTBR only upon binding to a tumor antigen (in this case, an EDB-containing fibronectin).

[0416] To further enhance tumor antigen-dependent LTBR activation, a 2:1 bispecific formulation with two binding sites targeting EDB (to increase antigen-mediated clustering) or two nonspecific binding sites, and one binding site targeting LTBR, ​​was designed (see [link to formulation]). Figures 1P to 1W and Figures 1A2 to 1A5 For LTBR binding sites, scFv fragments are used. Due to potential stability issues with scFv fragments, two different methods are used to stabilize them: scFv fragments derived from LTBRmAb1 are stabilized using an additional disulfide bond between VH and VL (Reiter et al., Nat Biotechnol. 14(10):1239-45(1996)), or by using a bound scFv platform (VH-VL; VL-VH) and fused to EDBmAb1 or B21M (isotype control antibody) via a (G4S)3 linker.

[0417] Figure 5A Showing COVA1456 ( Figure 1A4 (and Table 2), the 2:1 bispecific EDB / LTBR antibody effectively activated LTBR, ​​while the control bispecific antibody COVA1462 ( Figure 1A5 (and Table 2) cannot activate the LTBR. This indicates that clustering via binding to TAA (in this case, immobilized EDB-containing fibronectin) is a prerequisite for effective LTBR activation by the 2:1 bispecific antibody COVA1456. If EDB-containing fibronectin is absent, COVA1456 cannot activate the LTBR ( Figure 5B This supports the fact that the presence of EDB is essential for LTBR activation, and that tumor-specific activation of LTBR is achieved by bispecific antibodies targeting both LTBR and EDB in the extracellular matrix.

[0418] To demonstrate that the ability to activate LTBR in a TAA-dependent manner is not an inherent property of the disulfide-stabilized scFv derived from LTBRmAb1 used to construct COVA1456, COVA1456 and COVA1482 were compared in the same A549 NF-κB reporter gene assay. COVA1482 differs from COVA1456 only in the method used to stabilize the scFv. The scFv in COVA1482, also derived from LTBRmAb1, was stabilized using a binding platform. Figure 5C The results show that both COVA1482 and COVA1456 effectively activate the LTBR in an EDB-dependent manner. The corresponding isotype controls COVA1486 and COVA1462 do not activate the LTBR. Figure 5C These results indicate that the method used to stabilize the scFv fragment does not affect the ability of the bispecific antibody to activate the LTBR in a TAA-dependent manner. Surprisingly, the 2:1 bispecific EDB / LTBR antibody (COVA1482 or COVA1456) showed increased potency in inducing NF-κB signaling in this reporter gene assay. The mean EC50 of COVA1482 was calculated in several assays with the same experimental setup. 50 It is approximately 30 pM ± 10 pM, however in Figure 4A In the assay shown, COVA14121 (1:1 heterodimer) showed EC 50 The concentration is approximately 3 nM, indicating that 2:1 bispecific antibodies are 100 times more effective than 1:1 bispecific antibodies. This can be explained by the increased clustering of LTBR binding sites achieved by utilizing the two binding sites to TAA.

[0419] To investigate the effect of affinity for LTBR on the TAA-dependent activation of LTBR by such bispecific antibodies, lower affinity variants of the scFv fragment derived from LTBRmAb1 were generated (SEQ ID NO:53, KD≈60 nM and SEQ ID NO:54, KD≈600 nM) and used to construct a 2:1 bispecific antibody (COVA14107). Figure 1X and Table 2; and COVA14108, Figure 1Y (See Table 2). Bispecific antibodies were tested in the A549 NF-kB reporter gene assay to observe the effect of affinity on LTBR activation. Figure 5D This study showed that in this assay, lower affinity for LTBR corresponds to a lower ability of the bispecific antibody to activate LTBR in a TAA-dependent manner.

[0420] As described in Example 1, a mutation (WO2010151792) was introduced into the Fc region of some constructs to eliminate binding to protein A (for antibody purification) in order to facilitate the purification of the desired heterodimer. COVA14133 was generated without these mutations, and its activity was compared with COVA1482 to demonstrate that mutations in the Fc region do not affect the activity of the bispecific antibody. COVA14133 and COVA1482, along with their corresponding isotype controls COVA14136 and COVA1486, were compared in an A549 NF-κB reporter gene assay. Figure 5E This demonstrates that COVA14133 activates the LTBR in a TAA-dependent manner with similar efficiency to COVA1482, thus showing that mutations in the Fc do not affect the ability of bispecific antibodies to activate the LTBR.

[0421] In the presence of EDB ( Figure 5F Both COVA14133 (2:1EDBmAb1×LTBR mAb1) and COVA14116 (2:1EDBmAb1×LTα1β2) achieved efficient activation of LTBR. In the absence of the target isotype control molecule COVA14136 (2:1B21M×LTBR mAb1), no LTBR activation was observed; however, the non-target LTα1β2 control COVA14117 (2:1B21M×LTα1β2) showed activation independent of TAA binding. In the absence of EDB ( Figure 5G LTBR activation could not be detected by COVA14133 or its isotype control molecule COVA14136. In contrast, TAA-independent activation of LTBR by COVA14116 and COVA14117 was measured in the absence of EDB, demonstrating that tumor antigen-dependent activation of LTBR is very difficult to achieve by such antibody-LTα1β2 constructs.

[0422] In summary, COVA14133 demonstrates its superior ability to activate LTBR in a TAA-dependent manner.

[0423] Example 3: EDB-dependent in vitro LTBR activation - A375 / WI38VA subline 2RA co-culture cell assay

[0424] A375 / WI38VA subline 2RA co-culture assays were performed to verify whether LTBR activation in the presence of EDB+ fibronectin (produced by WI38VA cells and deposited in the extracellular matrix (Zardi, L. et al., EMBO J, 6, 2337-42 (1987)) led to the release of cytokines and chemokines, and the upregulation of the adhesion molecule ICAM-1 on A375 cells. WI38VA subline 2RA cells were seeded in 96-well plates at a density of 5000 cells / well. CCL75.1 TM Cells were incubated at 37°C / 5% CO2 in their growth medium (MEM (glutamine-free) + 10% heat-inactivated FBS + 0.1 mM NEAA + 2 mM L-Gln + 1 mM sodium pyruvate) for 48 hours. Triple 1:5 dilutions of the test compound were prepared in assay medium (DMEM + 10% heat-inactivated FBS) as 2-fold stock solutions (final test concentrations in the range of 40 nM to 0.5 pM). A375 cells were labeled with CellTrace violet (CTV, Invitrogen; Carlsbad, CA) before incubation with the co-culture containing the WI38VA subline 2RA cells. CRL-1619 TM For labeling, a concentration of 10 × 10⁻⁶ was used. 6 Cell suspensions of 100 cells / ml and 2.5 μM CTV in PBS in 5% FBS were incubated at room temperature for 5 minutes, protected from light. Cells were then washed and incubated at 0.4 × 10⁻⁶ cells / ml. 6 Resuspend the cells at a density of 100 cells / ml in assay medium. Carefully remove the medium from the plate containing 48-hour WI38VA subline 2RA culture, then add 50 μl of A375 cell suspension (20,000 cells / well; CTV+ or CTV-) to each well. Add 50 μl of serially diluted compound (final volume 100 μl per well) to the cells and incubate at 37°C / 5% CO2 for 24 hours.

[0425] After incubation for 24 or 72 hours, the supernatant was removed by centrifugation and stored for measurement of cytokines and chemokines using the MSD assay, or for use in the PBMC migration assay (incubation for 24 hours, Example 5). Cells were further processed by flow cytometry (incubation for 24 hours) for ICAM-1 measurement.

[0426] ICAM-1 was detected by flow cytometry.

[0427] Carefully remove any remaining culture medium from the 96-well plate, separate the cells from the Accutase, transfer them to a deep-well 96-well plate (three copies, pooled into one well), wash, resuspend in 100 μl of FACS buffer (PBS + 1% FBS + 0.1% NaN3) and transfer to a round-bottom 96-well plate. Antibodies, namely labeled anti-human ICAM-1 PE (clone 1H4, Thermo; Waltham, MA) or labeled isotype control antibody PE (MPC-11, BioLegend; San Diego, CA), and LIVE / DEAD immobilizable near-infrared staining agent (Invitrogen), are diluted as shown in Table 6 for single or combined staining.

[0428] Table 6: Dilution regimens for single or combined staining in FACS buffer

[0429]

[0430] Cells were centrifuged at 400×g for 4 minutes at 4°C, the supernatant was discarded, and 50 μl of antibody solution was prepared as described in Table 6. Cells and antibody were incubated in the dark at 4°C for 30 minutes. After incubation, 120 μl of the solution was added to each well, and the cells were centrifuged at 400×g for 4 minutes at 4°C. Cells were washed once with FACS buffer, centrifuged, and resuspended in 90 μl of FACS buffer. Cells were then fixed by adding 90 μl of 3.7% formalin solution to PBS and incubated in the dark on ice for 15 minutes. After fixation, cells were centrifuged at 400×g for 4 minutes at 4°C and resuspended in 100 μl of FACS buffer. Cells were measured at high flow rate in screening mode using a MACS Quant instrument, yielding 49 μl / well. Data were analyzed using FlowLogics software (version 700.2A) and plotted using GraphPad Prism.

[0431] Cytokine measurements were performed in the supernatant of processed cells using the MSD platform.

[0432] Several cytokines known to be controlled by NF-κB signaling were measured using an MSD platform and multiplexed MSD plates. Some examples of the measured cytokines are listed here:

[0433] ■RANTES: R-Plex antibody was used to collect RANTES (MSD);

[0434] ■I-TAC, IP-10, MIP-3b: Measured using 3-PLEX cytokine release assay (MSD);

[0435] ■IL-8, IP-10, MIP-3b: measured using the 3-PLEX cytokine release assay (MSD); and

[0436] ■IL-12p70, IL-6, TNF-α, MIP-3α, SDF-1α: Calculated using the 5-PLEX cytokine release assay (MSD)

[0437] The concentration of cytokines in the supernatant of treated cells was measured using the MSD platform according to the manufacturer's instructions. In short, the protocol involves the following steps:

[0438] (1) Plate preparation involves coating the provided plate with a linker-conjugated capture antibody. The plate is incubated overnight at 2–8°C with shaking. The next day, the plate is washed with PBST (PBS with 0.05% Tween-20) using a plate washer (Biotek; Winooski, VT).

[0439] (2) Preparation of calibrators, standards, and detection antibody solutions;

[0440] (3) Dilute the supernatant at a ratio of 1:3 or 1:5 depending on the availability of the material. Measure the supernatant after incubation for 24 hours or 72 hours (for I-TAC, MIP-3a, TNFα).

[0441] Measurement scheme :

[0442] Step 1: Add the sample or calibrator standard to the plate and incubate the plate at room temperature for 1 hour while shaking.

[0443] Step 2: Wash the plate and add the detection antibody. Incubate the plate at room temperature with shaking for 1 hour.

[0444] Step 3: Wash the plate and add 2× read buffer T. Analyze the plate on an MSD instrument.

[0445] - Data were analyzed using Mesoscale software (MSD discovery workbench program v 4.0.12.1). Dose-response curves were plotted using GraphPad Prism, including standard deviations from three replicates, and a nonlinear fit (log(agonist) versus response (variable slope - four parameters)) was applied, if applicable. To fit the data, the x-values ​​(compound concentrations) were transformed using the X = Log(x) function of GraphPad Prism.

[0446] Results – Detected by flow cytometry ICAM-1

[0447] Previously, it was shown that NF-κB signaling can lead to the upregulation of ICAM-1 on the cell surface (da Silva Antunes et al., Front Immunol, 9, 576, (2018)). Therefore, the level of ICAM-1 expression on the surface of A375 cells was measured after co-culture with the EDB / LTBR bispecific antibody. For example, Figure 6 The study showed upregulation of ICAM-1 after incubation with the EDB / LTBR bispecific antibody COVA1482. In contrast, the isotype control molecule COVA1486 did not lead to upregulation of ICAM-1. These findings indicate that the ability to cluster LTBR scFv via binding to EDB is a prerequisite for LTBR activation, and therefore, a prerequisite for ICAM-1 upregulation.

[0448] Results - Measurement of cytokines in the supernatant of treated cells

[0449] Several cytokines and chemokines expressed as a result of LTBR activation were measured in the supernatant of cocultures treated with the EDB / LTBR bispecific antibody and control molecule as described above. Figures 7A to 7J This illustrates a representative example of cytokine readings upregulated due to LTBR activation by COVA14133. Figure 7A :RANTES, Figure 7B IL-6 Figure 7C IL-8 Figure 7D MIP-3b Figure 7E IP-10 Figure 7F SDF-1a Figure 7G IL-12p70 Figure 7H I-TAC Figure 7I MIP-3a Figure 7J (TNFα). Untargeted LTBR mAb1-derived scFv in COVA14136 does not activate LTBR, ​​and therefore, the concentration of cytokines in the supernatant does not increase the aforementioned background. Background is represented by the levels achieved with B21M (COVA1440) and EDBmAb1 (COVA1452) antibodies, as shown as single concentrations in the figure. Figures 7E to 7JBoth COVA14133 (2:1EDBmAb1×LTBRmAb1) and COVA14116 (2:1EDBmAb1×LTα1β2) demonstrated efficient activation of the LTBR, ​​as measured by induction of cytokine release. No LTBR activation was observed with the non-targeted allotype control molecule COVA14136 (2:1B21M×LTBR mAb1); however, the non-targeted LTα1β2 control COVA14117 (2:1B21M×Ltα1β2) showed cytokine induction independent of TAA binding. Again, these data illustrate how difficult it is to achieve tumor antigen-dependent activation of the LTBR with this type of antibody-LTα1β2 construct.

[0450] In conclusion, the upregulation of ICAM-1 and cytokine secretion during LTBR activation confirm that LTBR activation can have the expected effects on cells.

[0451] In this embodiment, the molecule of the present invention demonstrates that it achieves efficient tumor-associated antigen (EDB-containing fibronectin)-dependent activation of the LTBR. Due to the widespread expression of the LTBR in normal tissues (Lukashev et al., Cancer Res., 66(19):9617-24(2006)), the molecule of the present invention has a significant advantage over the previously described LIGHT and LTα1β2 antibody fusions, as such previously described fusions have been shown herein to efficiently activate the LTBR even in the absence of a tumor-associated antigen, and therefore lack the expected tumor specificity for LTBR activation. In contrast, the multispecific binding molecule of the present invention surprisingly does indeed possess this expected tumor specificity.

[0452] Comparative Example 4: Mesothelin-dependent in vitro LTBR activation – using A549 NF-κB reporter gene cells and CHOK1- Co-culture cell assays using huMSLN or H226

[0453] In Examples 2 and 3, bispecific antibodies targeting EDB (tumor-associated antigens in the extracellular matrix) and LTBR were demonstrated to activate LTBR very effectively in a tumor antigen-dependent manner. To verify whether this finding applies to any tumor antigen, regardless of its location (deposited in the extracellular matrix or on the cell surface of tumor cells), a bispecific 2:1 antibody targeting mesothelin (MSLN) (a tumor-associated antigen expressed on different types of tumors (Hassan and Ho, European Journal of Cancer, 44:46-53 (2008))) and LTBR was designed and prepared as described in Example 1. COVA14146 is a 2:1 MSLN / LTBR bispecific antibody consisting of an anti-mesothelin antibody (MSLNmAb1) fused with an scFv fragment derived from LTBRmAb1. To demonstrate whether a bispecific antibody targeting LTBR and tumor-associated antigens (e.g., mesothelin) present on the cell surface of tumor cells can effectively activate LTBR in a tumor-dependent manner, co-cultured cells were used for assays. The co-culture assays used were A549 cell NF-κB luciferase reporter gene assays (described in Example 2) and H226 cells (mesothelioma cell line) known to express mesothelin (Fan et al., Mol. Canc. Ther. Vol. 1, 595–600 (2002)) and LTBR. CRL-5826).

[0454] Preparation of H226 cells

[0455] H226 cells (expressing approximately 200,000 copies of mesothelin and 10,000 copies of LTBR) suspension were seeded at 10,000 cells per well (in 75 μl assay medium: DMEM + 10% FBS-HI) in 96-well tissue culture plates and incubated at 37°C / 5% CO2 in their growth medium (MEM + 2 mM glutamine + 10% FBS-HI + 10 μg / ml puromycin and RPMI-1640 + 10% FBS + 1 mM sodium pyruvate, respectively) for 6 hours to allow cell attachment to the plate.

[0456] Preparation of compounds

[0457] The compounds were tested in a concentration range of 100 nM to 1.3 pM. Five consecutive dilutions of the compounds (4:1) were prepared in assay medium (DMEM + 10% FBS-HI) and stored at 4°C until use.

[0458] Preparation and addition of A549 reporter gene cells

[0459] A549 reporter cells were separated from Accutase / EDTA in cell culture flasks and transferred to assay medium (DMEM + 10% FBS-HI). After adding a total of 20,000 A549 reporter cells / well to a plate containing H226 cells, 50 μL of pre-diluted compound was added to each well and incubated at 37°C / 5% CO2 for 20 h.

[0460] Measurement of luminescence in treated co-cultures

[0461] After incubation for 20 hours, use Bio-Glo according to the manufacturer's instructions. TM Luciferase activity was detected using a luciferase assay system (Promega; Madison, WI). Luminescence was measured using a Tecan M1000Pro instrument with an integration time of 500 ms. The induction factor of LTBR signal transduction was calculated from the obtained relative light units (RLU) as follows: Induction factor = RLU 受刺激的细胞 / Average RLU 未受刺激的细胞 (Unstimulated cells were included as controls in each test plate).

[0462] Dose-response curves were plotted using GraphPad Prism, including standard deviation, and a nonlinear fit (log(agonist) versus response (variable slope - three parameters)) was applied, if applicable. To fit the data, the x values ​​(compound concentrations) were transformed using GraphPadPrism's X = Log(x) function.

[0463] Cytokine measurements were performed in the supernatant of processed cells using the MSD platform.

[0464] Several cytokines known to be controlled by NF-κB signaling can be measured using MSD platforms and multiplexed MSD plates. For example, this article describes a method for measuring RANTES using R-Plex antibody-based human RANTES (MSD).

[0465] The concentration of RANTES in the supernatant of treated cells was measured using the MSD platform according to the manufacturer's instructions. In short, the protocol involves the following steps:

[0466] (1) Plate preparation involves coating the provided plate with a linker-conjugated capture antibody. The plate is incubated overnight at 2–8°C with shaking. The next day, the plate is washed with PBST (PBS with 0.05% Tween-20) using a plate washer (Biotek; Winooski, VT).

[0467] (2) Preparation of calibrators, standards, and detection antibody solutions;

[0468] (3) Dilute the supernatant at a ratio of 1:3 or 1:5 depending on the availability of the material.

[0469] Measurement scheme :

[0470] Step 1: Add the sample or calibrator standard to the plate and incubate the plate at room temperature for 1 hour while shaking.

[0471] Step 2: Wash the plate and add the detection antibody. Incubate the plate at room temperature with shaking for 1 hour.

[0472] Step 3: Wash the plate and add 2× read buffer T. Analyze the plate on an MSD instrument.

[0473] - Analyze the data using Mesoscale software (MSD discovery workbench program v 4.0.12.1) and plot the graphs using GraphPad Prism.

[0474] Results - Mesothelin-dependent activation of LTBR in A549 reporter gene-producing cells / H226 co-culture was measured.

[0475] Co-culture assays were performed using A549 reporter cells and H226 cells to verify whether COVA14146 could activate LTBR in a more physiological system, where, due to its widespread expression (Lukashev et al., Cancer Res., 66(19):9617-24(2006)), LTBR was expected to be co-expressed on the cell surface of tumor cells along with mesothelin (and other tumor-associated antigens on the cell surface of tumor cells, such as EGFR). Figure 8A The diagram shows that under these conditions, COVA14146 does not effectively activate the LTBR. The concentration of RANTES secreted in the supernatant of treated cells was measured to confirm that COVA14146 does not effectively activate the LTBR. As expected, Figure 8B The results showed that the amount of RANTES secreted by cells treated with COVA14146 was the same as that secreted by cells treated with the same type control molecule COVA1486, thus confirming that LTBR cannot be activated under these conditions.

[0476] In summary, the data presented in Examples 2-4 indicate that fibronectin containing EDB (tumor-associated antigens deposited in the extracellular matrix of tumors); Figure 9A This can lead to efficient clustering of LTBRs, resulting in their efficient activation, which differs from antigens co-expressed with LTBRs on the surface of tumor cells (e.g., mesothelin). Figure 9AUnder the conditions shown, activation of the LTBR by the bispecific antibody of the present invention leads to the secretion of chemically induced chemokines and cytokines, as well as the overexpression of adhesion molecules (e.g., ICAM-1) on treated cells. In the absence of EDB-containing fibronectin ( Figure 9B The bispecific antibody of the present invention cannot activate the LTBR, ​​and therefore no expression of chemical inducers, chemokines, and cytokines, or overexpression of adhesion molecules was observed. Furthermore, it was demonstrated that tumor-associated antigens co-expressed with the LTBR on tumor cells are unsuitable for tumor-dependent activation of the LTBR by the bispecific antibody.

[0477] In summary, this paper demonstrates that targeting the LTBR with bispecific antibodies that bind to both the LTBR and a tumor-associated antigen (TAA) co-expressed with the LTBR on tumor cells, and that such TAAs do not activate the LTBR in a tumor-specific manner (Example 4), and that targeting the LTBR via a fusion protein containing a TAA-binding moiety and either the natural LTBR ligand LIGHT or LTα1β2 does indeed lead to LTBR activation, but not in a tumor-specific manner (Example 2). However, it is noteworthy and surprising that bispecific antibodies binding to one binding domain of the LTBR and another binding domain of fibronectin's EDB (a TAA present in the extracellular matrix) can activate the LTBR in a tumor-specific manner (Examples 2 and 3). Particularly favorable results were observed when such bispecific antibodies contained three binding domains, such as two binding domains targeting the EDB and one binding domain targeting the LTBR. Given their tumor specificity, this makes the bispecific antibodies of the present invention potential candidates for cancer immunotherapies of interest.

[0478] Those skilled in the art will understand that modifications can be made to the above embodiments without departing from the broad inventive concept of the present invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention, as defined in this specification.

[0479] Example 5: PBMC orientation towards conditioned medium from A375 / WI38VA subline 2RA co-cultured cells. Transwell migration

[0480] In Example 3, a bispecific antibody was demonstrated that targets EDB (tumor-associated antigen in the extracellular matrix) and LTBR and activates LTBR very effectively in a tumor antigen-dependent manner, thereby leading to the production of pro-inflammatory cytokines.

[0481] The purpose of this assay was to investigate whether the cytokines and chemokines generated during co-culture could attract PBMCs and cause their migration. Human PBMCs were isolated from the ESR amber layer by Ficoll Paque density gradient centrifugation, and A375 / WI38VA co-cultures were prepared and stimulated with EDB / LTBR bispecific antibody and control molecules as described in Example 3.

[0482] After incubation at 37°C / 5% CO2 for 24 hours, the supernatant of the stimulated co-culture was transferred to 96-well deep-well plates, and the assay medium (RPMI 1640 + 10% FBS + 1mM sodium pyruvate) was diluted 1:1. After dilution, the supernatant was centrifuged (500xg / 5min) and transferred to fresh 96-well deep-well plates to remove any cells or cell debris.

[0483] Recombinant SDF-1a (an effective chemical inducer) was used at a concentration of 40 ng / ml in the assay medium as a positive control to stimulate PBMC migration. 235 μl / well of conditioned medium, SDF-1a control, or assay medium was transferred for use in HTS. Migration assays were performed (in triplicate) on a carrier plate with a 5 μm pore polycarbonate membrane (Corning) that had been previously equilibrated at 37 °C / 5% CO2 in assay medium (RPMI 1640 + 10% FBS + 1 mM sodium pyruvate) for at least 1 hour. After the membrane insert was placed back into the carrier plate, 75 μl / well of PBMC suspension was sputtered at 4.67 × 10⁻⁶. 6 10 cells / ml were added to all wells of the migration assay plate, resulting in 350,000 cells / well. The plate was incubated at 37°C / 5% CO2 for 2 hours to allow PBMCs to migrate toward the conditioned medium.

[0484] After 2 hours of incubation, the plate insert was removed, and the migrated cells in the carrier plate were carefully resuspended and transferred to fresh U-bottom 96-well plates. The migrated cells were centrifuged (400 x g / 4 min), resuspended in 50 μl / well FACS buffer (PBS containing 1% FBS-HI, 0.1% sodium azide, and 1 mM EDTA), and measured directly using a MACS Quant instrument (high flow rate and fast mode). Data were analyzed using FlowLogics software (version 700.2A). Dose-response curves were plotted using GraphPad Prism, including standard deviations from three replicates. To fit the data, the x-values ​​(compound concentrations) were transformed using the X = Log(x) function of GraphPad Prism.

[0485] Results – PBMCs were observed in conditioned medium from A375 / WI38VA subline 2RA co-cultured cells. Transwell migration

[0486] In this embodiment, the study investigated that a mixture of cytokines and chemokines expressed upon LTBR activation in a co-culture assay (see Example 3) could induce PBMCs to migrate toward the cytokine and chemokine gradient. A transwell migration assay was established in which the supernatant of co-cultures stimulated with different concentrations of EDB / LTBR bispecific antibody was placed in the lower chamber, while freshly isolated human PBMCs were added to the upper chamber of the transwell plate. After a 2-hour incubation period, the migrating cells were counted and phenotyped by flow cytometry. Figure 10 Representative results of the migration assay are shown. PBMCs migrated from co-cultures stimulated with COVA14133 (EDB / LTBR bispecific antibody) to the supernatant in a dose-dependent manner; however, supernatant from co-cultures incubated with the non-targeted control molecule COVA14136 (isotype control / LTBR) did not induce PBMC migration. The LTα1β2 antibody fusion COVA14116 (EDB mAb1-LTα1β2; 2:1) and, to some extent, COVA14117 (B21M-LTα1β2; 2:1) did indeed induce PBMC migration. Migration of different immune cell subsets was confirmed by staining with immune cell markers to phenotypically classify the migrating cells. Migration of monocytes, eosinophils / neutrophils, basophils, NK cells, NKT cells, dendritic cells, and T cells was confirmed (data not shown). This embodiment demonstrates that EDB-dependent activation of the LTBR leads to the secretion of cytokines and chemokines, and shows that these factors can act as chemical inducers for immune cells. Figure 10 ).

[0487] Furthermore, this embodiment again demonstrates that the molecules of the present invention have a significant advantage over previously described LTα1β2 antibody fusions, as such previously described fusions have been shown herein to effectively activate the LTBR and lead to PBMC migration even in the absence of tumor-associated antigens, and therefore lack the desired tumor specificity for LTBR activation. In contrast, the multispecific binding molecules of the present invention surprisingly do indeed possess this desired tumor specificity.

[0488] Example 6: Effects of EDB-dependent LTBR-mediated endothelial activation on monocyte transport across the endothelial monolayer

[0489] As demonstrated in Examples 3 and 5, cytokines produced after EDB-dependent activation of the LTBR can cause PBMCs to migrate toward the cytokine gradient. The assays described herein aim to verify whether EDB-dependent activation of the LTBR on endothelial cells can lead to increased monocyte transport across the endothelial layer.

[0490] The mononuclear cells used in this assay were purified from EDTA-treated blood collected from healthy donors using the appropriate negative selection kit (Miltenyi Biotec) and at a concentration of 1.5 × 10⁻⁶. 6 HUVECs (human umbilical vein endothelial cells) were cultured for 48 hours in chamber slides coated with recombinant EDB+ fibronectin domain 7-B-8-9 (EDB+; SEQ ID NO: 51) supplemented with M199 medium (M199 medium, 20% FCS, hydrocortisone (0.1 μM), heparin (100 μg / ml), ECGS 15 μg / ml, vitamin C (10 μg / ml), penicillin / streptomycin (1% / 1%)). HUVECs were then stimulated with TNF (500 U / ml; positive control), EDB / LTBR bispecific antibody (COVA14133; 50 nM), or non-LTBRmAb1-derived scFv (COVA14136; 50 nM) and incubated for 2 days.

[0491] The flow measurement apparatus consisted of a heated microscope chamber (37°C) and a calibrated pump, through which flow was generated by perfusing a monocyte suspension (M199 medium, 0.1% BSA) + / - over the HUVEC monolayer. Flow rate was expressed as venule / capillary (0.05 Pa). The wash buffer was then pumped over the HUVECs for 10 minutes to remove the activating medium, thus equaling 20 minutes of total HUVEC exposure. Monocytes were then perfused over the HUVECs for 6 minutes (step 2), followed by perfusion of wash buffer for 50 minutes (step 3). This was performed at 0.1 Pa, which is standard for all monocyte recruitment protocols. Throughout steps 2–3, images of the captured monocytes were created using a phase-contrast microscope and camera. Individual images were recorded every 30 seconds in a fixed field and compiled into a short movie sequence, allowing analysis of individual monocytes over a large area. Monocytes adhering to the surface of the HUVECs had a phase-white / gray appearance, while those that had migrated had a phase-black appearance.

[0492] By playing a movie sequence, during the duration of the experiment, at 0.19mm 2 Within a defined area, adherent and migrating cells are counted within a fixed grid on each image. Each cell count is performed at fixed time points throughout the experiment.

[0493] The total number of adherent cells represents the sum of cells captured at each time point; a certain percentage of these will migrate (phase gray + black). Migration events (phase black) are the percentage of total monocytes (phase gray + black) captured per unit field flux. Monocytes typically remain adherent throughout the duration of co-culture and exhibit very few dissociation events.

[0494] All experiments were performed in triplicate and expressed as mean plus standard error (SEM). Statistical analysis of the hypothetical parameter distribution was performed using the Student's t-test. The p-values ​​for significance scores are shown in the figure below: *P<0.05, **P<0.01, ***P<0.005 (Bradfield PF et al., Blood. 1 Oct 2007; 110(7):2545-55).

[0495] Results—The effect of EDB-dependent LTBR-mediated endothelial activation on monocyte transport across the endothelial monolayer.

[0496] This example investigated the effect of EDB-dependent LTBR activation on endothelial cell monolayers on monocyte adhesion and migration, since endothelial cells have previously been shown to express LTBR on their surface (Lukashev et al., Cancer Res., 66(19): 9617-24 (2006)).

[0497] Figure 11A The results showed that more monocytes adhered to the HUVEC monolayer, which was activated with the non-targeted control molecule COVA14136 (isotype control / LTBR), and was grown in the presence of EDB-containing fibronectin and activated with COVA14133 (EDB / LTBR bispecific antibody), compared to the monolayer activated with the non-targeted control molecule COVA14136 (isotype control / LTBR).

[0498] Figure 11B The study showed that, compared with HUVECs incubated with the non-targeted control COVA14136, activation with COVA14133 resulted in increased not only monocyte adhesion but also increased monocyte migration through the HUVEC monolayer.

[0499] In summary, the results of this embodiment further confirm that the molecules of the present invention have significant advantages, as they can activate LTBR only in the presence of fibronectin containing EDB, unlike the previously described LTα1β2 antibody fusions, thereby providing the desired tumor specificity for LTBR activation.

[0500] sequence list

[0501] SEQ ID NO:1 (HC BHA 10 IgG1s pestle)

[0502] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0503] SEQ ID NO:2(LC BHA10)

[0504] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0505] SEQ ID NO:3(HC L19 IgG1s)

[0506] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0507] SEQ ID NO:4 (HC L19 IgG1s peptide)

[0508] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0509] SEQ ID NO:5(LC L19)

[0510] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0511] SEQ ID NO:6(HC B21M(RSV)IgG1s)

[0512] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0513] SEQ ID NO:7(HC B21M(RSV)IgG1s peptide)

[0514] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0515] SEQ ID NO:8[LC B21M(RSV)]

[0516] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0517] SEQ ID NO:9 HC(CBE11 IgG1s)

[0518] EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMYWFRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCAREENGNFYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0519] SEQ ID NO:10(LC CBE11)

[0520] DIQMTQSPSSLSASVGDRVTITCKAGQDIKSYLSWYQQKPGKAPKLLIYYATRLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQHGESPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0521] SEQ ID NO:11(HC BHA10 IgG1s)

[0522] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0523] SEQ ID NO:12(HC L19 IgG1s)

[0524] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0525] SEQ ID NO:13(HC CBE11 IgG1s)

[0526] EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMYWFRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYCAREENGNFYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGS

[0527] SEQ ID NO:14(HC B21M(RSV)IgG1s)

[0528] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0529] SEQ ID NO:15 (3xhmLIGHT fused with Fc, possessing IgG1s, club, and pA mutations)

[0530] RRSHEVNPAAHLTGANSLTGSGGPLLWETQLGLAFLGRLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRLGKRLVRLRDGTRSYFGAFMVGGGSGGGGGSGGGGGSGGGGGSRRRSHEVNPAAHLTGANSLTGSGGPLLWETQLGLAFLGRLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRLGKRLVRLRDGTRSYFGAFMVGGGSGGGGGSGGGGGSRRRSHEVNPAAHLTGANSLTGSG PLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLGKRLVRLRDGTRSYFGAFMVGGGSGGGSGGGGSDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0531] SEQ ID NO:16 (3xhmLIGHT single strand for fusion)

[0532] RRSHEVNPAAHLTGANSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVHLEAGEKVVVRLGKRLVRLRDGTRSYFGAFMVGGGSGGGGGSGGGGGSGGGGGSRRSHEVNPAAHLTGANSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVHLEAGEKVVVRLGKRLVRLRDGTRSYFGAFMVGGGSGGGGGSGGGGGSRRSHEVNPAAHLTGANSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVHLEAGEKVVVRLGKRLVRLRDGTRSYFGAFMVGGGSGGGGGSGGGGGSRRSHEVNPAAHLTGANSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKTGYYYIYSKVQLGGVGCPLGLAGTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVHLEAGEKVVVRLGKRLVRLRDGTRSYFGAFMV

[0533] SEQ ID NO:17 (LTa1b2 for fusion)

[0534] KPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0535] SEQ ID NO:18 (HC B21M, IgG1s fused with LTa1b2)

[0536] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAHSTLKPAAHL IGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGDPQGRSVTLRSSLYRAGGAYGPGTP ELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0537] SEQ ID NO:19 (HC B21M fused with LTa1b2, IgG1s, club, with pA mutation)

[0538] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAAGGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKAHSTLKPAAHL IGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGDPQGRSVTLRSSLYRAGGAYGPGTP ELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0539] SEQ ID NO:20 (HC L19, IgG1s fused with LTa1b2)

[0540] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0541] SEQ ID NO:21 (HC L19, IgG1s, fused with LTa1b2, with pA mutation)

[0542] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGAFALLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVGLSPGLPAAHLIGAPLKGQGLGWETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLRSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG

[0543] SEQ ID NO:22 [Bound scFv BHA10(VH-VL)]

[0544] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0545] SEQ ID NO:23 [Bound scFv BHA10(VL-VH)]

[0546] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0547] SEQ ID NO:24 [Binding connector (VH-VL)]

[0548] GGGSGGGSGCPPCGGGG

[0549] SEQ ID NO:25 [Disulfide bond-stabilized scFv BHA10 (VH-VL)]

[0550] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSARYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0551] SEQ ID NO:26 (HC B21M N-terminal bound BHA10(VH-VL), IgG1s, club-shaped, with pA mutation)

[0552] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGDGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPK SLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQITLKESGPTLVKPTQTLLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDT ATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0553] SEQ ID NO:27 (HCB21MN end-stitched BHA10(VL-VH), IgG1s, club-shaped, with pA mutation)

[0554] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGN VHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGCGTTTVVSSGGGGSGGGGSGGGGSGGGSGGGSQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTA TYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0555] SEQ ID NO:28 (HC B21M C-terminal bound BHA (VH-VL), IgG1s, club, with pA mutation)

[0556] QITLKESGPTLVKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGSGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0557] SEQ ID NO:29 (HC B21M C-terminal bound BHA(VL-VH), IgG1s, club, with pA mutation)

[0558] QITLKESGPTLVKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGSGGGSGIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0559] SEQ ID NO:30 (HC L19 N-terminal bound BHA10(VH-VL), IgG1s, club, with pA mutation)

[0560] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAP KSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLLESSGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0561] SEQ ID NO:31 (HC L19 N-terminal bound BHA10(VL-VH), IgG1s, club, with pA mutation)

[0562] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSGSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0563] SEQ ID NO:32 (HC L19 C-terminal bound BHA10(VH-VL), IgG1s, club, with pA mutation)

[0564] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGSGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0565] SEQ ID NO:33 (HC L19 C-terminal bound BHA10(VL-VH), IgG1s, club, with pA mutation)

[0566] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGSGGGSGIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGCAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKGGSGGSGGCPPCGSGGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGCGTTVTVSS

[0567] SEQ ID NO:34(HC L19 C-terminal bound (VL3_Y36F_S49Y_F87Y)BHA(VH-VL), IgG1s, pestle, (with pA mutation)

[0568] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0569] SEQ ID NO:35(HC L19 C-end binding (VH_CDR1_Y33A)BHA10(VH- (VL), IgG1s, pestle, with pA mutation)

[0570] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0571] SEQ ID NO:36(HC B21M C-terminal bound (VL3_Y36F_S49Y_F87Y)BHA(VH-VL), IgG1s, (Pepper, with pA mutation)

[0572] QITLKESGPTLVKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0573] SEQ ID NO:37(HC B21M C-end bound (VH_CDR1_Y33A)BHA10(VH-VL), IgG1s, pestle, with (with pA mutation)

[0574] QITLKESGPTLVKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0575] SEQ ID NO:38 (HC L19 C-terminal bound BHA10(VH-VL), IgG1s, club, no pA mutation)

[0576] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKF KGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0577] SEQ ID NO:39 (HC L19 C-terminal disulfide bond stable, BHA(VH-VL), IgG1s, club, without pA mutation)

[0578] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPP CREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKPSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0579] SEQ ID NO:40(HC B21M C-terminal disulfide bond stable, BHA10(VH-VL), IgG1s, club, without pA mutation) Change)

[0580] QITLKESGPTLVKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKG RVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGGSGGGGSGGGGSGIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0581] SEQ ID NO:41 (HC B21M C-terminal bound BHA10(VH-VL), IgG1s, club-shaped, without pA mutation)

[0582] QITLKESGPTLVKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVY TLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEK FKGRVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0583] SEQ ID NO:42 [(GGGGS)4 linker (for linking the Fv fragment in disulfide bond-stabilized scFv)]

[0584] GGGGSGGGGGSGGGGGSGGGGGS

[0585] SEQ ID NO:43(VH BHA10)

[0586] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSS

[0587] SEQ ID NO:44(VL BHA10)

[0588] DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIK

[0589] SEQ ID NO:45(VH L19)

[0590] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

[0591] SEQ ID NO:46(VL L19)

[0592] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

[0593] SEQ ID NO:47(VH CBE11)

[0594] EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMYWFRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCAREENGNFYYFDYWGQGTTVTVSS

[0595] SEQ ID NO:48(VL CBE11)

[0596] DIQMTQSPSSLSASVGDRVTITCKAGQDIKSYLSWYQQKPGKAPKLLIYYATRLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQHGESPWTFGGGTKLEIK

[0597] SEQ ID NO:49(VH B21M)

[0598] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSS

[0599] SEQ ID NO:50(VL B21M)

[0600] DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIK

[0601] SEQ ID NO:51 (Fibronectin domain 7B89)

[0602] PLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGNSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTHHHHHH

[0603] SEQ ID NO:52 (Fibronectin domain 789)

[0604] PLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGNSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTHHHHHH

[0605] SEQ ID NO:53[Bound scFv(VL3_Y36F_S49Y_F87Y)BHA10(VH-VL)]

[0606] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWFQQKPGKAPKSLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDTYPFTFGCGTKVEIK

[0607] SEQ ID NO:54[Bound scFv(VH_CDR1_Y33A)BHA10(VH-VL)]

[0608] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYALHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0609] SEQ ID NO:55 [Binding connector (VL-VH)]

[0610] GGSGGSGGCPPCGSGG

[0611] SEQ ID NO:56 (HC L19 C-terminal disulfide bond stable, BHA10(VH-VL), IgG1s, club, with pA mutation)

[0612] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPP CREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKPSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0613] SEQ ID NO:57(HC B21M C-terminal disulfide bond stable, BHA10(VH-VL), IgG1s, club, with pA mutation) Change)

[0614] QITLKESGPTLVKPTQTLLTTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLPGKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGQCLEWMGWIYPGNVHAQYNEKFKG RVTITADKSTSTAYMELSSLRSEDTAVYCARSWEGFPYWGQGTTVTVSSGGGGSGGGGSGGGGSGIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0615] SEQ ID NO:58(IgG1σFc)

[0616] DKTHTCPPCPAPEAAGASSVFLFPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGS

[0617] SEQ ID NO:59[(GGGGS)3 linker, scFv to Fc]

[0618] GGGGSGGGGSGGGGS

[0619] SEQ ID NO:60 (HCDR1 of VH BHA10)

[0620] TYYLH

[0621] SEQ ID NO:61 (HCDR2 of VH BHA10)

[0622] WIYPGNVHAQYNEKFKG

[0623] SEQ ID NO:62 (HCDR3 of VH BHA10)

[0624] SWEGFPY

[0625] SEQ ID NO:63 (LCDR1 of VL BHA10)

[0626] KASQNVGINVA

[0627] SEQ ID NO:64 (LCDR2 of VL BHA10)

[0628] SASYRYS

[0629] SEQ ID NO:65 (LCDR3 of VL BHA10)

[0630] QQYDTYPFT

[0631] SEQ ID NO:66 (HCDR1 of VH CBE11)

[0632] DYYMY

[0633] SEQ ID NO:67 (HCDR2 of VH CBE11)

[0634] TISDGGSYTYYPDSVK

[0635] SEQ ID NO:68 (HCDR3 of VH CBE11)

[0636] EENGNFYYFDY

[0637] SEQ ID NO:69 (LCDR1 of VL CBE11)

[0638] KAGQDIKSYLS

[0639] SEQ ID NO:70 (LCDR2 of VL CBE11)

[0640] YATRLAD

[0641] SEQ ID NO:71 (LCDR3 of VL CBE11)

[0642] LQHGESPWT

[0643] SEQ ID NO:72 (HCDR1 of VH L19)

[0644] SFSMS

[0645] SEQ ID NO:73 (HCDR2 of VH L19)

[0646] SISGSSGTTYYADSVKG

[0647] SEQ ID NO:74 (HCDR3 of VH L19)

[0648] PFPYFDY

[0649] SEQ ID NO:75 (LCDR1 of VL L19)

[0650] RASQSVSSSFLA

[0651] SEQ ID NO:76 (LCDR2 of VL L19)

[0652] YASSRAT

[0653] SEQ ID NO:77 (LCDR3 of VL L19)

[0654] QQTGRIPPT

[0655] SEQ ID NO:78(VH MSLNmAb1)

[0656] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS

[0657] SEQ ID NO:79(VL MSLNmAb1)

[0658] DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK

[0659] SEQ ID NO:80(MSLNmAb1 HC C-terminal bound BHA10(VH-VL), IgG1s, club-shaped, with pA mutation)

[0660] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVY LPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGKGGGGSGGGSGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRQAPGCGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSGGGSGGGSGCPPCGGGGDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQQKPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGCGTKVEIK

[0661] SEQ ID NO:81 (HC MSLNmAb1 IgG1s)

[0662] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0663] SEQ ID NO:82(LC MLSNmAb1)

[0664] DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0665] SEQ ID NO:83 (BHA10 HCDR1 low affinity variant)

[0666] TYALH

[0667] SEQ ID NO:84 (HC L19 IgG1s, with pA mutation)

[0668] VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0669] SEQ ID NO:85 (HC B21M(RSV)IgG1s, with pA mutation)

[0670] QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0671] SEQ ID NO:86 (MSLNmAb1 HC, IgG1s, with pA mutation)

[0672] Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ala Thr Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Ser Gly Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Gly Thr Gln Thr Tyr Ile Cys Asn Val His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Ala Glu Ala Ala Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser Ala Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly <110> Cilag GmBH International <120> Multispecific binding molecule and its uses <130> COV6002WOPCT1 <140> <141> <150> 62 / 946,452 <151> December 11, 2019 <160> 102 <170> PatentIn version 3.5 <210> 1 <211> 446 <212> PRT <213> Artificial sequence <220> <223> HC BHA 10 IgG1s stubs <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asn Val His Ala Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Trp Glu Gly Phe Pro Tyr Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Ser Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Ala Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 2 <211> 214 <212> PRT <213> Artificial sequence <220> <223> LC BHA10 <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Ile Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Ser Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Asp Thr Tyr Tyr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 3 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> HC L19 IgG1s knob <400> 3 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Ser Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Ala Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 4 <211> 446 <212> PRT <213> Artificial sequence <220> <223> HC L19 IgG1s臼 <400> 4 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Ser Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Ala Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 5 <211> 215 <212> PRT <213> Artificial sequence <220> <223> LC L19 <400> 5 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 85 90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 6 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> HC B21M (RSV) IgG1s Stalk <400> 6 [[ID= 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Leu Tyr Gly Phe Thr Tyr Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala 225 230 235 240 Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ala Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 7 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> HC B21M (RSV) IgG1s臼 <400> 7 Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser...

Claims

1. A bispecific binding molecule, said bispecific binding molecule comprising: (i) A first binding domain that specifically binds to the lymphotoxin β receptor (LTBR) and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) The VH comprises HCDR1 with the amino acid sequence SEQ ID NO:60, HCDR2 with the amino acid sequence SEQ ID NO:61, and HCDR3 with the amino acid sequence SEQ ID NO:62, and the VL comprises LCDR1 with the amino acid sequence SEQ ID NO:63, LCDR2 with the amino acid sequence SEQ ID NO:64, and LCDR3 with the amino acid sequence SEQ ID NO:65; (b) The VH comprises HCDR1 with the amino acid sequence SEQ ID NO:83, HCDR2 with the amino acid sequence SEQ ID NO:61, and HCDR3 with the amino acid sequence SEQ ID NO:62; and the VL comprises LCDR1 with the amino acid sequence SEQ ID NO:63, LCDR2 with the amino acid sequence SEQ ID NO:64, and LCDR3 with the amino acid sequence SEQ ID NO:65; or (c) The VH comprises HCDR1 with the amino acid sequence SEQ ID NO:66, HCDR2 with the amino acid sequence SEQ ID NO:67, and HCDR3 with the amino acid sequence SEQ ID NO:68; and the VL comprises LCDR1 with the amino acid sequence SEQ ID NO:69, LCDR2 with the amino acid sequence SEQ ID NO:70, and LCDR3 with the amino acid sequence SEQ ID NO:71; and (ii) A second binding domain that specifically binds to the additional domain B (EDB) of fibronectin, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1 of SEQ ID NO:72, HCDR2 of SEQ ID NO:73 and HCDR3 of SEQ ID NO:74, and the VL comprises LCDR1 of SEQ ID NO:75, LCDR2 of SEQ ID NO:76 and LCDR3 of SEQ ID NO:77; The bispecific binding molecule activates the LTBR when it binds to the EDB.

2. The bispecific binding molecule of claim 1, wherein the bispecific binding molecule activates LTBR in a tumor-specific manner.

3. The bispecific binding molecule according to claim 1, wherein the bispecific binding molecule comprises two antigen-binding domains.

4. The bispecific binding molecule according to claim 1, wherein the bispecific binding molecule comprises three antigen-binding domains.

5. The bispecific binding molecule according to claim 4, wherein the three antigen-binding domains include one binding domain that specifically binds to LTBR.

6. The bispecific binding molecule according to claim 4, wherein the three antigen-binding domains include two binding domains that specifically bind EDB.

7. The bispecific binding molecule of claim 4, wherein the binding domain that specifically binds to LTBR comprises a single-chain variable domain of the antibody.

8. The bispecific binding molecule according to claim 1, wherein (i) the VH comprises the amino acid sequence of SEQ ID NO:43 and the VL comprises the amino acid sequence of SEQ ID NO:44; or (ii) the VH comprises the amino acid sequence of SEQ ID NO:47 and the VL comprises the amino acid sequence of SEQ ID NO:

48.

9. The bispecific binding molecule according to claim 1, wherein the first binding domain comprises the amino acid sequence of SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:

25.

10. The bispecific binding molecule of claim 1, wherein the second binding domain that specifically binds to EDB comprises: (a) VH and VL, wherein VH contains the amino acid sequence of SEQ ID NO:45 and VL contains the amino acid sequence of SEQ ID NO:

46.

11. The bispecific binding molecule according to claim 1, wherein the bispecific binding molecule comprises: (a)(i) a first heavy chain comprising the amino acid sequence of SEQ ID NO:1, wherein the first heavy chain forms a binding domain 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, wherein the second heavy chain forms a binding domain with a second light chain comprising the amino acid sequence of SEQ ID NO:5; or (b)(i) a first heavy chain containing the amino acid sequence of SEQ ID NO:9, the first heavy chain forming a binding domain with a first light chain containing the amino acid sequence of SEQ ID NO:10, and (ii) a second heavy chain containing the amino acid sequence of SEQ ID NO:4, the second heavy chain forming a binding domain with a second light chain containing the amino acid sequence of SEQ ID NO:

5.

12. The bispecific binding molecule according to claim 1, wherein the bispecific binding molecule comprises any one of the following: (a)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:30, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain 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 of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain 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 of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain 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 of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain 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 of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain 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 of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain 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:38, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or (h)(i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:39, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:5; or (i) a scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:56, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:

5.

13. The bispecific binding molecule according to claim 1, wherein the bispecific binding molecule comprises (i) an scFv heavy chain fusion comprising the amino acid sequence of SEQ ID NO:38, wherein the heavy chain portion of the scFv heavy chain fusion forms a binding domain 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, wherein the heavy chain forms a binding domain with a light chain comprising the amino acid sequence of SEQ ID NO:

5.

14. One or more nucleic acid molecules, said nucleic acid molecules encoding a bispecific binding molecule according to any one of claims 1 to 13.

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

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

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

18. Use of the bispecific binding molecule according to any one of claims 1 to 13, one or more nucleic acid molecules according to claim 14, one or more carriers according to claim 15, or the pharmaceutical composition according to claim 17 for the manufacture of a medicament for the treatment of lung cancer in a subject of need.

19. A method for preparing a bispecific binding molecule, the method comprising expressing one or more nucleic acid molecules according to claim 14 or one or more vectors according to claim 15 in a host cell, and harvesting the bispecific binding molecule.

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