Anti-EGFR antibodies, anti-cMET antibodies, anti-VEGF antibodies, multispecific antibodies and uses thereof

Abstract

The present disclosure provides antibodies and fragments that target EGFR, VEGF, PD-L1, or cMET. The present disclosure also provides multispecific antibodies comprising a first variable domain capable of binding to epidermal growth factor receptor (EGFR), a second variable domain capable of binding to cMET, and a third variable domain capable of binding to PD-L1 or VEGF. The multispecific antibodies are effective in the treatment of cancer and / or other diseases, disorders, and conditions, the pathogenesis of which is mediated by EGFR, VEGF or PD-L1, and cMET.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 256,705, filed October 18, 2021, the contents of which are incorporated herein by reference.

[0002] Sequence Listing This application includes the following: sql The sequence listing contained in the Sequence Listing was submitted electronically as an XML file entitled "20221014.xml", is 140,305 bytes in size, and was created on October 14, 2022. The information contained in the Sequence Listing is incorporated herein by reference.

[0003] Field of Disclosure The present disclosure relates to antibodies that target epidermal growth factor receptor (EGFR), cMET, and the PD-L1 / VEGF axis, and uses of the antibodies to treat or prevent cancer and other diseases, disorders, and conditions whose etiology is mediated by EGFR, PD-L1 / VEGF, and / or cMET. [Background technology]

[0004] The epidermal growth factor (EGF) receptor (EGFR) is a cell surface receptor, also known as ErbB-1 receptor, ERBB, ERBB1, HER1, PIG61, mENA. EGFR is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: ErbB-1 (EGFR), ErbB-2 (HER2 / c-neu; Her2), ErbB-3 (Her3), and ErbB-4 (Her4). EGFR is a member of the type 1 tyrosine kinase family of growth factor receptors and plays an important role in cell growth, differentiation, and survival.

[0005] EGFR can be activated by specific ligands, including epidermal growth factor, amphiregulin, heparin-binding ETF, betacellulin, and transforming growth factor alpha (TGFα). Upon activation by growth factor ligands, the receptor changes from an inactive, mostly monomeric form to an active homodimer. In addition to forming homodimers after ligand binding, EGFR can pair with another member of the ErbB receptor family, such as ErbB-2, to form activated heterodimers in the absence of ligand binding.

[0006] Mutations involving EGFR have been identified in several types of cancer. First-generation TKIs, such as gefitinib and erlotinib, small molecule tyrosine kinase inhibitors (TKIs), block autophosphorylation of EGFR at the intracellular tyrosine kinase domain, thereby inhibiting downstream signaling events. Lung cancer cell lines with T790M mutation, such as H1975 (L858R / T790M) and H820 (del(E746, A750), T790M), are resistant to different generations of TKI molecules. Second-generation TKIs such as afatinib, dacomitinib, and neratinib have shown promising activity against EGFR T790M, but at the same time have shown limited efficacy due to dose-limiting toxicity from suppressing wild-type EGFR. The H1975-HGF xenograft model is resistant to erlotinib and afatinib, shown in Figure 13 of Janssen's patent US2018 / 0258173 A1. Osimertinib, as a third-generation TKI, has been approved for non-small cell lung cancer that has acquired EGFR T790M resistance mutations. However, patients treated with osimertinib eventually acquire drug resistance. Mutations of EGFR (C797S) are a frequent mechanism of resistance. Numerous studies have highlighted increased expression of cMET due to MET gene amplification or activation of cMet due to increased expression of HGF as the major resistance mechanisms for both first- and third-generation TKIs.

[0007] Both cetuximab and panitumumab target the extracellular portion of EGFR and function by blocking ligand binding, thereby inhibiting downstream events that lead to inhibition of cell proliferation. However, patients whose tumors contain other mutations usually do not benefit from cetuximab or panitumumab therapy. Gain-of-function mutations in KRAS alter the signaling properties of tumor cells by persistently sending growth signals even when EGFR is blocked. Side effects of current EGFR-targeted therapies targeting EGFR-overexpressing cancer cells suffer from toxicity due to basal expression of EGFR in other normal tissues outside the tumor. Activating mutations in the EGFR gene, mainly L858R mutations, exon 19 deletions, and exon 20 mutations, result in ligand-independent activation of EGFR kinase activity. Aberrant activation of both the EGFR and mesenchymal-epithelial transition factor (MET) signaling pathways has been implicated in promoting tumor cell growth and proliferation in lung cancer (Bean, Brennan et al. 2007, Engelman, Zejnullahu et al. 2007).

[0008] MET is the human receptor for human hepatocyte growth factor (HGF; also known as scatter factor), a member of the tyrosine kinase superfamily. cMET ligands are potent mitogens / morphogens that include HGF and its splicing isoforms (NK1, NK2). HGF expression is also associated with activation of the HGF / cMET signaling pathway, which is one of the mechanisms of tumor escape under selection by EGFR-targeted therapy. Ligand binding to cMET leads to receptor multimerization, phosphorylation of multiple tyrosine residues in the intracellular domain, and catalytic activation of downstream signaling. The HGF / cMET signaling pathway plays an important role in normal somatic development and wound healing. However, aberrant cMET activation in cancer leads to tumor progression, angiogenesis, invasive growth, and cancer metastasis. Dysregulation and / or hyperactivation of HGF or cMET in human cancers via overexpression, amplification, or mutation is associated with poor prognosis. cMET can be activated in HGF-related and HGF-independent manners. Overexpression of cMET, MET gene amplification or mutations have been reported in various cancers, including colorectal, lung, gastric, and renal cancers, and may promote ligand-independent receptor activation (Birchmeier, Birchmeier et al. 2003). The abundance of cMET may also induce homodimerization and heterodimerization, which subsequently activates intracellular signaling in the absence of ligand.

[0009] MET and EGFR are also co-expressed in many human tumors. Blocking one receptor tends to upregulate the other, frequently and often rapidly resulting in resistance to a single antitumor drug treatment (Engelman, Zejnullahu et al. 2007). Conversely, cMET-amplified lung cancer cells exposed to cMET inhibitors for a long time develop resistance via the EGFR pathway (McDermott, Pusapati et al. 2010). cMET / HGF signaling in resistance to EGFR-targeted therapy has fostered the development of molecules to treat resistance. Unfortunately, antibody-based approaches include anti-HGF antibodies, and anti-cMET antibodies have not been clinically effective (Lee, Sung et al. 2015). Furthermore, some in vivo studies have shown that some cMET small molecule inhibitors have potential side effects such as heart rate acceleration, myocardial degeneration, nephrotoxicity, and weight loss (Cui, Shen et al. 2013).

[0010] To address the challenge of blocking both the EGFR and cMET pathways, various multispecific EGFRxcMET antibodies have been developed. Samsung ME22S with a 2+2 single chain (sc) Fv-mAb fusion format (US2015030599), Merck with a SEED 1+1 multispecific antibody format (WO2017 / 076492), Genentech with a 2:1 scFv of cMET fused onto cetuximab, Roche Glycart with a 1+1 scFab cMET fused to cetuximab format (UWO2017 / 076492), Lilly LY3164530 with a 2+2 scFv-Fab-Fc / scFv-Fab format (US20130156772), Epimab with a FIT Ig 2+2 format, Merus MCLA-129 with a common light chain 1+1 format (US2020024892), and Janssen / Genmab 1+1 Various architectures exist, including the DuoBody format (US20180258173). Each molecule has a different range of efficacy, but still suffers from limited application to certain patient subsets due to the induction of resistance as well as the occurrence of dose-limiting adverse effects.

[0011] Programmed death ligand-1 (PD-L1) is expressed in 19.6%-65.3% of NSCLC (Pawelczyk, Piotrowska et al. 2019). Furthermore, the presence of EGFR mutations is linked to PD-L1 expression. EGFR activation by EGF stimulation, exon-19 deletion, and L858R mutation could also induce PD-L1 expression. Such EGFR activation can induce T cell apoptosis via the PD-L1 / VEGF axis in tumor cell and peripheral blood mononuclear cell co-culture systems. Therefore, inhibition of EGFR by EGFR-TKIs can release the inhibition of T cells and enhance the production of interferon-γ (Chen, Fang et al. 2015, Fan, Liu et al. 2015). cMET and programmed death-1 (PD-1) humanized polyspecific monoclonal antibodies have been developed to inhibit tumor progression, migration, metastasis, and angiogenesis by blocking cMET, and can also rescue systemic T cell function by blocking PD-1 in cancer cells overexpressing cMET and PD-L1. Furthermore, such BsAbs can bridge T cells and tumor cells, allowing T cells to directly target tumor cells (Sun, Wu et al. 2017). In other studies, multispecific cMET / PD-L1 CAR-T is more effective than monovalent cMET CAR-T for the treatment of hepatocellular carcinoma. In vivo experiments have demonstrated that cMET / PD-L1 CAR-T cells significantly inhibit tumor growth and improve survival persistence (Jiang, Li et al. 2021).

[0012] Currently, immunotherapy involving PD-1 and its respective ligand PD-L1 is promising. PD-L1 (also known as B7-H1 or CD274) is the cognate ligand of PD-1 that is overexpressed in various tumors. Binding of PD-1 and PD-L1 inhibits the activation, proliferation, and survival of NK cells and T cells, eventually leading to immune evasion of tumor cells. Recent studies have demonstrated that blocking the PD-1 / PD-L1 pathway can enhance endogenous antitumor immunity by restoring the function of T lymphocytes. Thus, manipulating the PD-1 / PD-L1 axis may be a promising therapeutic option for NSCLC. Anti-PD-1 / PD-L1 antibodies may be an optional therapy for EGFR-TKI-resistant patients, especially those with EGFR-TKI-resistant NSCLC harboring EGFR mutations.

[0013] To obtain better strategies to contain EGFR+ cancers, alternative therapeutic approaches are being developed. The interaction between cancer cells and their microenvironment is important for the development and progression of solid tumors (Holmgren, O'Reilly et al. 1995). Tumor growth and metastasis are critically dependent on the development and / or remodeling of the microvasculature (Folkman 1995). Inflammatory breast cancer is pathologically characterized by high vascularity and increased microvessel density due to the high expression of angiogenic factors such as VEGF, a key mediator of angiogenesis and involved in endothelial and tumor cell proliferation, motility, and vascular permeability (Kaumaya and Foy 2012). The transition between dormancy and active growth in tumorigenesis appears to be triggered by an "angiogenic switch" (Holmgren, O'Reilly et al. 1995). This angiogenic switch has been recently demonstrated in several types of cancer. VEGF constitutes one of the most proangiogenic factors known today (Troiani, Martinelli et al. 2012). In many different types of cancer, VEGF gene expression and secretion levels are increased (Fukumura, Xavier et al. 1998). VEGF acts as an angiogenic factor as well as an important promoter of metastasis through VEGF receptor (VEGFR)-1 and / or VEGFR-2 signaling. While endothelial cells respond through VEGFR-2 activation, infiltrating cells such as macrophages are activated through VEGFR-1 signaling, which is also involved in the recruitment of endothelial progenitor cells in neovasculature (Kaumaya and Foy 2012).

[0014] Angiogenesis is involved in the pathogenesis of various diseases, including solid tumors, intraocular neovascular syndromes such as proliferative retinopathy or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis (Klagsbrun and D'Amore 1991, Folkman and Shing 1992). In the case of solid tumors, neovascularization allows tumor cells to acquire a growth advantage and growth autonomy compared to normal cells. Thus, in breast cancer as well as several other tumors, a correlation has been observed between the density of microvessels in tumor sections and patient survival (Weidner, Semple et al. 1991, Horak, Leek et al. 1992, Macchiarini, Fontanini et al. 1992).

[0015] To address one or more challenges in human tumors with anti-EGFR, cMET, PD-L1 / VEGF, and HGF immunotherapy, the present disclosure provides novel anti-EGFR, anti-cMET, and anti-VEGF antibodies. The present disclosure also provides novel multispecific antibodies, such as multispecific antibodies that include a first variable domain capable of binding to EGFR (e.g., the extracellular domain of EGFR), a second variable domain capable of binding to cMET (e.g., the extracellular domain of cMET), and a third variable domain capable of blocking PD-L1 or VEGF.

[0016] The multifunctional antibodies of the present disclosure that bind cMET, PD-L1 / VEGF, and EGFR with high affinity can provide one or more of the following advantages. For example, the multifunctional antibodies of the present disclosure can effectively neutralize cMET activation by HGF and EGFR activation by ligands of the EGF and HGF families, and / or provide superior activity in the internalization and / or degradation of cMET and EGFR (both wild-type and mutant) compared to single-agent combinations. Such multifunctional antibodies are needed as effective pharmacological interventions for certain cancers. Such multifunctional antibodies can prevent the potential for heterodimer clustering between EGFR, cMET, and HER family members. Such multifunctional antibodies can block the engagement of PD-1 and PD-L1 to enhance the activity of nearby immune cells. In particular, the multifunctional antibodies of the present disclosure can (a) more effectively treat cancers characterized by one or more KRAS and Exon20 mutations; (b) exhibit superior activity in preventing or delaying the development of resistance to other cMET and / or EGFR inhibitors, including but not limited to erlotinib, gefitinib, lapatinib, and vemurafenib, compared to related combinations of single agents; (c) induce minimal or no measurable agonistic EGFR and cMET activity; (d) block the binding of PD-L1 and PD-1; or block angiogenic VEGF activity; or (f) exhibit in vivo stability, physical and chemical stability, including but not limited to, thermal stability, solubility, low self-association, and pharmacokinetic properties acceptable for development and / or use in the treatment of cancer. Summary of the Invention

[0017] The disclosure provides antibodies or antigen-binding fragments thereof against EGFR, PD-L1 / VEGF, and cMET, nucleic acids encoding such antibodies and fragments, methods for preparing the antibodies and fragments, and methods for treating diseases, such as EGFR-, PD-L1 / VEGF-, and cMET-mediated diseases or disorders, e.g., human cancers, including lung, head and neck, renal, liver, gastric, colorectal, triple-negative breast, pancreatic, and neuroendocrine cancers.

[0018] In one aspect, the disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof. In some embodiments, the disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 95, 96, and 97; SEQ ID NOs: 95, 96, and 98; SEQ ID NOs: 95, 96, and 105; SEQ ID NOs: 102, 100, and 101; and SEQ ID NOs: 102, 103, and 104, respectively.

[0019] In some embodiments, the disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof comprising at least one antibody single domain selected from SEQ ID NOs: 5-12 or antigen-binding fragments thereof. In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof comprises a tandem antibody single domain heavy chain, optionally linked via a linker. In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof comprises a tandem antibody single domain heavy chain or antigen-binding fragment thereof selected from SEQ ID NOs: 13-18, wherein two EGFR-binding VHO sequences are linked via a linker.

[0020] In some embodiments, the disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof comprising at least one antibody single domain having at least 85% identity to any one of SEQ ID NOs: 5-12, or antigen-binding fragments thereof. In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof comprises a tandem antibody single domain heavy chain having at least 85% identity to any one of SEQ ID NOs: 13-18, or antigen-binding fragments thereof.

[0021] In some embodiments, the disclosure provides an anti-EGFR antibody, or antigen-binding fragment thereof, that binds to one or more epitopes on EGFR (e.g., human EGFR) recognized by the anti-EGFR antibody, or antigen-binding fragment thereof, comprising at least one antibody single domain selected from SEQ ID NOs:5-12, or comprising a tandem antibody single domain heavy chain selected from SEQ ID NOs:13-18.

[0022] In some embodiments, the disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof comprising a human antibody heavy chain SEQ ID NO:1 and a human antibody light chain SEQ ID NO:2; or a human antibody heavy chain SEQ ID NO:3, and a human antibody light chain SEQ ID NO:4.

[0023] In another aspect, the disclosure provides an anti-cMET antibody or antigen-binding fragment thereof. In some embodiments, the disclosure provides an anti-cMET antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, where HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 106, 107, and 133; SEQ ID NOs: 111, 112, and 113; SEQ ID NOs: 111, 114, and 113; SEQ ID NOs: 99, 118, and 119; SEQ ID NOs: 99, 120, and 119; SEQ ID NOs: 99, 121, and 119; and SEQ ID NOs: 99, 122, and 119, respectively.

[0024] In some embodiments, the disclosure provides an anti-cMET antibody or antigen-binding fragment thereof comprising a light chain variable region comprising three CDRs designated LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NOs: 108, 109, and 110; SEQ ID NOs: 115, 116, and 117; and SEQ ID NOs: 123, 124, and 125, respectively.

[0025] In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, comprising an antibody heavy chain sequence selected from SEQ ID NOs: 24, 28-29, and 34-37, and an antibody light chain sequence selected from SEQ ID NOs: 26, 31-32, and 39-40. In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, comprising an antibody heavy chain sequence selected from SEQ ID NOs: 23, 27, and 33, and an antibody light chain sequence selected from SEQ ID NOs: 25, 30, and 38. In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, comprising at least one cMET-binding VHO (variable heavy chain only) sequence selected from SEQ ID NOs: 41-44.

[0026] In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, comprising an antibody heavy chain sequence having at least 85% identity to any one of SEQ ID NOs: 23, 24, 27-29, and 33-37, and an antibody light chain sequence having at least 85% identity to any one of SEQ ID NOs: 25, 26, 30-32, and 38-40. In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, comprising at least one cMET-binding VHO sequence having at least 85% identity to any one of SEQ ID NOs: 41-44.

[0027] In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, that binds to one or more epitopes on cMET recognized by an anti-cMET antibody, or antigen-binding fragment thereof, comprising an antibody heavy chain sequence selected from SEQ ID NOs: 23, 24, 27-29, and 33-37, and an antibody light chain sequence selected from SEQ ID NOs: 25, 26, 30-32, and 38-40. In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, that binds to one or more epitopes on cMET recognized by an anti-cMET antibody, or antigen-binding fragment thereof, comprising at least one cMET-binding VHO sequence selected from SEQ ID NOs: 41-44.

[0028] In some embodiments, the disclosure provides human antibody heavy chain SEQ ID NO:23 and human antibody light chain SEQ ID NO:25; human antibody heavy chain SEQ ID NO:23 and human antibody light chain SEQ ID NO:26; human antibody heavy chain SEQ ID NO:24 and human antibody light chain SEQ ID NO:25; human antibody heavy chain SEQ ID NO:24 and human antibody light chain SEQ ID NO:26; human antibody heavy chain SEQ ID NO:27 and human antibody light chain SEQ ID NO:30; human antibody heavy chain SEQ ID NO:27 and human antibody light chain SEQ ID NO:31; human antibody heavy chain SEQ ID NO:27 and human antibody light chain SEQ ID NO:32; human antibody heavy chain SEQ ID NO:28 and human antibody light chain SEQ ID NO:30; human antibody heavy chain SEQ ID NO:28 and human antibody light chain SEQ ID NO:31; human antibody heavy chain SEQ ID NO:28 and human antibody light chain SEQ ID NO:32; human antibody heavy chain SEQ ID NO:29 and human antibody light chain SEQ ID NO:30; human antibody heavy chain SEQ ID NO:29 and human antibody light chain SEQ ID NO:31; human antibody heavy chain SEQ ID NO:29 and human antibody light chain SEQ ID NO:32; human antibody heavy chain SEQ ID NO:33 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:33 and The present invention provides an anti-cMET antibody or antigen-binding fragment thereof, comprising a human antibody heavy chain and a human antibody light chain selected from human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:33 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:34 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:34 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:34 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:35 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:35 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:35 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:37 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:37 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:37 and human antibody light chain SEQ ID NO:40.

[0029] In another aspect, the present disclosure provides an anti-PDL-1 antibody or an antigen-binding fragment thereof, comprising an amino acid sequence selected from SEQ ID NOs: 71-72.

[0030] In some embodiments, the disclosure provides an anti-PD-L1 antibody, or antigen-binding fragment thereof, comprising an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 71-72.

[0031] In some embodiments, the disclosure provides an anti-PD-L1 antibody, or antigen-binding fragment thereof, that binds to one or more epitopes on PD-L1 recognized by the anti-PD-L1 antibody, or antigen-binding fragment thereof, comprising an amino acid sequence selected from SEQ ID NO:71-72.

[0032] In another aspect, the disclosure provides an anti-VEGF antibody or antigen-binding fragment thereof. In some embodiments, the disclosure provides an anti-VEGF antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 129, 130, and 131; SEQ ID NOs: 129, 132, and 131, respectively.

[0033] In some embodiments, the disclosure provides an anti-VEGF antibody or antigen-binding fragment thereof comprising a light chain variable region comprising three CDRs designated LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 are SEQ ID NOs: 126, 127, and 128, respectively.

[0034] In some embodiments, the disclosure provides an anti-VEGF antibody, or antigen-binding fragment thereof, comprising an amino acid sequence selected from SEQ ID NOs: 73-76.

[0035] In some embodiments, the disclosure provides an anti-VEGF antibody, or antigen-binding fragment thereof, comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs:73-76.

[0036] In some embodiments, the disclosure provides an anti-VEGF antibody, or antigen-binding fragment thereof, that binds to one or more epitopes on VEGF recognized by the anti-VEGF antibody, or antigen-binding fragment thereof, comprising an amino acid sequence selected from SEQ ID NOs: 73-76.

[0037] In another aspect, the disclosure provides multispecific antibodies that bind EGFR and cMET, as well as VEGF or PD-L1, and exhibit one or more desirable functional properties, including, for example, high affinity specific binding to human EGFR and cMET, the ability to block EGFR ligands, such as EGF, from binding to EGFR, the ability to block cMET ligands, such as HGF, from binding to cMET, the ability to bind to PD-L1 or VEGF, and the ability to block PD-1 from binding to PD-L1.

[0038] In some embodiments, bivalent anti-cMET antibodies that bind cMET can result in tumor cell proliferation. Thus, the multispecific antibodies disclosed herein preferably have monovalent cMET binding (i.e., one Fab arm that binds to an epitope of cMET).

[0039] Some embodiments provide a multispecific antibody having a cMET valency of 1.

[0040] Some embodiments provide multispecific antibodies with one or two EGFR binding valencies.

[0041] Some embodiments provide multispecific antibodies with one or two PD-L1 or PD-1 binding valencies.

[0042] In some embodiments, the disclosure provides a trispecific antibody comprising an EGFR arm capable of binding EGFR (e.g., human EGFR), a cMET arm capable of binding cMET (e.g., human cMET), and a third variable domain capable of binding PD-L1 (e.g., human PD-L1) or VEGF (e.g., human VEGF). The antibody can be a full-length antibody in an IgG1 format, with a 2:1:1 or 2:1:2 anti-EGFR, anti-cMET, and anti-PD-L1 or anti-VEGF stoichiometry.

[0043] In some embodiments, the disclosure provides a trispecific antibody comprising an EGFR arm comprising a first variable domain capable of binding to EGFR (e.g., the extracellular domain of EGFR), a cMET arm comprising a second variable domain capable of binding to cMET (e.g., the extracellular domain of cMET), and a third variable domain capable of binding to PD-L1 or VEGF. The antibody may be a full-length antibody in IgG1 format, with a stoichiometry of 2:1:1 or 2:1:2 anti-EGFR, anti-cMET, and anti-PD-L1 or anti-VEGF.

[0044] In some embodiments, the disclosure provides multispecific antibodies comprising binding arms capable of targeting EGFR, which may comprise a human IgG heavy chain fusion comprising an amino acid sequence N-to-C-terminal, including but not limited to, sequence A-shielding A-linker A-protease sequence A-linker B-VHO-A linker C targeting EGFR-VHO-B linker D-Fc targeting EGFR. In some embodiments, the disclosure provides multispecific antibodies comprising binding arms capable of targeting both EGFR and PD-L1 / VEGF, comprising a human IgG heavy chain fusion comprising signal sequence Ashielding-A linker A-protease sequence A-linker VHO-A targeting EGFR-linker VHO-B targeting EGFR-linker D-Fc-anti-PD-L1 / VEGF. The IgG may have a human IgG1, IgG2, IgG3, and / or IgG4 Fc framework.

[0045] In some embodiments, the disclosure provides multispecific antibodies comprising a binding arm capable of targeting cMET, which may include, but is not limited to, a human IgG heavy chain fusion comprising an amino acid sequence from the N-to-C-terminus that targets cMET, a signal sequence C-masking C-linker E-protease sequence B-linker F-IgG1 heavy chain fusion, and a human IgG light chain fusion comprising an amino acid sequence from the N-to-C-terminus that targets signal sequence D-masking D-linker G-protease sequence C-linker H-IgG light chain targeting cMET. In some embodiments, the disclosure provides a multispecific antibody comprising a human IgG heavy chain fusion comprising the amino acid sequence from the N to C terminus, the binding arm capable of targeting both cMET and PD-L1 / VEGF, signal sequence C-shielding C-linker E-protease sequence B-linker F-IgG1 heavy chain-anti-PD-L1 / VEGF targeting cMET, and a human IgG light chain fusion comprising the amino acid sequence from the N to C terminus, signal sequence D-shielding D-linker G-protease sequence C-linker H-IgG1 light chain-anti-PD-L1 / VEGF targeting cMET. In some embodiments, the disclosure provides a multispecific antibody comprising a binding arm capable of targeting both cMET and PD-L1 / VEGF, the multispecific antibody comprising a human IgG heavy chain fusion comprising the amino acid sequence from the N to C terminus, signal sequence A-shielding-A linker A-protease sequence A-linker VHO-A targeting cMET-linker VHO-B targeting cMET-linker D-Fc-anti-PD-L1 / VEGF. The IgG may have a human IgG1, IgG2, IgG3, and / or IgG4 Fc framework.

[0046] The shields (shields A, B, C, D) can be the same or different. The linkers (linkers A, B, C, D, E, F, G, H) can be the same or different. The protease sequences (protease sequences A, B, C) can be the same or different.

[0047] Some embodiments provide a multispecific antibody of the present disclosure appended with an immune checkpoint modulating domain to enhance immune cell activity against the tumor, in some embodiments the immune modulating domain is selected from the group consisting of a B7-1 polypeptide, a PD-L1 polypeptide, an anti-PD-1 binding domain, and an anti-PD-L1 binding domain.

[0048] In some embodiments, the disclosure provides a multispecific antibody comprising a binding arm capable of targeting EGFR, a binding arm capable of targeting cMet, and a binding arm capable of targeting VEGF, wherein the EGFR arm, the cMET arm, and the VEGF arm each comprise an amino acid sequence selected from SEQ ID NOs: 83-94. In some embodiments, the disclosure provides a multispecific antibody comprising an amino acid sequence as set forth in Table 10.

[0049] In some embodiments, the disclosure provides multispecific antibodies comprising a binding arm capable of targeting EGFR, wherein the binding arm comprises a human IgG1 Fab heavy chain sequence selected from SEQ ID NOs: 1 and 3, a light chain sequence selected from SEQ ID NOs: 2 and 4, and / or one or more single domain VHO sequences selected from SEQ ID NOs: 5-12, and / or one or more tandem single domain VHO sequences selected from SEQ ID NOs: 13-18.

[0050] In some embodiments, the disclosure provides a multispecific antibody comprising a binding arm capable of targeting cMET, wherein the binding arm comprises a human IgG1 Fab heavy chain sequence selected from SEQ ID NOs: 23-24, 27-29, and 33-37, a light chain sequence selected from SEQ ID NOs: 25-26, 30-32, and 38-40, and / or one or more single domain VHO sequences selected from SEQ ID NOs: 41-44.

[0051] It is recognized that EGFR interacts with many other cell surface markers that contribute to the development of cancer (Wang, Ma et al. 2015, Kennedy, Hastings et al. 2016). In some embodiments, the present disclosure provides a multispecific antibody that can effectively inhibit the association of EGFR receptor with a HER family receptor selected from HER2, HER3, HER4 and corresponding downstream signaling factors. In some embodiments, the present disclosure provides a multispecific antibody that includes a binding arm capable of targeting EGFR, wherein the binding arm includes a human IgG1 Fab heavy chain sequence selected from SEQ ID NOs: 1 and 3, and a light chain sequence selected from SEQ ID NOs: 2 and 4. In some embodiments, the present disclosure provides a multispecific antibody that includes a binding arm capable of targeting EGFR, wherein the binding arm includes one or more single domain VHO sequences selected from SEQ ID NOs: 5-18.

[0052] Heterodimerization of cMET with other receptors contributes to the development of cancer (Viticchie and Muller 2015). In some embodiments, the disclosure provides multispecific antibodies that can effectively inhibit the association of cMET receptor with one or more of RTKs such as Plexin B1 family members, CD44 family members, integrin family receptors including a6b4, a5b1, a3b1, a2b1, and death receptors such as Ron, IGFR, RET, Fas, and DR5, and corresponding downstream signaling factors. In some embodiments, the disclosure provides multispecific antibodies that include a binding arm capable of targeting cMET, wherein the binding arm includes a heavy chain sequence selected from SEQ ID NOs: 23-24, 27-29, 33-37, and a light chain sequence selected from SEQ ID NOs: 25-26, 30-32, and 38-40. In some embodiments, the disclosure provides a multispecific antibody comprising a binding arm capable of targeting cMET, wherein the binding arm comprises one or more single domain VHO sequences selected from SEQ ID NOs: 41-44.

[0053] Some embodiments provide multispecific antibodies with one or more masked epitopes, shields, or caps (e.g., shields A, B, C, D) that can be removed by proteases and / or other in situ specific enzymes found in the tumor microenvironment. The presence of masked epitopes is to minimize systemic toxicity induced by the multispecific antibody.

[0054] In some embodiments, the disclosure provides multispecific antibodies comprising one or more shields or caps that mask cMET and / or EGFR mAb binding. In some embodiments, the shields or caps for the antibodies are selected from SEQ ID NOs: 45-51. In some embodiments, the shields or caps that mask cMET and / or EGFR single domain binding are selected from SEQ ID NOs: 52-61. In some embodiments, the disclosure provides multispecific antibodies, wherein the shields or caps are attached via a protease substrate linker and optionally a peptide linker or spacer. The protease substrate linker is selected from SEQ ID NOs: 62-69.

[0055] In some embodiments, an antibody of the disclosure (e.g., an anti-EGFR, anti-cMET, anti-PD-L1, anti-VEGF, or multispecific antibody) may be a whole antibody, an antibody fragment, an antibody mimetic, a humanized antibody, a single chain antibody, an immunoconjugate, a defucosylated antibody, or a multispecific antibody. The antibody fragment may be selected from the group consisting of a UniBody, a domain antibody, and a VHO domain.

[0056] In some embodiments, the antibodies or fragments of the present disclosure disclosed herein (e.g., anti-EGFR, anti-cMET, anti-PD-L1, anti-VEGF, or multispecific antibodies) may be human, humanized, or chimeric antibodies or antigen-binding fragments.

[0057] In some embodiments, an antibody of the disclosure (e.g., an anti-EGFR, anti-cMET, anti-PD-L1, anti-VEGF, or multispecific antibody) may be a full-length IgG1, IgG2, IgG3, or IgG4 antibody, or an antigen-binding fragment thereof, such as a Fab, F(ab')2, or scFv fragment. The antibody scaffold may be modified to affect functionality, for example to eliminate residual effector functions.

[0058] In some embodiments, the present disclosure provides an immunoconjugate comprising an antibody or fragment disclosed herein and a therapeutic agent, hi some embodiments, the therapeutic agent carries a cytotoxin or a radioisotope.

[0059] Some embodiments provide multispecific antibodies that may be in a human IgG1, IgG2, IgG3, and / or IgG4 framework. Multispecific antibodies can be engineered to have hinge regions with enhanced protease stability. Multispecific antibodies can also be engineered to have shorter and longer half-lives.

[0060] In some embodiments, the heavy chain of the multispecific antibody comprises the constant region of an IgG1 antibody, preferably a human IgG1 antibody. The CH2 region of said IgG1 constant region can be engineered to alter the ADCC, ADCP, and / or CDC activity of the antibody. In a preferred embodiment, said alteration results in enhanced ADCC (antibody-dependent cellular cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), and / or CDC activity. In a preferred embodiment, the CH3 region of the multispecific antibody is engineered to promote heterodimerization of heavy chains comprising a first heavy chain that binds EGFR and a second heavy chain that binds cMET.

[0061] In one embodiment, the multispecific antibodies are capable of inducing higher levels of down-modulation of EGFR and cMET when compared to their parental mAbs. This multispecific antibody activity can reduce the viability of tumor cells driven by the EGFR and cMET signaling cascades.

[0062] Some embodiments provide multispecific antibodies that can simultaneously target and bind to human EGFR and cMET with high affinity and effectively block EGFR at the protein level. The multispecific antibodies have the ability to bind to both cMET and EGFR proteins or to one target protein without affecting the binding of another target protein and to simultaneously bind to cMET and EGFR. The multispecific antibodies inhibit the proliferation of vascular endothelial cells, human lung cancer cells, human breast cancer cells, human pancreatic cancer cells, and / or human gastric cancer cells.

[0063] In some embodiments, the disclosure provides a composition comprising an antibody or fragment of the disclosure (e.g., an anti-EGFR, anti-cMET, anti-PD-L1, anti-VEGF, or a multispecific antibody) and a carrier.

[0064] In some embodiments, the disclosure provides a pharmaceutical composition comprising an antibody or fragment of the disclosure (e.g., an anti-EGFR, anti-cMET, anti-PD-L1, anti-VEGF, or a multispecific antibody) and a pharma- ceutically acceptable carrier.

[0065] Some embodiments provide a composition comprising a multispecific antibody disclosed herein. In some embodiments, the multispecific antibody may be present in the composition at a concentration of 10 mg / mL to 250 mg / mL.

[0066] In some embodiments, the composition of the present disclosure further comprises at least one buffering agent, at least one stabilizer, and / or at least one surfactant. In some embodiments, the composition is liquid. In some embodiments, the composition is formulated for subcutaneous injection. In some embodiments, the composition is sterile. In some embodiments, the composition further comprises histidine hydrochloride, trehalose dihydrate, methionine, and / or polysorbate.

[0067] In some embodiments, the disclosure provides a method for treating or preventing a disease associated with target cells expressing cMET, EGFR, and PD-L1 / VEGF, comprising administering to a subject an antibody of the disclosure, such as an anti-cMETxanti-EGFRxPD-L1 / VEGF multispecific antibody, or an antigen-binding portion thereof, in an amount effective to treat or prevent the disease. In some examples, the disease treated or prevented is a human cancer. In some examples, the disease treated or prevented includes lung cancer, head and neck cancer, colorectal cancer, gastric cancer, breast cancer, intestinal cancer, neuroendocrine, glioblastoma multiforme, and pancreatic cancer.

[0068] In some embodiments, the disclosure provides an antibody of the disclosure, e.g., an anti-cMETxanti-EGFRxPD-L1 / VEGF multispecific antibody, or an antigen-binding portion thereof, for use in treating or preventing a cancer associated with target cells expressing cMET, PD-L1 / VEGF, and EGFR. In some embodiments, the disease to be treated or prevented is a human cancer. In some embodiments, the disease to be treated or prevented comprises lung cancer, head and neck cancer, colorectal cancer, gastric cancer, intestinal cancer, neuroendocrine, glioblastoma multiforme, breast cancer, and pancreatic cancer.

[0069] In some embodiments, the disclosure provides for the use of an antibody of the disclosure, e.g., an anti-cMETxanti-EGFRxPD-L1 / VEGF multispecific antibody, or an antigen-binding portion thereof, for the manufacture of a medicament for use in treating or preventing a cancer associated with target cells expressing cMET, PD-L1 / VEGF, and EGFR. In some embodiments, the disease treated or prevented by the medicament of the disclosure is a human cancer. In some embodiments, the disease treated or prevented by the medicament of the disclosure includes lung cancer, head and neck cancer, colorectal cancer, gastric cancer, intestinal cancer, neuroendocrine, breast, glioblastoma multiforme, and pancreatic cancer.

[0070] In some embodiments, the type of cancer treated or prevented by the antibodies or fragments of the present disclosure may be a conventional cancer, preferably selected from lung cancer, breast cancer, pancreatic cancer, and gastric cancer.

[0071] Some embodiments provide multispecific antibodies that can be developed into combination regimens using high dose chemotherapy together with EGFR inhibitors to determine the best synergistic partners.

[0072] Some embodiments provide multispecific antibodies that can be developed into combination regimens using high dose chemotherapy together with cMET inhibitors to determine the best synergistic partners.

[0073] Some embodiments provide multispecific antibodies of the present disclosure that may be used to treat tumors that are resistant to EGFR tyrosine kinase inhibitors, including, but not limited to, for example, erlotinib, gefitinib, osimertinib, dacomitinib, or afatinib; analogs of erlotinib, gefitinib, osimertinib, dacomitinib, or afatinib; or combinations of one or more of each compound and / or analogs thereof.

[0074] Some embodiments provide multispecific antibodies of the present disclosure that may be used to treat tumors that are resistant to treatment with a cMET tyrosine kinase inhibitor, including, but not limited to, for example, crizotinib, cabozantinib, tivantinib, teptinib; analogs of crizotinib, cabozantinib, tivantinib, teptinib; or combinations of one or more of each compound and / or analogs thereof.

[0075] In some embodiments, the disclosure provides an isolated nucleic acid molecule encoding a heavy or light chain of an antibody or antigen-binding portion thereof of the disclosure, hi some embodiments, the disclosure provides an expression vector comprising one or more of such nucleic acids, and a host cell comprising one or more of such expression vectors.

[0076] In some embodiments, the disclosure provides hybridomas expressing an antibody or antigen-binding portion of the disclosure.

[0077] In some embodiments, the disclosure provides an isolated nucleic acid molecule encoding a heavy or light chain of an isolated multispecific antibody or antigen-binding portion that binds to an epitope on human EGFR and cMET, and PD-L1 or VEGF, hi some embodiments, the disclosure provides an expression vector comprising such a nucleic acid molecule, and a host cell comprising such an expression vector.

[0078] Some embodiments provide a method of producing a multispecific antibody disclosed herein, comprising culturing a recombinant expression transformant disclosed herein and obtaining the multispecific antibody from the culture.

[0079] Some embodiments provide for the application of a multispecific antibody disclosed herein in the manufacture of a medicament for the treatment or prevention of cancer.

[0080] Some embodiments provide nucleic acids encoding multispecific antibodies that target cMET with a mask and EGFR with another mask.

[0081] In some embodiments, the disclosure provides a method for preparing an anti-cMETxAnti-EGFRxPD-L1 / VEGF multispecific antibody, the method comprising obtaining a host cell comprising one or more nucleic acid molecules encoding an antibody of the disclosure, growing the host cell in a host cell culture, providing host cell culture conditions in which the one or more nucleic acid molecules are expressed, and recovering the antibody from the host cell or host cell culture.

[0082] In some embodiments, the disclosure provides a cDNA encoding an isolated multispecific antibody, antigen-binding portion, antibody fragment, or multispecific antibody mimetic. In some embodiments, the disclosure provides expressing the cDNA into a phage such that the multispecific antibody, antigen-binding portion thereof, antibody fragment, or multispecific antibody mimetic (e.g., anti-cMET, anti-PD-L1 / VEGF, and anti-EGFR multispecific antibodies) encoded by the cDNA is displayed on the surface of the phage; selecting a phage displaying the multispecific antibody, antigen-binding portion, antibody fragment, or multispecific antibody mimetic; recovering a nucleic acid molecule from the selected phage that encodes the multispecific antibody, antigen-binding portion, antibody fragment, or multispecific antibody mimetic; expressing the recovered nucleic acid molecule in a host cell; and recovering the multispecific antibody, antigen-binding portion, antibody fragment, or multispecific antibody mimetic from the host cell.

[0083] In some embodiments, the present disclosure provides a method for producing the multispecific antibodies disclosed herein. Recombinant DNA encoding the parent antibodies of the multispecific antibodies is prepared by DNA recombinant technology and then transfected into mammalian cells to express the parent antibodies. After purification, identification, and screening, the multispecific antibodies are generated using controlled Fab arm exchange or other multispecific antibody generation processes, for example, to generate multispecific antibodies that exhibit the biological effect of simultaneous binding to EGFR and cMET. The multispecific antibody affinity and blocking efficiency are identified through the completion of in vitro experiments.

[0084] These and other embodiments of the present disclosure are described in greater detail herein. [Brief description of the drawings]

[0085] [Figure 1]Figure 1 shows the profile of inhibitors of EGFR and cMET signaling pathways in cancer. Binding of EGF to EGFR and HGF to cMET results in phosphorylation of specific tyrosine residues and subsequent activation of these receptors. Overexpression of EGFR and cMET RTK in certain cancers leads to activation of downstream signaling pathways PI3K / Akt and MAPK (RAS-RAF, MEK-ERK / MAPK). Induction of these signaling cascades leads to stimulation of cancer cell survival through dysregulation of cell death pathways. Several inhibitors block these pathways by binding to the tyrosine kinase domain or ligands to inactivate the receptors. TKIs and mAbs of EGFR and cMET signaling pathways are shown in boxes with targets marked with arrows for inhibition or activation as shown in the figure. [Figure 2A] Figure 2 shows that 7D VH hits bind EGFR and block EGFR-EGF binding using ELISA. Figure 2A shows that 7D VH hits (7D VH1, 7D VH2, 7D VH3, 7D VH4, 7D VH5, and 7D VH6) bound to the EGFR extracellular domain (ECD) in an ELISA format. There was no binding by gp120 mAb. Cetuximab and 7D VHO are hits that bound to EGFR ECD in an ELISA format. In this EGFR binding experiment, the EC50 values ​​in ng / mL were: cetuximab - 10 ng / mL; 7D VH1 (Fv set forth in SEQ ID NO: 5) - 14 ng / mL; 7D VH2 (Fv set forth in SEQ ID NO: 6) - 3 ng / mL; 7D VH3 (Fv set forth in SEQ ID NO: 7) - 3 ng / mL; 7D VH4 (Fv set forth in SEQ ID NO: 8) - 3 ng / mL; 7D VH5 (Fv set forth in SEQ ID NO: 9) - 3 ng / mL; and 7D VH6 (Fv set forth in SEQ ID NO: 10) - 2 ng / mL. [Figure 2B] FIG. 2B shows the binding of TAVO412E (also referred to herein as "TAVO412") to recombinant human EGFR ECD in an ELISA format with an EC50 value of 0.059 nM. [Figure 2C]Figure 2C shows the binding of TAVO412E to recombinant cynomolgus monkey EGFR ECD in an ELISA format with an EC50 value of 0.109 nM. In Figures 2A, 2B, and 2C, the y-axis represents absorbance at 450 nm reflecting ELISA binding levels, and the x-axis represents the concentration of test reagent. [Figure 2D] Figure 2D shows that 7D VH hits blocked EGFR ECD binding to EGF in HCC827 cells. There was no blocking by gp120 mAb. In this EGF ligand-EGFR binding blocking experiment, the EC50 values ​​are (in ng / mL): cetuximab -65ng / mL; 7D VH1 (Fv set forth in SEQ ID NO:5) -25ng / mL; 7D VH2 (Fv set forth in SEQ ID NO:6) -23ng / mL; 7D VH3 (Fv set forth in SEQ ID NO:7) -31ng / mL; 7D VH4 (Fv set forth in SEQ ID NO:8) -38ng / mL; 7D VH5 (Fv set forth in SEQ ID NO:9) -34ng / mL; and 7D VH6 (Fv set forth in SEQ ID NO:10) -36ng / mL. In Figure 2D, the y-axis represents the geometric mean fluorescence intensity (gMFI), which reflects the binding level on the cells, and the x-axis represents the concentration of the test reagent. [Figure 2E] Figure 2E shows that TAVO412E blocked EGFR ECD binding to EGF in an ELISA format with an IC50 value of 1.53 nM. In Figure 2E, the y-axis represents the absorbance at 450 nm, which reflects the level of EGF binding to EGFR by ELISA, and the x-axis represents the concentration of the test reagent. [Figure 3A]FIG. 3 shows that cMET hits bind to cMET and block cMET-HGF binding using ELISA. In FIGS. 3A-3G, the y-axis represents absorbance at 450 nm reflecting ELISA binding levels, and the x-axis represents the concentration of test reagent. FIG. 3A shows cMET hits bound to the cMET extracellular domain (ECD) in an ELISA format. Test article and sequence information are shown in the table below. Amivantamab analogs use JNJ-61186372 heavy and light chain amino acid sequences with associated hypofucosylation. The terms amivantamab, amivantamab analog, JNJ-61186372, or JNJ-6372 all refer to the same amino acid sequences with the same associated hypofucosylation. [Table 1] Figure 3A shows onartuzumab and EV1 (heavy chain Fv set forth in SEQ ID NO:24, light chain Fv set forth in SEQ ID NO:26), TV1 (heavy chain Fv set forth in SEQ ID NO:28, light chain Fv set forth in SEQ ID NO:30), TV4 (heavy chain Fv set forth in SEQ ID NO:29, light chain Fv set forth in SEQ ID NO:32) hits bound to cMET ECD in an ELISA assay. In this cMET binding experiment, the EC50 values ​​(in ng / mL) were about 25 ng / mL for onartuzumab, about 17.5 ng / mL for EV1, about 18.5 ng / mL for TV1, and about 18.0 ng / mL for TV4. [Figure 3B] Figure 3B shows that TAVO412E has potent binding to recombinant human cMET with an EC50 value of 0.234 nM. There was no binding to human cMET by the isotype mAb. [Figure 3C] Figure 3C shows that TAVO412E had strong binding to recombinant cynomolgus cMET with an EC50 value of 0.595 nM. There was no binding to cynomolgus cMET by the isotype mAb. A schematic of the ligand blocking assay is shown in Figure 3E. Streptavidin was coated onto an ELISA plate. Biotinylated cMET ECD was then added to this layer. [Figure 3D]Antibodies and ligands were then added to compete for binding to the cMET ECD: the primary antibody was a rabbit polyclonal anti-HGF Ab, and the secondary antibody was an HRP-labeled anti-rabbit antibody for detection. [Figure 3E] The assay format can be reformatted for use in a cMETHGF blocking assay, as described below. [Figure 3F] Figure 3F shows that cMET hits blocked cMET ECD binding to HGF in an ELISA format. There was no blocking by gp120 mAb. In this cMET-HGF blocking binding experiment, the EC50 values ​​(in ng / mL) were: Onartuzumab - 93 ng / mL; EV1 - 93 ng / mL; TV1 - 122 ng / mL; TV4 - 108 ng / mL. [Figure 3G] FIG. 3G shows that TAVO412E blocked the binding of human hepatocyte growth factor (HGF) to recombinant human cMET with an IC50 value of 8.05 nM. [Figure 4A] Figure 4 shows TAVO412E binding to VEGF and blocking VEGF-VEGFR binding using an ELISA assay. In Figures 4A-C, the y-axis represents absorbance at 450 nm reflecting ELISA binding levels, and the x-axis represents the concentration of test reagent. Figure 4A shows that TAVO412E bound to recombinant human VEGF165 in an ELISA format with an EC50 value of 0.084 nM. There was no binding by the isotype mAb. [Figure 4B] FIG. 4B shows the binding of TAVO412E to recombinant cynomolgus VEGF165 in an ELISA format with an EC50 value of 0.346 nM. [Figure 4C] FIG. 4C shows that TAVO412E blocked binding of recombinant human VEGF165 to recombinant human VEGFR with an IC50 value of 14.8 nM. [Figure 5A]FIG. 5 shows the structural design for an anti-cMET×anti-EGFR multispecific antibody. The anti-cMET×anti-EGFR multispecific antibody is illustrated showing the EGFR binding arm in black, the cMET binding arm in dark grey, and the VEGF binding arm in light grey. FIG. 5A shows that the EGFR binding arm can have a valency of one or two VHO domains. The cMET binding arm can have a valency of one Fab domain. The VEGF binding arm can have a valency of one to two domains. The EGFR VHO domain can be on the same heavy chain as the N- or C-terminal fusion of Fc, as a tandem Fc fusion molecule on the Fc, or as a C-terminal fusion on the cMET heavy chain. [Figure 5B] FIG. 5B shows that the EGFR binding arm can have a valency of one or two VHO domains. The cMET binding arm can have a valency of one or two VHO domains on the Fc domain. The VEGF binding arm can have a valency of one to two domains. The EGFR VHO domain can be on the same heavy chain as an N- or C-terminal fusion to Fc, as a tandem fusion molecule on Fc, or as a C-terminal fusion on a cMET VHO heavy chain fusion. The cMET VHO domain can be on the same heavy chain as an N-terminal fusion to Fc, as a tandem fusion molecule on Fc, or as an N-terminal fusion on an EGFR VHO heavy chain fusion molecule. [Figure 6A] Figure 6 shows the binding of TAVO412E to CD16a, CD32a, CD64, and C1q. In Figures 6A-D, the y-axis represents absorbance at 450 nm reflecting ELISA binding levels, and the x-axis represents the concentration of test reagent. Figure 6A shows that TAVO412E binds to recombinant human CD16a in an ELISA format with an EC50 value of 0.46 nM, compared to an isotype mAb with human IgG1 with an EC50 value of 3.7 nM. [Figure 6B] FIG. 6B shows that TAVO412E bound to recombinant human CD32a in an ELISA format with an EC50 value of 2.9 nM, whereas an isotype mAb with human IgG1 had an EC50 value of 14.0 nM. [Figure 6C] FIG. 6C shows that TAVO412E bound to recombinant human CD64 in an ELISA format with an EC50 value of 0.16 nM, whereas the human IgG1-bearing isotype mAb bound with an EC50 value of 0.12 nM. [Figure 6D] FIG. 6D shows that TAVO412E bound recombinant human C1q in an ELISA format with an EC50 value of 14.2 nM compared to an isotype mAb with human IgG1 with an EC50 value of 14.1 nM. [Figure 7A] Figure 7 shows inhibition of EGFR ligand binding to EGFR in H292 cells. Figure 7A shows the assay format of the FACS-based assay used to characterize ligand blockade in H292 cells (EGFR:cMET ratio 365000 vs. 64000). Anti-cMET x anti-EGFR multispecific antibodies were added to compete with 0.2 mg / mL EGF to block binding to the cells. EGF was detected using an AF488 nm labeled rabbit anti-EGF antibody. Figure 7B shows that gMFI was measured to determine the level of EGF binding in the presence of competing mAbs. [Figure 7B] In Figure 7B, the y-axis represents gMFI, which reflects the binding level to H292 cells, and the x-axis represents the concentration of test reagent. In this assay, competitive cMET antibody does not significantly affect EGF binding to H292 cell line. EGFR antibody binds to EGFR and competes with EGF for binding to EGFR. [Figure 8A] Figure 8 shows inhibition of EGFR ligand binding to EGFR in HCC827 cells. Figure 8A shows the assay format of the FACS-based assay used to characterize ligand blockade in HCC827 cells (EGFR:cMET ratio 420,000 to 204,000). [Figure 8B]Figure 8B shows that gMFI was measured to determine the level of EGF binding in the presence of competitive mAb.In Figure 8B, y-axis represents gMFI, which reflects the binding level to HCC827 cells, and x-axis represents the concentration of test reagent.In this assay, competitive cMET antibody does not significantly affect EGF binding to HCC827 cell line.EGFR antibody binds to EGFR and competes with EGF for binding to EGFR. In this EGFR-EGF inhibition experiment, the EC50 values ​​in ng / mL for the EGFRxcMet hits were: 7D VH6xTV4 - 0.63 nM; 7D VH6xEV1 - 0.63 nM; 7D VH4xTV4 - 0.93 nM; 7D VH4xEV1 - 0.81 nM; cetuximabxgp120 - 0.60 nM; cetuximab - 0.33 nM; 7D VH4-Fc - 0.18 nM; and 7D VH6-Fc - 0.23 nM. [Figure 9A] FIG. 9 shows the inhibition of EGFR phosphorylation in NCI-H1975 cells using Western blotting. Figure 9A: The top panel shows western blot lanes corresponding to (1) medium only, (2) EGF only, (3) 7D VH4-Fc (SEQ ID NO: 8), (4) gp120 (heavy chain Fv, SEQ ID NO: 82 and light chain Fv, SEQ ID NO: 81), (5) 7D VH6-Fc (SEQ ID NO: 10), (6) EV1 (heavy chain Fv, SEQ ID NO: 24 and light chain Fv, SEQ ID NO: 26), (7) TV4 (heavy chain Fv, SEQ ID NO: 29 and light chain Fv, SEQ ID NO: 32), (8) 7D VH4 x EV1 bispecific antibody, (9) 7D VH4 x TV4 bispecific antibody; (10) 7D VH6 x EV1 bispecific antibody, (11) 7D VH6 x TV4 bispecific antibody, and (12) cetuximab x gp120 bispecific antibody (monovalent EGFR binding arm). Integration values ​​are normalized to b-actin levels in each lane. All four candidate BsAbs were able to inhibit EGFR phosphorylation, and showed similar inhibitory effects to one armed cetuximab × gp120. [Figure 9B]Figure 9B: The top panel shows Western blot lanes corresponding to (1) medium only; (2) EGF only; (3) 7D VH4xEV1; (4) 7D VH4xTV4; (5) 7D VH6xEV1; (6) 7D VH6xTV4; (7) cetuximabxgp120; (8) gp120; (9) 7D VH4xgp120; (10) 7D VH6xgp120. All four candidate BsAbs (7D VH4xEV1; 7D VH4xTV4; 7D VH6xEV1; 7D VH6xTV4) were able to inhibit EGFR phosphorylation and had similar inhibitory effects with one-arm cetuximabxgp120. [Figure 9C] Figure 9C shows inhibition of EGFR phosphorylation in H292 and HCC827 cells using Western blot. Figure 9C: Results for H292 cells with Western blot lanes corresponding to (1) media only; (2) EGF only (3) 7D VH4 x EV1; (4) 7D VH4 x TV4; (5) 7D VH6 x EV1; (6) 7D VH6 x TV4; (7) cetuximab x gp120; (8) gp120; (9) 7D VH4 x gp120; (10) 7D VH6 x gp120. [Figure 9D] Figure 9D: Results for HCC827 cells with Western blot lanes corresponding to (1) medium only; (2) EGF only (3) 7D VH4xEV1; (4) 7D VH4xTV4; (5) 7D VH6xEV1; (6) 7D VH6xTV4; (7) cetuximabxgp120; (8) gp120; (9) 7D VH4xgp120; (10) 7D VH6xgp120. All four candidates (7D VH4xEV1; 7D VH4xTV4; 7D VH6xEV1; 7D VH6xTV4)) BsAbs were able to inhibit EGFR phosphorylation and showed similar inhibitory effects with one armed cetuximabxgp120 in H292 cells but not in HCC827 cells. [Figure 10A]FIG. 10 shows the utility of TAVO412E in the non-small cell lung cancer cell line HCC827, as shown by cell binding, blocking EGF binding to EGFR on HCC827 cells, and blocking HGF binding to cMET on HCC827 cells. In FIG. 10A-C, the y-axis represents the gMFI value reflecting the level of binding to HCC827 cells, and the x-axis represents the concentration of the test reagent. FIG. 10A shows that TAVO412E had an EC50 value of 1.04 nM for binding to HCC827 cells. Isotype mAbs did not bind to HCC827 cells. [Figure 10B] Figure 10B shows that TAVO412E had an IC50 value of 2.56 nM for blocking EGF binding to EGFR on HCC827 cells. Isotype mAb did not block EGF binding to EGFR on HCC827 cells. [Figure 10C] Figure 10C shows that TAVO412E had an IC50 value of 0.28 nM for blocking HGF binding to cMET on HCC827 cells. Isotype mAb did not block HGF binding to cMET on HCC827 cells. [Figure 11A] Figure 11 shows inhibition of EGFR and cMET phosphorylation in H292 and HCC827 cells. In Figures 11A-B, the y-axis is shown as a percentage of EGFR phosphorylation exhibited by control mAb, and the x-axis is the concentration of test substance. Figure 11A shows that TAVO412E inhibited EGFR phosphorylation in H292 cells in the presence of EGF with an IC50 value of 0.79 nM. Isotype mAbs did not inhibit EGFR phosphorylation. [Figure 11B] Figure 11B shows that TAVO412E inhibited EGFR phosphorylation in H292 cells in the presence of EGF and HGF with an IC50 value of 0.78 nM. Isotype mAbs did not inhibit EGFR phosphorylation. [Figure 11C]In Figures 11C-D, the y-axis is shown as the percentage of cMET phosphorylation shown in the control mAb, and the x-axis is the concentration of the test substance. Figure 11C shows that TAVO412E inhibited cMET phosphorylation in HCC827 cells in the presence of HGF with an IC50 value of 1.41 nM. Isotype mAbs did not inhibit cMET phosphorylation. [Figure 11D] Figure 11D shows that TAVO412E inhibits cMET phosphorylation in HCC827 cells in the presence of HGF and EGF with an IC50 value of 1.99 nM. Isotype mAbs did not inhibit cMET phosphorylation. [Figure 12A] Figure 12 shows the inhibition of HCC827 cell proliferation. In Figures 12A-B, the y-axis is shown as percent viability, and the x-axis is the concentration of the test substance. Figure 12A shows that TAVO412E inhibited the proliferation of HCC827 cells with an IC50 value of 1.76 nM. Isotype mAb did not inhibit the proliferation of HCC827 cells. [Figure 12B] Figure 11B shows that TAVO412E inhibited the proliferation of HCC827 cells in the presence of EGF and HGF with an IC50 value of 1.39 nM. Isotype mAb did not inhibit the proliferation of HCC827 cells. [Figure 13A] Figure 13 shows inhibition of cMET phosphorylation in H1975, HCC827, and H292 cells as detected by Western blot. Figure 13A shows the results for HCI-H1975, Figure 13B shows the results for HCC827, and Figure 13C shows the results for H292 cells. [Figure 13B] Each Western blot lane corresponded to: (1) medium only; (2) HGF only; (3) 7D VH4 x EV1; (4) 7D VH4 x TV4; (5) 7D VH6 x EV1; (6) 7D VH6 x TV4; (7) JNJ6372 cMET (cMET heavy chain Fv SEQ ID NO: 77 and cMET light chain Fv SEQ ID NO: 78) x gp120; (8) gp120; (9) EV1 x gp120; (10) TV1 x gp120. [Figure 13C]Four BsAbs (7D VH4 x EV1; 7D VH4 x TV4; 7D VH6 x EV1; 7D VH6 x TV4) had significantly more inhibitory effects than their monovalent parental Abs in HCC827, H292 and NCI-H1975. [Figure 14A] Figure 14 shows the Fc effector function of TAVO412E on HCC827 cells. In Figures 14A-B, the y-axis is shown as the level of ADCC (Figure 14A) or ADCP (Figure 14B) activation, and the x-axis is the concentration of the test substance. In Figures 14C-E, the y-axis is shown as the percentage lysis, and the x-axis is the concentration of the test substance. Figure 14A shows the ADCC reporter activity induced by TAVO412E in the presence of HCC827 cells with an IC50 value of 0.022 nM. Isotype mAb did not induce ADCP reporter activity of HCC827 cells. [Figure 14B] Figure 14B shows that TAVO412E had ADCP reporter activity against HCC827 cells with an IC50 value of about 0.27 nM. Isotype mAb had no ADCP reporter activity in HCC827 cells. [Figure 14C] Figure 14C shows that TAVO412E had ADCC killing activity against HCC827 cells with an IC50 value of 0.12 nM. The isotype mAb had no ADCP reporter activity in HCC827 cells. [Figure 14D] Figure 14D shows that TAVO412E had ADCP killing activity against HCC827 cells with an IC50 value of 0.16 nM. The isotype mAb had no ADCP reporter activity in HCC827 cells. [Figure 14E] Figure 14E shows that TAVO412E had CDC killing activity against HCC827 cells with an IC50 value of 3.76 nM. The isotype mAb had no ADCP reporter activity in HCC827 cells. [Figure 15A]The antitumor activity of TAVO412E against the non-small cell lung cancer cell line NCI-H1975 is shown in Figure 15. Figure 15A shows 42% NCI-H1975 tumor growth inhibition at 1 mg / kg, 76% at 3 mg / kg, and 94% at 10 mg / kg on day 13. TAVO412E had a dose-dependent tumor growth inhibition in H1975 cells. [Figure 15B] Figure 15B shows that TAVO412E induced degradation of EGFR and reduced EGFR phosphorylation in tumors excised from an in vivo NCI-H1975 xenograft model. TAVO412 shown in Western blot refers to TAVO412E. [Figure 15C] Figure 15C shows that TAVO412E induces degradation of cMET as well as a decrease in cMET phosphorylation in tumors excised from an in vivo NCI-H1975 xenograft model. TAVO412 shown in Western blot refers to TAVO412E. [Figure 15D] Figure 15D shows a bar graph representation of the results for control isotype mAb and TAVO412E in Figures 15B and C. TAVO412E reduced the levels of total and phosphorylated forms of cMET and EGFR in tumors resected from the in vivo NCI-H1975 xenograft model experiment. [Figure 16A] The antitumor activity of TAVO412E against the NSCLC line HCC827 is shown in Figure 16. Figure 16A shows 45% HCC827 tumor growth inhibition at 1 mg / kg, 79% at 3 mg / kg, and 94% at 10 mg / kg on day 13. TAVO412E had dose-dependent tumor growth inhibition in HCC827 xenografts. [Figure 16B] Figure 16B shows that TAVO412E induces degradation of EGFR and cMET in tumors excised from an in vivo HCC827 xenograft model experiment. TAVO412 shown in Western blot refers to TAVO412E. [Figure 16C]Figure 16C shows a bar graph representation of the quantitative results for control isotype mAb and TAVO412E in Figure 16B. TAVO412E reduced the levels of total and phosphorylated forms of cMET and EGFR in tumors resected from an in vivo HCC827 xenograft model experiment. [Figure 17A] The antitumor activity of TAVO412E against triple-negative breast cancer (TNBC) cell line MDA-MB-468 is shown in Figure 17. Figure 17A shows TAVO412E bound to MDA-MB-468 with an EC50 value for binding of 1.11 nM. TAVO412E had dose-dependent tumor growth inhibition in MDA-MB-468 xenografts. In Figure 17A, the y-axis is the gMFI for cell binding, and the x-axis is the concentration of test substance. [Figure 17B] In Figure 17B-C, the y-axis is the percentage of EGFR phosphorylation, and the x-axis is the concentration of the test substance. Figure 17B shows that TAVO412E inhibited human EGFR phosphorylation in MDA-MB-468 cells in the presence of human EGF with an IC50 value of 9.08 nM. Isotype mAb did not inhibit human EGFR phosphorylation. [Figure 17C] Figure 17C shows that TAVO412E inhibited human EGFR phosphorylation in MDA-MB-468 cells in the presence of human EGF and human HGF with an IC50 value of 8.50 nM. Isotype mAbs did not inhibit EGFR phosphorylation. [Figure 18A] Figure 18 shows the antitumor activity of TAVO412E against TNBC cell line MDA-MB-231. Figure 18A shows TAVO412E bound to MDA-MB-231 with an EC50 value for binding of 0.37 nM. In Figure 18A, the y-axis is the gMFI for cell binding, and the x-axis is the concentration of the test substance. [Figure 18B]In Figures 18B and D, the y-axis is the luminescence expressed as relative light units (RLU) upon reporter probe activation, and the x-axis is the concentration of test substance. Figure 18B shows that TAVO412E had ADCP reporter activity in the presence of MDA-MB-231 cells with an EC50 value of 0.087 nM. The isotype mAb had no ADCP reporter assay response. [Figure 18C] In Figures 18C, E, and F, the y-axis is percent lysis and the x-axis is the concentration of the test substance. Figure 18C shows that TAVO412E had ADCP killing of MDA-MB-231 cells with an EC50 value of 0.156 nM. The isotype mAb had no ADCP killing response. [Figure 18D] Figure 18D shows TAVO412E induced ADCC reporter activity in the presence of MDA-MB-231 cells with an EC50 value of 0.13 nM. The isotype mAb had no ADCP reporter assay response. [Figure 18E] Figure 18E shows that TAVO412E had ADCC killing of MDA-MB-231 cells with an EC50 value of 0.12 nM. The isotype mAb had no ADCC killing response. [Figure 18F] Figure 18F shows that TAVO412E had CDC killing of MDA-MB-231 cells with an EC50 value of 1.22 nM. Isotype mAbs had no CDC killing response. [Figure 19A] The antitumor activity of TAVO412E against the triple-negative breast cancer cell line MDA-MB-231 is shown in Figure 19. Figure 19A shows 62% MDA-MB-231 tumor growth inhibition at 10 mg / kg dosing on day 20. [Figure 19B] Figure 19B shows that TAVO412E induces degradation of EGFR and cMET in tumors excised from an in vivo MDA-MB-231 xenograft model experiment. TAVO412 shown in Western blot refers to TAVO412E. [Figure 19C]Figure 19C shows a bar graph representation of the quantitative results for control isotype mAb and TAVO412E in Figure 19B. TAVO412E reduced the levels of cMET and all forms of EGFR in an in vivo MDA-MB-231 xenograft model experiment. [Figure 20A] Demonstration of the utility of TAVO412E in gastric cancer cell lines SNU-5 and MKN-45 as shown by cell binding, blocking HGF binding to cMET on MKN45 cells, and inhibiting proliferation of SNU-5 cells. In Figures 20A-C, the y-axis is the gMFI value of cell binding and the x-axis is the concentration of test substance. In Figure 20D, the y-axis is percent viability and the x-axis is the concentration of test substance. Figure 20A shows that TAVO412E had an EC50 value of 1.78 nM for binding to MKN45 cells. Isotype mAbs did not bind to MKN45 cells. [Figure 20B] Figure 20B shows that TAVO412E had an IC50 value of 0.28 nM for blocking HGF binding to cMET on MKN45 cells. Isotype mAb did not block HGF binding to cMET on MKN45 cells. [Figure 20C] Figure 20C shows that TAVO412E had an EC50 value of 1.99 nM for binding to SNU-5 cells. The isotype mAb did not bind to SNU-5 cells. [Figure 20D] Figure 20D shows that TAVO412E had an IC50 value for inhibition of SNU-5 cell proliferation of 2.66 nM. Isotype mAbs did not inhibit SNU-5 cell proliferation. [Figure 21A]FIG. 21 shows the in vitro antitumor activity of TAVO412E against gastric cancer cell line SNU-5. The experimental protocol was similar to that described in Example 16. In FIG. 21A-B, the y-axis is the RLU value of ADCC reporter probe assay response, and the x-axis is the concentration of test substance. In FIG. 21C, the y-axis is the percent lysis, and the x-axis is the concentration of test substance. FIG. 21A shows the ADCC reporter activity induced by TAVO412E in the presence of SNU-5 cells with an EC50 value of 0.18 nM. Isotype mAb had no ADCC reporter assay response. [Figure 21B] Figure 21B shows that TAVO412E had ADCP reporter activity on SNU-5 cells with an EC50 value of 0.20 nM. The isotype mAb had no ADCP reporter assay response. [Figure 21C] Figure 21C shows that TAVO412E had CDC killing of SNU-5 cells with an EC50 value of 1.19 nM. The isotype mAb had no CDC killing response. [Figure 22A] Figure 22 shows the in vivo antitumor activity of TAVO412E against the gastric cancer cell line MKN45. Figure 22A shows 70% MKN-45 tumor growth inhibition at 3 mg / kg on day 21. [Figure 22B] Figure 22B shows that TAVO412E induced degradation of EGFR and cMET in tumors excised from an in vivo MKN45 xenograft model experiment. TAVO412 shown in Western blot referred to TAVO412E. [Figure 22C] Figure 22C shows a bar graph representation of the quantitative results for control isotype mAb and TAVO412E in Figure 22B. TAVO412E reduced the levels of cMET and all forms of EGFR in an in vivo MKN45 xenograft model experiment. [Figure 23A]Demonstration of the utility of TAVO412E in vitro in pancreatic ductal adenocarcinoma cell line BxPC-3 as shown by cell binding, ADCC reporter assay, and ADCP reporter assay. In FIG. 23A, the y-axis is gMFI of cell binding and the x-axis is the concentration of test article. In FIG. 23B-C, the y-axis is RLU of ADCC reporter probe assay response and the x-axis is the concentration of test article. FIG. 23A shows that TAVO412E had an EC50 value of 0.90 nM for binding to BxPC-3 cells. Isotype mAbs did not bind to BxPC-3 cells. [Figure 23B] Figure 23B shows that TAVO412E had an EC50 value of 0.20 nM in the ADCC reporter assay on BxPC-3 cells. The isotype mAb had no ADCC reporter assay activation on BxPC-3 cells. [Figure 23C] Figure 23C shows that TAVO412E had an EC50 value of 0.65 nM in the ADCP reporter assay on BxPC-3 cells. The isotype mAb showed no ADCP reporter assay activation on BxPC-3 cells. [Figure 24A] Figure 24 shows in vitro inhibition of EGFR and cMET phosphorylation in BxPC-3 cells. Experiments were performed similarly to those described in Example 12. In Figures 24A-D, the y-axis is shown as the value of percent receptor phosphorylation exhibited by the control mAb, and the x-axis is the concentration of the test substance. Figure 24A shows that TAVO412E inhibited EGFR phosphorylation in BxPC-3 cells in the presence of recombinant human EGF with an IC50 value of 3.45 nM. Isotype mAbs did not inhibit EGFR phosphorylation. [Figure 24B] Figure 24B shows that TAVO412E inhibited cMET phosphorylation in BxPC-3 cells in the presence of recombinant human HGF with an IC50 value of 1.18 nM. Isotype mAbs did not inhibit cMET phosphorylation. [Figure 24C]Figure 24C shows that TAVO412E inhibited EGFR phosphorylation in BxPC-3 cells in the presence of recombinant human EGF and recombinant human HGF with an IC50 value of 1.13 nM. Isotype mAbs did not inhibit EGFR phosphorylation. [Figure 24D] Figure 24D shows that TAVO412E inhibited cMET phosphorylation in BxPC-3 cells in the presence of recombinant human EGF and recombinant human HGF with an IC50 value of 0.44 nM. Isotype mAbs did not inhibit cMET phosphorylation. [Figure 25A] Figure 25 shows the in vivo antitumor activity of TAVO412E against the pancreatic ductal adenocarcinoma cell line BxPC-3. Figure 25A shows that TAVO412E treatment resulted in 80% BxPC-3 tumor growth inhibition at day 34 at 10 mg / kg. [Figure 25B] Figure 25B shows that TAVO412E induced degradation of EGFR and cMET in tumors in an in vivo BxPC-3 xenograft model experiment. TAVO412 shown in Western blot refers to TAVO412E. [Figure 25C] Figure 25C shows a bar graph representation of the results for control isotype mAb and TAVO412E in Figure 25B. TAVO412E reduced levels of cMET and all forms of EGFR in an in vivo BxPC-3 xenograft model experiment. [Figure 26A] Figure 26 shows the antitumor activity of TAVO412E against liver cancer cell line HCC9810 in vitro, triple negative breast cancer cell line HCC70 in vivo, and head and neck cancer cell line FaDu in vivo. Figure 26A shows that TAVO412E had ADCC activity against HCC9810 cell line with an EC50 value of 0.098nM. In Figure 26A, the y-axis is the percent lysis and the x-axis is the concentration of the test substance. [Figure 26B] FIG. 26B shows that TAVO412E treatment resulted in HCC-70 tumor growth inhibition at day 21 of 26% at the 10 mg / kg dose. [Figure 26C]FIG. 26C shows that TAVO412E treatment resulted in FaDu tumor growth inhibition at Day 21 of 95% at a dose of 10 mg / kg. [Figure 27A] Demonstration of the in vitro antitumor activity of TAVO412E in head and neck esophageal squamous cell carcinoma cell line KYSE-150 as shown by cell binding, ADCC reporter assay, and ADCC killing assay. In Figure 27A, the y-axis is the gMFI for cell binding, and the x-axis is the concentration of test substance. Figure 27A shows that TAVO412E has an EC50 value for binding to KYSE-150 cells of 0.39nM. Isotype mAb does not bind to KYSE-150 cells. [Figure 27B] In Figure 27B, the y-axis is the RLU of the ADCC reporter probe assay, and the x-axis is the concentration of the test substance. Figure 27B shows that TAVO412E had an EC50 value of 0.15nM for the ADCC reporter assay on KYSE-150 cells. Isotype mAb did not show ADCC reporter assay activation on KYSE-150 cells. [Figure 27C] In Figure 27C, the y-axis is percent lysis and the x-axis is the concentration of the test substance. Figure 27C shows that TAVO412E had an EC50 value of 0.038 nM for ADCC killing response against KYSE-150 cells. Isotype mAb had no ADCC killing response against KYSE-150 cells. [Figure 28A] Demonstration of TAVO412E antitumor in vitro activity in mesothelioma cancer cell line NCI-H226 as shown by cell binding, ADCC reporter assay, and ADCC killing assay. In Figure 28A, the y-axis is gMFI for cell binding, and the x-axis is the concentration of test substance. Figure 28A shows that TAVO412E had an EC50 value of 0.78nM for binding to NCI-H226 cells. Isotype mAb did not bind to NCI-H226 cells. [Figure 28B]In Figure 28B, the y-axis is the RLU of the ADCC reporter probe assay, and the x-axis is the concentration of the test substance. Figure 28B shows that TAVO412E had an EC50 value of 0.17nM for the ADCC reporter assay on NCI-H226 cells. Isotype mAb had no ADCC reporter assay activation on NCI-H226 cells. [Figure 28C] In Figure 28C, the y-axis is percent lysis and the x-axis is the concentration of the test substance. Figure 28C shows that TAVO412E had an EC50 value of 0.025 nM for ADCC killing of NCI-H226 cells. Isotype mAb did not show ADCC killing response against NCI-H226 cells. [Figure 29A] Demonstration of TAVO412E antitumor in vitro activity in colon cancer cell line HT-29 as shown by cell binding, ADCC reporter assay, and ADCC killing assay. In Figure 29A, the y-axis is the gMFI for cell binding, and the x-axis is the concentration of test substance. Figure 29A shows that TAVO412E has an EC50 value of 0.23nM for binding to HT-29 cells. Isotype mAb does not bind to HT-29 cells. [Figure 29B] In Figure 29B, the y-axis is the RLU of the ADCC reporter probe assay, and the x-axis is the concentration of the test substance. Figure 28B shows that TAVO412E had an EC50 value of 0.078nM for the ADCC reporter assay on HT-29 cells. Isotype mAb had no ADCC reporter assay activation on HT-29 cells. [Figure 29C] In Figure 29C, the y-axis is percent lysis and the x-axis is the concentration of the test substance. Figure 27C shows that TAVO412E had an EC50 value of 0.023 nM for ADCC killing response against HT-29 cells. Isotype mAb did not show ADCC killing response against HT-29 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] definition All publications cited in this specification, including but not limited to patents and patent applications, are hereby incorporated by reference as if fully set forth.

[0087] It is to be understood that the terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. 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 disclosure pertains.

[0088] Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present disclosure, exemplary materials and methods are described herein. In describing and claiming the present disclosure, the following terminology is used.

[0089] As used in this specification and the appended claims, the singular terms "a," "an," and "the" encompass plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0090] "Antibody(s)" or "antibody" is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, multispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen binding site of the required specificity.

[0091] A "full-length antibody molecule" comprises two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (V H ) and a heavy chain constant region (comprising domains CH1, hinge, CH2, and CH3). Each light chain comprises a light chain variable region (V L) and the light chain constant region (CL). H Area and V L The region is further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with framework regions (FRs). H and V L consists of three CDR and four FR portions, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0092] A "complementarity determining region (CDR)" is an "antigen binding site" in an antibody. CDRs are: (i) a complementarity determining region (CDR), H (HCDR1, HCDR2, HCDR3) and V L Three of the LCDR regions (LCDR1, LCDR2, and LCDR3) are defined based on sequence variability (Wu and Kabat 1970) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) "hypervariable region," "HVR," or "HV"; H Three of these (H1, H2, H3) and V L Three of these (L1, L2, L3) refer to antibody variable regions that are hypervariable as defined by Chothia and Lesk (Chothia and Lesk 1987). The International ImmunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definitions of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations has been described (Lefranc, Pommie et al. 2003). The terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include CDRs defined by any of the above methods, Kabat, Chothia or IMGT, unless expressly stated otherwise herein.

[0093] Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, depending on the heavy chain constant region amino acid sequence. IgA and IgG are further classified into isotypes, IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of its constant region.

[0094] An "antibody fragment," "antigen-binding fragment," or "antigen-binding portion" refers to an antibody that comprises heavy chain complementarity determining regions (HCDRs) 1, 2, and 3, light chain complementarity determining regions (LCDRs) 1, 2, and 3, heavy chain variable region (V H ), or the light chain variable region (V L Antibody fragments refer to a portion of an immunoglobulin molecule that retains the heavy and / or light chain antigen-binding sites, such as a single V (ab')2, Fd, and Fv fragment. Antibody fragments include the well-known Fab, F(ab')2, Fd, and Fv fragments, as well as a single V H Domain Antivirus (dAb) consists of domains. H and V L The domains are linked together via synthetic linkers to form various types of single chain antibody designs, including V H / V L The domains may be paired internally or in the middle, in which case V H and V L The domains can be expressed by separate single chain antibody constructs to form a monovalent antigen binding site, such as a single chain Fv (scFv) or diabody (e.g., as described in WO1998 / 44001, WO1988 / 01649, WO1994 / 13804, and WO1992 / 01047).

[0095] "Monoclonal antibody" refers to an antibody population having a single amino acid composition in each heavy and light chain, except for possible known modifications such as removal of the C-terminal lysine from the antibody heavy chain. A monoclonal antibody typically binds to one antigenic epitope, except that a multispecific monoclonal antibody binds to multiple, e.g., two, different antigenic epitopes. A monoclonal antibody may have heterogeneous glycosylation within the antibody population. A monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent, or multivalent. Multispecific antibodies are included in the term monoclonal antibody.

[0096] An "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigen specificities. An "isolated antibody" includes antibodies that have been isolated to greater degrees of purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0097] "Humanized antibody" refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from a human immunoglobulin sequence. Humanized antibodies may contain substitutions in the framework such that the framework is not an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0098] "Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and antigen binding sites are derived from sequences of human origin and are optimized to have a minimal immune response when administered to a human subject. If the antibody contains a constant region or a portion of a constant region, the constant region also is derived from sequences of human origin.

[0099] "Anti-target" refers to an antibody or antibody domain capable of binding to a specific target molecule such as EGFR (i.e., an anti-EGFR is an antibody or antibody domain capable of binding to EGFR). The style "EGFR" refers to the EGFR protein or EGFR gene product. The style "EGFR" refers to the EGFR gene.

[0100] "cMETxEGFRxPD-L1 / VEGF" refers to a multispecific antibody or antibody fragment capable of binding to cMET, EGFR, and PD-L1 or VEGF. The process of generating a multispecific antibody requires recombinant modification of the amino acid sequences of the parent mAbs. Although the amino acid sequences of the CH1, CL, and Fc domains of each parent mAb are not the same, there is no significant difference in binding between cMETxEGFR and EGFRxcMET multispecific antibodies. The cMETxEGFRxPD-L1 / VEGF multispecific may be a different structural isomer from PD-L1 / VEGF with a different structure-function activity profile.

[0101] Throughout this specification, the numbering of amino acid residues in antibody constant regions is according to the EU index as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise specified.

[0102] Conventional one-letter and three-letter amino acid codes are used herein, as set forth in Table 1.

[0103] [Table 2]

[0104] Polypeptides, nucleic acids, fusion proteins, and other compositions provided herein may include polypeptides, nucleic acids, fusion proteins, etc., having a recited percent identity to the amino acid or DNA sequences provided herein. The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by alignment and comparison of the sequences. "Percent identity", "percent homology", "sequence identity" or "sequence homology", etc., refer to the percent of identical residues between amino acids or nucleotides in the compared molecules, and are calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.In calculating percent identity, the sequences being compared are typically aligned in a manner that gives the maximum agreement between the sequences.

[0105] The constant region sequence of mammalian IgG heavy chains is designated in the sequence CH1-hinge-CH2-CH3. The IgG "hinge", "hinge region" or "hinge domain" is generally defined as including Glu216 and terminating at Pro230 in human IgG1 according to the EU index, but functionally the flexible part of the chain is considered to include additional residues called the upper and lower hinge regions, such as Glu216 to Gly237, the lower hinge being referred to as residues 233-239 of the Fc region, where FcγR binding is generally due. Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain S-S bonds. Although the boundaries may vary slightly, as numbered according to the EU index, the CH1 region is generally considered to be the V region of the immunoglobulin heavy chain molecule. H The Fc domain includes the first (most amino terminal) constant region of an immunoglobulin heavy chain, adjacent to the amino terminus of the hinge region and, for example, from about EU position 118-215. The Fc domain extends from amino acid 231 to amino acid 447; the CH2 domain is from about Ala231 to Lys340 or Gly341, and the CH3 is from about Gly341 or Gln342 to Lys447. The IgG heavy chain constant region residues of the CH1 region terminate with Lys. An Fc domain-containing molecule includes at least the CH2 and CH3 domains of an antibody constant region, and thus includes at least the region of the IgG heavy chain constant region from about Ala231 to Lys447. An Fc domain-containing molecule can optionally include at least a portion of a hinge region.

[0106] "Epitope" refers to a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active (polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes may be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. In the case of discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are in close proximity in three-dimensional space through folding of the protein molecule. Antibody "epitopes" depend on the methodology used to identify the epitope.

[0107] "Leader sequence" as used herein includes any signal peptide that can be processed by a mammalian cell, including the human B2M leader. Such sequences are well known in the art.

[0108] A "cleavable linker" is a peptide substrate that is cleavable by an enzyme. Operatively, the cleavable linker, when cleaved by an enzyme, allows activation of the present blocking antibody having a masking domain. Preferably, the cleavable linker is selected so that activation occurs at a desired site of action, which may be a site in or near a target cell (e.g., a carcinoma cell) or tissue. For example, the cleavable linker is a peptide substrate specific for an enzyme that is specific or highly expressed at the site of action, such that the cleavage rate of the cleavable linker at the target site is greater than the cleavage rate at sites other than the target site.

[0109] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The terms also include polypeptides having co-translational (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as, for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and the like.

[0110] Furthermore, as used herein, "polypeptide" refers to a protein containing modifications such as deletions, additions, and substitutions (generally conservative in nature, as known to those of skill in the art) to the native sequence, provided that the protein maintains a desired activity. These modifications may be deliberate, as by site-directed mutagenesis, or may be accidental, such as by mutations of hosts which produce the protein, or by errors resulting from PCR amplification or other recombinant DNA methods.

[0111] The term "masking domain" or "shielding" or "cap" in the present disclosure refers to a protein domain that can be fused to an antibody and can mask the antibody in binding to its antigen. The shielding domain can shield the antibody from recognizing its target epitope, so that the antibody remains in an inactive shielded antibody form. Removal of the shielding domain exposes the variable domain of the antibody, which can bind to and act on its target.

[0112] The term "recombinant," as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, which is not related by its origin or manipulation to all or a portion of the polynucleotide sequence associated in nature. The term "recombinant," as used with respect to a protein or polypeptide, refers to a polypeptide produced by expression from a recombinant polynucleotide. The term "recombinant," as used with respect to a host cell or virus, refers to a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein with respect to a material (e.g., a cell, a nucleic acid, a protein, or a vector) to refer to that the material has been modified by the introduction of heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).

[0113] The terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein to include polymeric forms of nucleotides, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule.

[0114] "Vector" refers to a polynucleotide that can be replicated within a biological system or transferred between such systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selection markers, which function to facilitate the replication or maintenance of these polynucleotides in biological systems (e.g., cells, viruses, animals, plants, and reconstituted biological systems that utilize biological components capable of replicating the vector). Vector polynucleotides can be DNA or RNA molecules, cDNA, or hybrids thereof, single-stranded or double-stranded.

[0115] An "expression vector" refers to a vector that can be utilized to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector in a biological system or engineered biological system.

[0116] As used herein, the term "heterologous" used in reference to a nucleic acid sequence, protein, or polypeptide means that these molecules do not naturally occur in the cell from which the heterologous nucleic acid sequence, protein, or polypeptide is derived. For example, a nucleic acid sequence encoding a human polypeptide inserted into a cell that is not a human cell is a heterologous nucleic acid sequence in that context. Heterologous nucleic acids can be derived from different organisms or animal species, but such nucleic acids do not have to be derived from separate organisms to be heterologous. For example, in some cases, a synthetic nucleic acid sequence or a polypeptide encoded therefrom can be heterologous to the cell into which it is introduced, in that the cell did not previously contain the synthetic nucleic acid. Thus, a synthetic nucleic acid sequence or a polypeptide encoded therefrom can be considered heterologous to a human cell, even if, for example, one or more components of the synthetic nucleic acid sequence or the polypeptide encoded therefrom are originally derived from a human cell.

[0117] "Host cell", as used herein, refers to an in vivo or in vitro eukaryotic cell, or a cell derived from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which can be or has been used as a recipient of a nucleic acid (e.g., an expression vector comprising a nucleotide sequence encoding a multimeric polypeptide of the present disclosure), and includes the progeny of the original cell genetically modified by the nucleic acid. It is understood that the progeny of a single cell will not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutation. A "recombinant host cell" (also called a "genetically modified host cell") is a host cell into which a heterologous nucleic acid, e.g., an expression vector, has been introduced. For example, a genetically modified eukaryotic host cell is genetically modified by introducing into a suitable eukaryotic host cell a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid not normally found in the eukaryotic host cell.

[0118] "Specific binding" or "specifically binds" or "binds" refers to an antibody that binds to a particular antigen with greater affinity than to other antigens. Typically, the equilibrium dissociation constant for binding (K D ) is about 1×10 -8 M or less, for example, about 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 M or less, typically its K for binding to a non-specific antigen (e.g., BSA, casein) D At least 100 times smaller than K D , and "specifically binds". D can be measured using standard procedures.

[0119] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in the sense of completely or partially preventing a disease or its symptoms, and / or therapeutic, in the sense of partially or completely curing a disease and / or side effects resulting from a disease. "Treatment", as used herein, encompasses any treatment of a disease in a mammal, e.g., a human, and includes (a) preventing a disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0120] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), and the like.

[0121] "Therapeutically effective amount" or "effective amount" refers to the amount of a drug, or the combined amount of two drugs, that when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease. A "therapeutically effective amount" varies depending on the drug, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0122] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to described embodiments, as such may, of course, vary, and the terminology used herein is for the purpose of describing embodiments only, and is not intended to be limiting.

[0123] The following examples provide further details that are not intended to limit the scope of the disclosure.

[0124] Multispecific antibody formats In some embodiments, the present disclosure provides multispecific antibodies that simultaneously target two or more of human cMET, EGFR, and PD-L1 / VEGF. In some embodiments, the multispecific antibodies comprise one or two sets of light chains and zero, one or two sets of heavy chains. The structures of the light and heavy chains from each parent antibody and the various multispecific formats are shown in Figure 5.

[0125] In some embodiments, the present disclosure provides shielding combinations that can form intermolecular interactions to block binding of the Fab arms to their respective epitopes. These intermolecular interactions can include association of a region of the heavy chain shielding fusion with a region of the light chain shielding fusion.

[0126] In some embodiments, the disclosure provides a multispecific antibody comprising a human IgG1 heavy chain fusion comprising, N to C-terminus: signal sequence A-shielding A-linker A-protease sequence A-linker B-IgG1 heavy chain; and N to C-terminus: signal sequence B-shielding B-linker B-protease sequence B-linker C-IgG1 light chain. In one embodiment, the human IgG1 heavy chain fusion comprises, N to C-terminus: signal sequence A-shielding A-linker A-protease sequence A-linker B-IgG1 heavy chain-SD; and the human IgG1 light chain fusion comprises, N to C-terminus: signal sequence A-shielding A-linker A-protease sequence A-linker B-IgG1 heavy chain-SD. SD refers to a single domain capable of binding to PD-L1 / VEGF (either PD-L1 or VEGF). Shielding A can be the same as or different from shielding B, linker A can be the same as or different from linker B, and protease sequence B can be the same as or different from protease sequence A.

[0127] In some embodiments, the disclosure provides multispecific antibodies comprising an anti-EGFR arm (e.g., an IgG1 heavy chain and / or an IgG1 light chain) that comprises a first variable domain that targets EGFR, an anti-cMET arm (e.g., an IgG1 heavy chain and / or an IgG1 light chain) that comprises a second variable domain that targets cMET, and an anti-PD-L1 arm that comprises a third variable domain that targets PD-L1, or an anti-VEGF arm that comprises a third variable domain that targets VEGF.

[0128] In some embodiments, the multispecific antibody comprises a monovalent binding arm capable of targeting EGFR comprising a human IgG1 heavy chain fusion comprising the N-terminus to C-terminus, signal sequence A-shielding-A linker A-protease sequence A-linker B-IgG1 heavy chain targeting EGFR-anti-PD-L1 or anti-VEGF; and a monovalent binding arm comprising a human IgG1 light chain fusion comprising the N-terminus to C-terminus, signal sequence B-shielding-B linker B-protease sequence B-linker C-IgG1 light chain targeting EGFR-anti-PD-L1 or anti-VEGF.

[0129] In some embodiments, the multispecific antibody comprises a monovalent binding arm capable of targeting cMET comprising a human IgG1 heavy chain fusion comprising the N- to C-terminus of signal sequence A-shielding-A linker A-protease sequence A-linker B-IgG1 heavy chain targeting cMET-anti-PD-L1 or anti-VEGF; and a monovalent binding arm comprising a human IgG1 light chain fusion comprising the N- to C-terminus of amino acid sequence signal sequence B-shielding-B linker B-protease sequence B-linker C-IgG1 light chain targeting cMET-anti-PD-L1 or anti-VEGF.

[0130] In some embodiments, the multispecific conjugate comprises one, two or more VHOs capable of binding monovalent or bivalent binding arms capable of targeting EGFR comprising a human IgG1 heavy chain fusion comprising from the N to C terminus: signal A sequence-A shielding-A linker A-protease sequence A-linker B-EGFR-C-Fc-anti-PD-L1 or anti-VEGF, where the two or more VHOs are optionally connected by one or more linkers or spacers.

[0131] In some embodiments, the multispecific antibody comprises, from N- to C-terminus, a monovalent or bivalent binding arm capable of targeting EGFR comprising a human IgG1 heavy chain fusion comprising signal sequence A, EGFR-linkerC-Fc-anti-PD-L1 or anti-VEGF, where the two or more VHOs are optionally connected by one or more linkers.

[0132] In some embodiments, the multispecific antibody comprises a monovalent binding arm capable of targeting cMET comprising a human IgG1 heavy and light chain fusion with one single domain anti-PD-L1 or anti-VEGF.

[0133] The present disclosure provides multispecific antibodies that can be generated using well-established point mutations in the CH1, CH2, and CH3 domains via controlled Fab arm exchange or via co-expression. In some embodiments, all constructs are symmetric, so that there is no preference in the selection of point mutations in each parent antibody.

[0134] Leader sequence In certain embodiments, the leader peptide is selected to drive secretion of the multispecific antibodies described in this disclosure into the cell culture supernatant as the respective secreted parent antibody proteins. Any leader peptide for any known secreted protein / peptide can be used.

[0135] As used herein, a "leader peptide" or "signal peptide" comprises a short peptide, usually 16-30 amino acids in length, that is present at the N-terminus of most newly synthesized proteins that are destined for the secretory pathway. Lead peptides are highly heterogeneous in sequence, and although many prokaryotic and eukaryotic lead peptides are functionally interchangeable between different species, the efficiency of protein secretion may be strongly determined by the sequence of the lead / signal peptide.

[0136] In certain embodiments, the leader peptide is derived from a protein that resides inside a particular organelle (such as the endoplasmic reticulum, Golgi, or endosome), is secreted from the cell, or is inserted into most cell membranes.

[0137] In certain embodiments, the leader peptide is derived from a eukaryotic protein.

[0138] In certain embodiments, the leader peptide is derived from a secreted protein, eg, a protein that is secreted outside the cell.

[0139] In certain embodiments, the leader peptide is derived from a transmembrane protein.

[0140] In certain embodiments, a leader peptide comprises a stretch of amino acids that is recognized and cleaved by a signal peptidase.

[0141] In certain embodiments, the leader peptide does not include a cleavage recognition sequence for a signal peptidase.

[0142] In certain embodiments, the leader peptide is a signal peptide of tissue plasminogen activator (tPA), herpes simplex virus glycoprotein D (HSV gD), growth hormone, cytokine, lipoprotein export signal, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR, or the S. cerevisiae mating factor alpha-1 signal peptide.

[0143] In some embodiments, a leader sequence described herein can be a mammalian CD4 or CD8 leader sequence, including, but not limited to, for example, a human CD4 or CD8 leader sequence, a non-human primate CD4 or CD8 leader sequence, a rodent CD4 or CD8 leader sequence, etc. In some embodiments, the CD4 or CD8 leader comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a human CD4 or CD8 leader sequence.

[0144] Anti-EGFR antibodies, anti-cMET antibodies, and multispecific antibodies In some embodiments, the disclosure provides therapeutic cMET, EGFR, and PD-L1 / VEGF antibodies and antigen-binding fragments. In some embodiments, the masking is attached to the Fab domains of each of the cMET, EGFR, and PD-L1 / VEGF antibodies and antigen-binding fragments via a protease-cleavable linker sequence to create a masked cMETxEGFRxPD-L1 / VEGF multispecific antibody. The cMET, EGFR, and PD-L1 / VEGF targets of such therapeutic antibodies have different expression levels in pathological sites and normal tissues. The masked cMETxEGFRxPD-L1 / VEGF multispecific antibody remains inactive in normal tissues due to the inhibitory effect of the masking domain on the CDR binding domains. The masking domain is cleaved by proteases at the disease site, and the masked cMETxEGFRxPD-L1 / VEGF multispecific antibody is converted to an active cMETxEGFRxPD-L1 / VEGF multispecific antibody.

[0145] In some embodiments, therapeutic antibodies and fragments applicable to the shielded cMETxEGFRxPD-L1 / VEGF multispecific antibody design of the present disclosure include full-length antibodies comprising two heavy chains and two light chains. The antibodies may be human or humanized. Humanized antibodies include chimeric and CDR-grafted antibodies. Chimeric antibodies are antibodies comprising a non-human antibody variable region linked to a human constant region. CDR-grafted antibodies are antibodies comprising CDRs from a non-human "donor" antibody linked to framework regions from a human "recipient" antibody. Exemplary human or humanized antibodies include IgG, IgM, IgE, IgA, and IgD antibodies. The antibodies may be of any class (such as IgG, IgM, IgE, IgA, IgD) or isotype. For example, a human antibody may comprise an IgG Fc domain, such as at least one of the isotypes IgG1, IgG2, IgG3, or IgG4.

[0146] In some embodiments, the disclosure provides human antibody heavy and light chain sequences that form the CDR binding regions that bind to cMET and EGFR, respectively.

[0147] In one aspect, the present disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof. In some embodiments, the present disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 95, 96, and 97; SEQ ID NOs: 95, 96, and 98; SEQ ID NOs: 95, 96, and 105; SEQ ID NOs: 102, 100, and 101; and SEQ ID NOs: 102, 103, and 104, respectively.

[0148] In some embodiments, the disclosure provides an anti-EGFR antibody, or antigen-binding fragment thereof, comprising at least one antibody single domain, or antigen-binding fragment thereof, selected from SEQ ID NOs: 5-12. In some embodiments, the anti-EGFR antibody, or antigen-binding fragment thereof, comprises a tandem antibody single domain heavy chain, or antigen-binding fragment thereof, selected from SEQ ID NOs: 13-18, in which two EGFR-binding VHO sequences are linked via a linker.

[0149] In some embodiments, the disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof comprising at least one antibody single domain having at least 85% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs:5-12 or antigen-binding fragments thereof. In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof comprises a tandem antibody single domain heavy chain having at least 85% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs:13-18 or antigen-binding fragments thereof.

[0150] In some embodiments, the disclosure provides an anti-EGFR antibody, or antigen-binding fragment thereof, that binds to one or more epitopes on human EGFR recognized by the anti-EGFR antibody, or antigen-binding fragment thereof, comprising at least one antibody single domain selected from SEQ ID NOs:5-12, or comprising a tandem antibody single domain heavy chain selected from SEQ ID NOs:13-18.

[0151] In some embodiments, the disclosure provides an anti-EGFR antibody or antigen-binding fragment thereof comprising a human antibody heavy chain SEQ ID NO:1 and a human antibody light chain SEQ ID NO:2; or a human antibody heavy chain SEQ ID NO:3 and a human antibody light chain SEQ ID NO:4.

[0152] As non-limiting examples, the present disclosure provides anti-EGFR heavy and light chain variable region amino acid sequences shown as SEQ ID NOs: 1-18, with the specific CDRs shown in Table 2.

[0153] [Table 3-1]

[0154] [Table 3-2]

[0155] [Table 3-3]

[0156] [Table 3-4]

[0157] In some embodiments, the multispecific antibody disclosed herein comprises an anti-EGFR antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 95, 96, and 97; SEQ ID NOs: 95, 96, and 98; SEQ ID NOs: 95, 96, and 105; SEQ ID NOs: 102, 100, and 101; and SEQ ID NOs: 102, 103, and 104, respectively.

[0158] In some embodiments, the anti-EGFR arm of a multispecific antibody disclosed herein comprises human antibody heavy chain SEQ ID NO:1 and human antibody light chain SEQ ID NO:2; human antibody heavy chain SEQ ID NO:3 and human antibody light chain SEQ ID NO:4. In some embodiments, a multispecific antibody of the disclosure comprises an EGFR-binding VHO sequence selected from SEQ ID NO:5-12 linked to an Fc using a linker selected from SEQ ID NO:19-22. In some embodiments, a multispecific antibody of the disclosure comprises an EGFR-binding VHO sequence selected from SEQ ID NO:13-18 linked to an Fc using a linker selected from SEQ ID NO:19-22. The selection of the linker sequence shown in SEQ ID NO:20 allows for a favorable developability profile.

[0159] [Table 4]

[0160] In another aspect, the disclosure provides an anti-cMET antibody or antigen-binding fragment thereof. In some embodiments, the disclosure provides a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are set forth in SEQ ID NOs: 106, 107, and 133; SEQ ID NOs: 111, 112, and 113; SEQ ID NOs: 111, 114, and 113; SEQ ID NOs: 99, 118, and 119; SEQ ID NOs: 99, 120, and 119; SEQ ID NOs: 99, 121, and 122; 19; a heavy chain variable region selected from SEQ ID NOs: 99, 122, and 119; and a light chain variable region comprising three CDRs designated LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NOs: 108, 109, and 110; SEQ ID NOs: 115, 116, and 117; and SEQ ID NOs: 123, 124, and 125, respectively.

[0161] In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, comprising a human antibody heavy chain sequence selected from SEQ ID NOs: 23, 24, 27-29, and 33-37, and a human antibody light chain sequence selected from SEQ ID NOs: 25, 26, 30-32, and 38-40. In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, comprising at least one cMET-binding VHO sequence selected from SEQ ID NOs: 41-44.

[0162] In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, comprising a human antibody heavy chain sequence having at least 85% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs: 23, 24, 27-29, and 33-37, and a human antibody light chain sequence having at least 85% identity to any one of SEQ ID NOs: 25, 26, 30-32, and 38-40. In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, comprising at least one cMET-binding VHO sequence having at least 85% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs: 41-44.

[0163] In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, that binds to one or more epitopes on cMET recognized by an anti-cMET antibody, or antigen-binding fragment thereof, comprising an antibody heavy chain sequence selected from SEQ ID NOs: 23, 24, 27-29, and 33-37, and an antibody light chain sequence selected from SEQ ID NOs: 25, 26, 30-32, and 38-40. In some embodiments, the disclosure provides an anti-cMET antibody, or antigen-binding fragment thereof, that binds to one or more epitopes on cMET recognized by an anti-cMET antibody, or antigen-binding fragment thereof, comprising at least one cMET-binding VHO sequence selected from SEQ ID NOs: 41-44.

[0164] In some embodiments, the disclosure provides a method for the preparation of human antibody heavy chain SEQ ID NO:23 and human antibody light chain SEQ ID NO:25; human antibody heavy chain SEQ ID NO:23 and human antibody light chain SEQ ID NO:26; human antibody heavy chain SEQ ID NO:24 and human antibody light chain SEQ ID NO:25; human antibody heavy chain SEQ ID NO:24 and human antibody light chain SEQ ID NO:26; human antibody heavy chain SEQ ID NO:27 and human antibody light chain SEQ ID NO:30; human antibody heavy chain SEQ ID NO:27 and human antibody light chain SEQ ID NO:31; human antibody heavy chain SEQ ID NO:27 and human antibody light chain SEQ ID NO:32; human antibody heavy chain SEQ ID NO:28 and human antibody light chain SEQ ID NO:30; human antibody heavy chain SEQ ID NO:28 and human antibody light chain SEQ ID NO:31; human antibody heavy chain SEQ ID NO:28 and human antibody light chain SEQ ID NO:32; human antibody heavy chain SEQ ID NO:29 and human antibody light chain SEQ ID NO:30; human antibody heavy chain SEQ ID NO:29 and human antibody light chain SEQ ID NO:31; human antibody heavy chain SEQ ID NO:29 and human antibody light chain SEQ ID NO:32; human antibody heavy chain SEQ ID NO:33 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:33 and human antibody light chain SEQ ID NO:38 human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:33 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:34 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:34 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:34 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:35 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:35 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:35 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:37 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:37 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:37 and human antibody light chain SEQ ID NO:40.

[0165] As non-limiting examples, the disclosure provides anti-cMET heavy and light chain variable region amino acid sequences set forth as SEQ ID NOs:23-44, and the specific CDRs set forth in Table 4.

[0166] [Table 5-1]

[0167] [Table 5-2]

[0168] [Table 5-3]

[0169] In some embodiments, the multispecific antibodies disclosed herein comprise a heavy chain variable region comprising three complementarity determining regions (CDRs), designated HCDR1, HCDR2, and HCDR3, which are set forth in SEQ ID NOs: 106, 107, and 133; SEQ ID NOs: 111, 112, and 113; SEQ ID NOs: 111, 114, and 113; SEQ ID NOs: 99, 118, and 119; SEQ ID NOs: 99, 120, and 119; SEQ ID NO: 99, and a light chain variable region comprising three CDRs designated LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NOs: 108, 109, and 110; SEQ ID NOs: 115, 116, and 117; and SEQ ID NOs: 123, 124, and 125, respectively.

[0170] In some embodiments, the anti-cMET arm of a multispecific antibody disclosed herein is represented by the following sequences: human antibody heavy chain SEQ ID NO:23 and human antibody light chain SEQ ID NO:25; human antibody heavy chain SEQ ID NO:23 and human antibody light chain SEQ ID NO:26; human antibody heavy chain SEQ ID NO:24 and human antibody light chain SEQ ID NO:25; human antibody heavy chain SEQ ID NO:24 and human antibody light chain SEQ ID NO:26; human antibody heavy chain SEQ ID NO:27 and human antibody light chain SEQ ID NO:30; human antibody heavy chain SEQ ID NO:27 and human antibody light chain SEQ ID NO:31; No. 31; human antibody heavy chain SEQ ID NO: 27 and human antibody light chain SEQ ID NO: 32; human antibody heavy chain SEQ ID NO: 28 and human antibody light chain SEQ ID NO: 30; human antibody heavy chain SEQ ID NO: 28 and human antibody light chain SEQ ID NO: 31; human antibody heavy chain SEQ ID NO: 28 and human antibody light chain SEQ ID NO: 32; human antibody heavy chain SEQ ID NO: 29 and human antibody light chain SEQ ID NO: 30; human antibody heavy chain SEQ ID NO: 29 and human antibody light chain SEQ ID NO: 31; human antibody heavy chain SEQ ID NO: 29 and human antibody light chain SEQ ID NO: 32; human antibody heavy chain sequences SEQ ID NO:33 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:33 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:33 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:34 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:34 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:34 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:35 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:35 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:35 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:39; human antibody heavy chain SEQ ID NO:36 and human antibody light chain SEQ ID NO:40; human antibody heavy chain SEQ ID NO:37 and human antibody light chain SEQ ID NO:38; human antibody heavy chain SEQ ID NO:37 and human antibody light chain SEQ ID NO:39; or human antibody heavy chain SEQ ID NO:37 and human antibody light chain SEQ ID NO:40.

[0171] In some embodiments, a multispecific antibody of the disclosure comprises a cMET-binding VHO sequence selected from SEQ ID NOs: 41-44, linked to an Fc using a linker selected from SEQ ID NOs: 19-22.

[0172] shielding In some embodiments of the present disclosure, the therapeutic multispecific cMETxEGFR antibody comprises an anti-cMET antibody arm comprising a masking domain and an anti-EGFR antibody arm comprising a masking domain. Long-term administration of anti-cMET or anti-EGFR biologics is a major risk factor for patients. Therefore, the safety profile and therapeutic window of each antibody arm can be increased by converting the anti-cMET and / or anti-EGFR antibody arm into a shielded arm with a masking domain.

[0173] Shielding or masking domains are sequences that can shield the multispecific antibody CDRs from binding to cMET and EGFR. As non-limiting examples, the present disclosure provides shielding or masking peptide sequences shown as SEQ ID NOs: 45-51. In some embodiments, SEQ ID NO: 45 pairs with SEQ ID NO: 48; SEQ ID NO: 46 pairs with SEQ ID NO: 48; SEQ ID NO: 47 pairs with SEQ ID NO: 48; SEQ ID NO: 50 pairs with SEQ ID NO: 51, and SEQ ID NO: 49 can be paired with itself as either an N-terminal heavy chain or an N-terminal light chain fusion.

[0174] [Table 6]

[0175] In some embodiments, the disclosure provides a heavy chain variable region that can be used as a shielding domain, comprising three complementarity determining regions (CDRs), termed HCDR1, HCDR2, and HCDR3, where HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 134, 135, and 136; SEQ ID NOs: 134, 135, and 137; SEQ ID NOs: 134, 138, and 136; SEQ ID NOs: 134, 138, and 137; SEQ ID NOs: 139, 135, and 136; SEQ ID NOs: 139, 138, and 137; and SEQ ID NOs: 140, 141, and 136, respectively.

[0176] As non-limiting examples, the disclosure provides the amino acid sequences of the shielding domains shown as SEQ ID NOs: 5-12 or 13-18 for EGFR VHO mAbs and either SEQ ID NOs: 41-44 for cMET VHO mAbs or SEQ ID NOs: 23-40 for cMET mAbs.

[0177] [Table 7-1]

[0178] [Table 7-2]

[0179] As a non-limiting example, the shielding domain can be fused to the N- or C-terminus of the anti-EGFR and anti-cMET binding arms using the linkers listed in SEQ ID NOs: 19-22.

[0180] Protease-cleavable linker The protease cleavable linker that links the shielding domain to the antibody heavy or light chain is a peptide substrate that can be cleaved by a protease. This sequence includes one or more protease substrate sequences and an optional linker spacer sequence. The shielding sequence exists as a pair of sequences that can be fused to either the heavy or light chain. For each of the two Fab arm domains of the antibody, the shielding sequence is fused to the N-terminus of the antibody heavy chain via one protease cleavable linker, and the complementary sequence is fused to the N-terminus of the antibody light chain via another protease cleavable linker. Alternatively, linkers can be used to link single domain anti-EGFR, anti-cMET, anti-VEGF, and anti-PD-L1 molecules together.

[0181] Many disease tissues, including tumor microenvironments and inflammatory sites, are abundant with various types of proteases whose overexpression correlates with disease progression. In disease tissues, the protease-cleavable linker sequence of the shielded antibody is recognized by the appropriate type of protease, which releases the shielding from the antibody chain. In some embodiments, the protease can cleave one of the two protease-cleavable linkers or the two protease-cleavable linker sequences, and thus the shielding domain is inactive. In either case, the shielding domain cannot interfere with or block the binding of the Fab arm to its target antigen. As a result, the shielded antibody is converted into an active antibody that binds to its target and exerts its functional activity.

[0182] In some embodiments, the protease-cleavable linker sequences connecting the two shielding domains to the two Fab domains in a shielding antibody comprise the same sequence that is cleaved by the same type of protease.

[0183] In some embodiments, the protease-cleavable linker sequence connecting the two masking domains and the two Fab domains in a shielding antibody comprises different sequences having substrate sequences that are cleaved by different types of proteases.

[0184] Among the matrix metalloproteinase (MMP) family, MMP2 and MMP9 are upregulated in many types of cancer, including breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, and lung cancer. Moreover, the expression and activity of MMP2 and MMP9 also correlate with the progression of many autoimmune and inflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, chronic obstructive pulmonary disease, inflammatory bowel disease, and osteoporosis (Lin, Lu et al., 2020). The present disclosure provides protease-cleavable linker sequences, including substrate peptide sequences cleaved by MMP2 and MMP9. As a non-limiting example, the present disclosure provides MMP2 and MMP9 cleavable substrate peptide sequences shown as SEQ ID NOs: 62-66. As a non-limiting example, the present disclosure provides MMP3 cleavable substrate peptide sequence shown as SEQ ID NO: 67.

[0185] Urokinase plasminogen activator (uPA) has been reported to be overexpressed in many types of cancer, especially breast cancer (B Anys-Paluchowski, Witzel et al. 2019). uPA is a serine protease that can catalyze the conversion of plasminogen to plasmin, which can degrade basement membranes or extracellular matrices. Degradation of the matrix can promote tumor cell migration and invasion into surrounding tissues. The present disclosure provides protease-cleavable linker sequences that include substrate peptide sequences cleaved by uPA. As a non-limiting example, the present disclosure provides uPA-cleavable substrate peptide sequences shown as SEQ ID NOs: 68 and 69.

[0186] [Table 8]

[0187] Protease-cleavable linkers of the disclosure can include, for example, one or more linker peptides interposed between the masking sequence and the protease substrate peptide sequence and / or between the protease substrate peptide sequence and the antibody chain.

[0188] Linker Suitable linkers (also called "spacers") can be readily selected and can be any of a number of suitable lengths, such as from 1 amino acid to 30 amino acids (e.g., any particular integer between 1 and 30, or from 1 amino acid (e.g., Gly) to about 20 amino acids, 2 to 15, 3 to 12, 4 to 10, 5 to 9, 6 to 8, or 7 to 8 amino acids). Exemplary linkers are shown in Table 3.

[0189] An exemplary linker is a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n and (GGGS) n where n is at least one integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20), glycine-alanine polymers, alanine-serine polymers, alanine-proline polymers, immunoglobulin isotype and subtype hinges (which may include IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM), and other flexible linkers known in the art. Both Gly and Ser are relatively unstructured and thus function as neutral tethers between components.

[0190] In certain embodiments, the linker is a glycine polymer. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (Scheraga 2008). Exemplary linkers may include amino acid sequences including, but not limited to, GGS; GGSG; GGSGG; GGGGS; GGSSG; GGGSG; GGSSG; GSSSG, and the like.

[0191] In certain embodiments, the linker is an alanine-proline polymer. An exemplary linker is: (AP) nwhere n is at least one integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0192] In certain embodiments, the linker is a rigid linker (Chen, Zaro et al. 2013). An exemplary rigid linker is a proline-rich sequence (XP). n X may comprise an amino acid sequence including, but not limited to, (EAAAK), where X represents any amino acid, preferably Ala, Lys, or Glu, and where n is at least one integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. Exemplary rigid linkers also include (EAAAK) n wherein n is at least one integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0193] Anti-PD-L1 antibody and anti-VEGF antibody In specific embodiments, an immunomodulatory domain of the disclosure is a PD-L1 polypeptide. Optionally, the PD-L1 polypeptide of a multimeric polypeptide of the disclosure comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 19-290 of SEQ ID NO:70 of the PD-L1 amino acid sequence. FTVTVPKDLYVVYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (sequence number 70).

[0194] In certain embodiments, suitable immunomodulatory domains of the present disclosure include PD-L1 peptides, Ig variable domains of anti-PD-L1, or scFv formats. Optionally, the single chain Fv polypeptide of an anti-PD-L1 antibody of a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the single chain Fv polypeptide of an anti-PD-L1 antibody as SEQ ID NO:71 or SEQ ID NO:72.

[0195] In some embodiments, the disclosure provides an anti-VEGF antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) designated HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 129, 130, and 131; and a light chain variable region comprising three CDRs designated LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 are SEQ ID NOs: 126, 127, and 128, respectively.

[0196] In some cases, the single chain Fv polypeptide of an anti-VEGF antibody of a multimeric polypeptide of the disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the single chain Fv polypeptide of an anti-VEGF antibody set forth in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:76.

[0197] In some embodiments, the multispecific antibody disclosed herein comprises an anti-VEGF antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three complementarity determining regions (CDRs), designated HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 129, 130, and 131; SEQ ID NOs: 129, 132, and 131; and a light chain variable region comprising three CDRs, designated LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 are SEQ ID NOs: 126, 127, and 128, respectively.

[0198] In some embodiments, the disclosure provides a control bispecific Ab Fv sequence for amivantamab: the EGFR binding arm is set forth in SEQ ID NO: 79 for the heavy chain Fv and SEQ ID NO: 80 for the light chain Fv; the cMET binding arm is set forth in SEQ ID NO: 77 for the heavy chain Fv and SEQ ID NO: 78 for the light chain Fv. A null control Ab Fv sequence for anti-gp120 mAb v12 comprises a heavy chain Fv (SEQ ID NO: 82) and a light chain (set forth in SEQ ID NO: 81).

[0199] [Table 9-1]

[0200] [Table 9-2]

[0201] [Table 9-3]

[0202] In some embodiments, a multispecific antibody, such as a shielded cMETxEGFR multispecific antibody, may comprise a modified Fc region, where the modified Fc region comprises at least one amino acid modification relative to the native Fc region. In some embodiments, a multispecific antibody, such as a shielded cMETxEGFR multispecific antibody, is provided with a modified Fc region, where the naturally occurring Fc region is modified to extend the half-life of the antibody, e.g., serum half-life or half-life as measured by an in vitro assay, when compared to the parent native antibody in a biological environment. Exemplary mutations that may be made alone or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.

[0203] In certain embodiments, increased half-life can be achieved by engineering M252Y / S254T / T256E mutations in the IgG1 Fc residue numbering according to the EU index (Dall'Acqua, Kiener et al. 2006).

[0204] In certain embodiments, increased half-life can also be achieved by engineering M428L / N434S mutations in the IgG1 Fc (Zalevsky, Chamberlain et al. 2010).

[0205] In certain embodiments, increased half-life can also be achieved by engineering T250Q / M428L mutations in the IgG1 Fc (Hinton, Xiong et al. 2006).

[0206] In certain embodiments, increased half-life can also be achieved by engineering the N434A mutation in the IgG1 Fc (Shields, Namenuk et al. 20011).

[0207] In certain embodiments, increased half-life can also be achieved by engineering T307A / E380A / N434A mutations in the IgG1 Fc (Petkova, Akilesh et al. 2006).

[0208] The efficacy of Fc engineering on extending antibody half-life can be evaluated in PK studies in mice compared to antibodies with native IgG Fc.

[0209] In some embodiments, the shielded cMETxEGFR multispecific antibody has a naturally occurring Fc region modified to provide a modified Fc region that enhances the antibody resistance to proteolysis by proteases that cleave the wild-type antibody between or at residues 222-237 (EU numbering).

[0210] In certain embodiments, resistance to proteolysis can be achieved by engineering an E233P mutation in the hinge region that deletes G236 when compared to the parent native antibody, with residue numbering according to the EU index (Kinder, Greenplate et al. 2013).

[0211] If effector function is undesirable, the antibodies of the disclosure can be further engineered to introduce at least one mutation in the antibody Fc that reduces binding of the antibody to an activating Fc receptor (FcR) and / or reduces an Fc effector function, such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).

[0212] Fc positions that can be mutated to reduce antibody binding to activating FcRs and subsequently reduce effector function are those described, for example, in (Xu, Alegre et al. 2000) (Vafa, Gilliland et al. 2014) (Vafa, Gilliland et al. 2014) (Bolt, Routledge et al. 1993, Shields, Namenuk et al. 2001, Chu, Vostiar et al. 2008). Fc mutations with minimal ADCC, ADCP, CDC, and / or Fc-mediated cell activation have also been described as sigma mutations of IgG1, IgG2 and IgG4 (Tam, McCarthy et al. 2017). Exemplary mutations, which may be made alone or in combination, are K214T, E233P, L234V, L234A, G236 deletion, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S mutations in IgG1, IgG2, IgG3 or IgG4.

[0213] Exemplary combination mutations that can be made to decrease ADCC include L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgG1, IgG2, IgG3 or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327 on IgG1, These are G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgG1, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4. A hybrid IgG2 / 4 Fc region may also be used, such as an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.

[0214] In some embodiments, shielded cMETxEGFR multispecific antibodies are provided with modified Fc regions, in which the naturally occurring Fc region has been modified to promote the generation of multispecific antibodies by Fc heterodimerization.

[0215] In certain embodiments, Fc heterodimerization can be achieved by engineering F405L and K409R mutations on two parent antibodies to generate multispecific antibodies in a process known as Fab arm exchange (Labrijn, Meesters et al. 2014).

[0216] In certain embodiments, Fc heterodimerization can also be achieved by Fc mutations to promote a knob-in-hole strategy (see, for example, WO2006 / 028936): an amino acid with a small side chain (hole) is introduced into one Fc domain and an amino acid with a large side chain (knob) is introduced into the other Fc domain. After co-expression of the two heavy chains, heterodimers form as a result of preferential interaction of the heavy chain with the "hole" with the heavy chain with the "knob" (Ridgway, Presta et al. 1996). Exemplary Fc mutation pairs forming knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S / L368A / Y407V. Instead of co-expression, controlled Fab arm exchange can be applied to generate multispecific antibodies from separate transfection and purification of the corresponding parent antibodies.

[0217] In certain embodiments, Fc heterodimerization can also be achieved by Fc mutations to promote electrostatically matched interaction strategies (Gunasekaran, Pentony et al. 2010). Mutations can be engineered to generate positively charged residues in one Fc domain and negatively charged residues in the other Fc domain, as described in US Patent Application Publication Nos. 2010 / 0015133; 2009 / 0182127; 2010 / 028637 or 2011 / 0123532. Heavy chain heterodimerization is formed by electrostatically matched interactions between two mutant Fcs.

[0218] In some embodiments, the shielded cMETxEGFRxPD-L1 / VEGF multispecific antibody is provided with a modified Fc region, where the naturally occurring Fc region is modified to promote antibody multimerization upon interaction with a cell surface receptor, but such engineered antibodies exist as monomers in solution. Fc mutations that facilitate antibody multimerization include, but are not limited to, E345R, E430G, E345R / E430G, and E345R / E430G / Y440R mutations, as described in (Diebolder, Beurskens et al. 2014). Such mutations may also include, but are not limited to, T437R, T437R / K248E, and T437R / K338A mutations, as described in (Zhang, Armstrong et al. 2017).

[0219] Antibodies of the present disclosure that further comprise conservative modifications are within the scope of the present disclosure. "Conservative modifications" refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative substitutions are those in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Additionally, any naturally occurring residue in the polypeptide can be substituted with alanine, as previously described for alanine scanning mutagenesis. Amino acid substitutions to the antibodies of the present disclosure can be made by known methods, for example, PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, libraries of variants can be generated using, for example, random (NNK) or non-random codons, such as DVK codons, that code for 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants can be tested for their characteristics using the assays described herein.

[0220] The antibodies of the present disclosure can be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, and / or non-naturally occurring covalent modifications such as the addition of polyethylene glycol moieties (PEGylation) and lipidation. Such modifications can occur in vivo or in vitro. For example, the antibodies of the present disclosure can be conjugated to polyethylene glycol (PEGylated) to improve their pharmacokinetic profile. Conjugation can be performed by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics without interfering with function.

[0221] The antibodies of the present disclosure may be modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity, and / or other desirable biological or biophysical properties. Antibody stability is influenced by many factors, including (1) core packing of individual domains, which affects intrinsic stability, (2) protein / protein interface interactions, which affect HC and LC pairing, (3) burial of polar and charged residues, (4) H-bond network of polar and charged residues, and (5) distribution of surface charge and polar residues among other intra- and intermolecular forces (Worn and Pluckthun 2001). Potential structure-destabilizing residues may be identified based on the crystal structure of the antibody, or in certain cases by molecular modeling, and the effect of the residues on antibody stability may be tested by generating and evaluating modifications with mutations at the identified residues. Thermal transition midpoints (T) measured by differential scanning calorimetry may be used to determine the stability of the antibody. m ) is one way to increase antibody stability. m The degree of degradation correlates with its stability and inversely correlates with its susceptibility to unfolding and denaturation in solution and with the degradation process, which depends on the tendency of the protein to unfold. Several studies have found a correlation between the ranking of the physical stability of formulations as measured by DSC as thermal stability and physical stability measured by other methods. Formulation studies have shown that Fab T m It has been suggested that this may have implications for the long-term physical stability of the corresponding mAb.

[0222] The antibodies of the present disclosure may have amino acid substitutions in the Fc region that improve manufacturing and drug stability. For example, IgG1 has a H224S (or H224Q) at hinge 221-DKTHTC-226 (European numbering) mutation to prevent rapid induced cleavage. IgG4 has a S228P mutation to prevent half-antibody exchange.

[0223] Trispecific cMet×EGFR×VEGF antibody The amino acid sequences of the specific components for making the cMetxEGFRxVEGF molecule are illustrated in the table below.

[0224] [Table 10-1]

[0225] [Table 10-2]

[0226] [Table 10-3]

[0227] [Table 10-4]

[0228] The amino acid sequence composition of certain multispecific agents is highlighted in the table below. The sequences of TAVO412A through TAVO412H are prepared via co-expression of open reading frames as shown in the table below.

[0229] [Table 11]

[0230] Antibody expression and purification In some embodiments, an antibody, such as a multispecific antibody of the disclosure, can be encoded by a single nucleic acid (e.g., a single nucleic acid comprising nucleotide sequences encoding light and heavy chain polypeptides of the multispecific antibody) or by two or more separate nucleic acids, each encoding a different portion of a parent antibody.

[0231] The nucleic acids described herein can be inserted into a vector, e.g., a nucleic acid expression vector and / or a targeting vector. Such vectors can be used in a variety of ways, e.g., for expression of the shielded antibodies with masking domains described herein in cells or transgenic animals. The vector is typically selected to be functional in the host cell in which it is used. The nucleic acid molecules encoding the shielded antibodies with masking domains described herein can be amplified / expressed in prokaryotic, yeast, insect (baculovirus systems) and / or eukaryotic host cells. The choice of host cell depends, in part, on whether the multispecific antibodies described herein are post-translationally modified (e.g., glycosylated and / or phosphorylated). If so, yeast, insect, or mammalian host cells are preferred. Expression vectors typically contain one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element.

[0232] In most cases, a leader or signal sequence is engineered at the N-terminus of the shielded cMETxEGFRxPD-L1 / VEGF multispecific antibody described herein to direct its secretion. Secretion of the shielded cMETxEGFRxPD-L1 / VEGF multispecific antibody from a host cell results in removal of the signal peptide from the antibody. Thus, the mature shielded cMETxEGFRxPD-L1 / VEGF multispecific antibody lacks any leader or signal sequence. In some cases, such as when glycosylation is desired in a eukaryotic host cell expression system, various presequences can be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of the signal peptide can be changed or a presequence can be added, which can also affect glycosylation.

[0233] The present disclosure further provides a cell (e.g., an isolated or purified cell) comprising a nucleic acid or vector of the present disclosure. The cell may be any type of cell that can be transformed with a nucleic acid or vector of the present disclosure to produce the polypeptide encoded thereby. To express the shielded cMETxEGFRxPD-L1 / VEGF multispecific antibodies described herein, DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences.

[0234] Methods for introducing nucleic acids and vectors into isolated cells, and for culturing and selecting in vitro transformed host cells, are known in the art and include the use of calcium chloride mediated transformation, transduction, conjugation, tripartite conjugation, DEAE, dextran mediated transfection, infection, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, direct microinjection into single cells, and electroporation.

[0235] After introducing a nucleic acid or vector of this disclosure into a cell, the cell is cultured under conditions appropriate for expression of the encoded sequence. The antibody, antigen-binding fragment, or portion of the antibody can then be isolated from the cell.

[0236] In certain embodiments, two or more vectors that together encode a shielded cMETxEGFRxPD-L1 / VEGF multispecific antibody as described herein may be introduced into a cell.

[0237] In certain embodiments, purification of the masked cMETxEGFRxPD-L1 / VEGF multispecific antibodies described herein that have been secreted into the cell culture medium can be achieved using a variety of techniques, including affinity chromatography, immunoaffinity or ion exchange chromatography, molecular sieve chromatography, preparative gel electrophoresis or isoelectric focusing, chromatofocusing, and high pressure liquid chromatography. For example, antibodies that include an Fc region can be purified by affinity chromatography with Protein A, which selectively binds to the Fc region.

[0238] Modified forms of the shielded cMETxEGFRxPD-L1 / VEGF multispecific antibodies can be prepared with affinity tags, such as hexahistidine or other small peptides, e.g., FLAG (Eastman Kodak Co., New Haven, Conn.) or Myc (Invitrogen), at either their C-terminus or N-terminus, and purified by one-step affinity columns. For example, polyhistidine binds with great affinity and specificity to nickel, and thus a nickel affinity column (such as a Qiagen® nickel column) can be used for purification of polyhistidine-tagged selective binding agents. In some cases, one or more purification steps may be used.

[0239] Effects on binding and functional activity of multispecific antibodies In some embodiments, the masking domain on the shielded cMETxEGFRxPD-L1 / VEGF multispecific antibody can inhibit or block the ability of the Fab arms to bind to their respective antigens, cMET, EGFR, and PD-L1 / VEGF. The masking domain can reduce the maximum binding capacity of the shielded multispecific antibody in binding to its respective antigen. The masking domain can also reduce the binding affinity of the shielded multispecific antibody in binding to its respective antigen.

[0240] When the masking domain is cleaved by the protease, the masked antibody is converted into an active multispecific antibody with the antibody's ability to bind to its target restored. Removal of the masking domain from the masked multispecific antibody can be achieved by in vitro protease cleavage assays using recombinant or purified proteases. Removal of the masking domain from the masked multispecific antibody can also be achieved in vivo by overexpressed proteases at the disease site. Removal of the masking domain can be assessed by comparing the molecular weight of the heavy and light chains of the masked antibody with the masking domain to an active antibody without the masking domain by SDS-PAGE, IEX, or HIC analysis.

[0241] In vitro and cell-based assays are well described in the art for use in determining blocked antibodies, active antibodies, and converted antibodies after protease cleavage upon binding to their antigen. For example, antibody binding can be determined by ELISA by immobilizing recombinant or purified antigen, sequestering the antibody with the immobilized antigen, and measuring the amount of bound antibody. This can also be performed using a Biacore® instrument for kinetic analysis of binding interactions. In cell-based binding assays, antibody binding can be determined by flow cytometry by incubating the antibody with cells expressing the antigen on their cell surface and measuring the amount of antibody bound to the cell surface antigen.

[0242] Pharmaceutical Compositions In some embodiments, the antibodies of the present disclosure, e.g., shielded cMETxEGFRxPD-L1 / VEGF multispecific antibodies for use according to the present disclosure, can be formulated into compositions, particularly pharmaceutical compositions, for use in the methods herein. Such compositions comprise a therapeutically or prophylactically effective amount of the multispecific antibodies described in this disclosure in admixture with a suitable carrier (e.g., a pharma- ceutically acceptable agent). Typically, the multispecific antibodies described in this disclosure are sufficiently purified for administration to animals prior to formulation in a pharmaceutical composition.

[0243] Pharmaceutically acceptable agents include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavoring agents and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonicity agents, co-solvents, wetting agents, complexing agents, buffers, antibacterial agents, and surfactants.

[0244] The compositions may be in liquid form or in lyophilized or lyophilized form and may include one or more cryoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents.

[0245] The composition may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into animals by any route available to those of skill in the art, including intra-articular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intracemall), intraventricular, intramuscular, intraocular, intra-arterial, intralesional, intrarectal, transdermal, oral, and inhalation routes.

[0246] The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentrations of the product (e.g., bolus, depot effect), sustained release, and / or increased stability or half-life in a particular local environment.

[0247] How to use In some embodiments, the antibodies of the present disclosure, such as the masked cMETxEGFRxPD-L1 / VEGF multispecific antibodies described herein, are useful in the treatment of gastric, lung, pancreatic, colorectal, and / or other cancers. In contrast to the corresponding therapeutic antibodies, the masked cMETxEGFRxPD-L1 / VEGF multispecific antibodies may have comparable efficacy in treating these diseases due to the conversion of the masked antibody into an active antibody specifically at the disease site by removal of the masking domain by proteases overexpressed at the disease site. However, the masked antibodies may have reduced systemic toxicity due to masking of the antibody activity by the masking domain in normal tissues that lack sufficient proteases required to cleave the masking domain. In short, the masked multispecific antibodies described herein are as effective as the corresponding therapeutic antibodies in the treatment of diseases, but may have a significantly improved safety profile. Due to the improved safety profile, increased levels of dosing comprising the masked multispecific antibodies may be administered to patients with improved treatment efficacy.

[0248] In some embodiments, the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a shielded cMETxEGFRxPD-L1 / VEGF multispecific antibody. The disclosure also provides the use of a shielded multispecific antibody provided herein in a method of treating cancer; and the use of a shielded cMETxEGFRxPD-L1 / VEGF multispecific antibody provided herein in the manufacture of a medicament for use in cancer. Exemplary cancers include, but are not limited to, non-small cell lung cancer, female breast cancer, pancreatic cancer, colorectal cancer, and peritoneal cancer.

[0249] All combinations of the various elements described herein are within the scope of the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0250] The present disclosure will be better understood from the following Experimental Details, however, those skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the present disclosure. EXAMPLES

[0251] Example 1: Expression and purification of anti-EGFR and anti-cMET antibodies Anti-cMET and anti-EGFR antibodies were generated and used to evaluate multispecific antibodies. Heavy and light chain constructs expressing anti-EGFR, anti-cMET, anti-cMET x anti-VEGF, and anti-EGFR x anti-VEGF parental mAbs were prepared. Plasmids encoding the heavy and light chains of these anti-cMET, anti-VEGF, and anti-EGFR antibodies were co-transfected into Expi293F cells according to the transfection kit instructions (Thermo Scientific). Five days after transfection, cells were spun down and the supernatant was passed through a 0.2 μm filter. Purification of the expressed antibodies from the supernatant was achieved by affinity chromatography on a Protein A agarose column (GE Healthcare Life Sciences). The purified antibodies were buffer exchanged into DPBS, pH 7.2 by dialysis, and the protein concentration was determined by UV absorption at 280 nm. The cMetxEGFRxVEGF Ab in a human IgG1 backbone with knob-in-hole mutations was expressed in a Chinese Hamster Ovary (CHO) cell line and purified by standard Protein A affinity capture followed by iron exchange chromatography. The protein was monomeric by SEC and pure by SDS-PAGE.

[0252] Example 2: Expression and purification of masked anti-EGFR and anti-cMET parent antibodies The shielded multispecific antibodies were evaluated using anti-cMET and anti-EGFR antibodies. Heavy and light chain constructs expressing the shielded anti-cMET and anti-EGFR parental mAbs were prepared. Plasmids encoding the heavy and light chains of these shielded anti-cMET and anti-EGFR antibodies were co-transfected into Expi293F cells according to the transfection kit instructions (Thermo Scientific). Five days after transfection, the cells were spun down and the supernatant was passed through a 0.2 μm filter. Purification of the expressed antibodies from the supernatant was achieved by affinity chromatography on a Protein A agarose column (GE Healthcare Life Sciences). The purified antibodies were buffer exchanged into DPBS, pH 7.2 by dialysis and the protein concentration was determined by UV absorption at 280 nm.

[0253] Example 3: Digestion of a Shielding Antibody Containing a Masking Domain with Proteinases An in vitro protease cleavage assay is set up to evaluate whether the shielding domain can be removed from the masked Ab by proteases. For MMP2, recombinant human MMP2 is activated by incubation with p-aminophenylmercuric acetate (APMA) according to the manufacturer's (R&D Systems) instructions. 10 mg of masked antibody is incubated with 50 ng of activated MMP2 overnight at 37°C, and digestion of the masked monoclonal antibody is evaluated by SDS-PAGE under reducing conditions. It is observed that the molecular weight of the heavy and light chains of the digested masked Ab is slightly smaller compared to the corresponding undigested pro-antibody. Upon protease treatment, the molecular weight of the uncapped mAb is closer to that of the unshielded bispecific Ab and the parent unshielded mAb.

[0254] Example 4: EGFR binding and EGF blocking by EGFR hit antibodies As shown in Figure 2A, an ELISA-based binding assay was used to assess binding to EGFR by anti-EGFR mAbs. In this assay, human EGFR was coated onto a plate and then EGFR VHO mAbs were added. After washing, the presence of EGFR was detected by an HRP-conjugated anti-His secondary antibody (BioLegend). The results show that anti-EGFR VHO mAbs (7D VH1-7D VH6) and cetuximab as a positive control can bind to EGFR. TAVO412E, an anti-cMET, anti-EGFR, anti-VEGF trispecific antibody, can bind to human EGFR with an EC50 value of 0.059 nM and to cynomolgus monkey EGFR with an EC50 value of 0.109 nM (Figures 2B, 2C). The results of an EGF ligand-EGFR blocking assay using the assay format exemplified in Figure 3E are shown in Figure 2D. The receptor used was EGFR and the ligand used was EGF at 1 mg / mL. Similarly, these same molecules can block EGF from binding to EGFR. TAVO412E was able to block human EGF binding to EGFR with an IC50 value of 1.5 nM.

[0255] Example 5: Binding of cMET and Blocking of HGF Binding by cMET Hit Antibodies Anti-cMET mAbs were assessed for binding to cMET using an ELISA-based binding assay, as shown in Figures 3A, 3B, and 3C. The results showed that anti-cMET mAbs bound to cMET. TAVO412E bound to human cMET with an EC50 value of 0.234 nM and to cynomolgus cMET with an EC50 value of 0.59 nM. These anti-cMET mAbs also blocked HGF binding to cMET using the protocol as shown in Figure 3E, with data generated as shown in Figures 3F and 3G. In this assay, human HGF was coated on the plate, and then cMET mAb was added. After washing, the presence of cMET was detected by an HRP-conjugated anti-His secondary antibody (BioLegend). The receptor used was cMET, and the ligand used was HGF. TAVO412E was able to block human HGF binding to cMET with an IC50 value of 8.0 nM.

[0256] Example 6: VEGF binding and VEGF blocking by TAVO412E An ELISA-based binding assay was used to assess the binding of TAVO412E to VEGF. In this assay, human VEGF165 was coated onto a plate, and then TAVO412E dilutions were added. After washing away nonspecific binding, the presence of bound TAVO412E was detected by an HRP-conjugated anti-Fc secondary antibody (BioLegend). TAVO412E bound human VEGF165 with an EC50 value of 0.085 nM (Figure 4A) and cynomolgus VEGF165 with an EC50 value of 0.346 nM (Figure 4B). Similarly, TAVO412E was able to inhibit the binding of VEGF to VEGFR with an IC50 value of 14.8 nM (Figure 4C).

[0257] Example 7: Design of trispecific antibody TAVO412 FIG. 5 shows the structural design for anti-cMET×anti-EGFR×anti-VEGF multispecific antibodies. The anti-cMET×anti-EGFR multispecific antibodies shown in FIG. 5 are illustrated with the EGFR binding arm in black, the cMET binding arm in dark grey, and the VEGF binding arm in light grey. FIG. 5A shows that the EGFR binding arm can have a valency of one or two VHO domains. The cMET binding arm can have a valency of one Fab domain. The VEGF binding arm can have a valency of one to two domains. The EGFR VHO domain can be on the same heavy chain as the N- and C-terminal fusions of Fc, as a tandem Fc fusion molecule on the Fc, or as a C-terminal fusion on the cMET heavy chain. FIG. 5B shows that the EGFR binding arm can have a valency of one or two VHO domains. The cMET binding arm can have a valency of one or two VHO domains on the Fc domain. The cMET VHO domain can be on the same heavy chain as an N-terminal fusion to Fc, as a tandem fusion molecule on Fc, or as an N-terminal fusion on an EGFR VHO heavy chain fusion molecule.

[0258] AVO412E is a humanized antibody of the IgG1 subclass, with one heavy and one light chain (kappa) and one chain and two nanobody domains fused to an IgG1 Fc with a carboxy-terminal single-chain Fv. The three chains are stabilized by multiple disulfide bonds. TAVO412E is a glycoprotein in which the constant region of each heavy chain has a single N-linked glycan site. To generate the heterodimeric Fc, the clinically validated knob-in-hole technology was used for TAVO412. The EGFR-VEGF binding arm has an Fc with the knob mutation T366W, and the cMET arm has an Fc with the hole mutations Y407V, L368A, and T366S. The heavy chains formed the heterodimeric Fc using knob-in-hole mutations. To enhance Fc effector function, both heavy chains have the following clinically confirmed mutations: F243L, R292P, Y300L, V305I, P396L. TAVO412 has an EGFR arm with tandem anti-EGFR VHO domains, anti-cMET Fab, and anti-VEGF scFv domains, as shown in the box in Figure 5A. The linkers used to optimize the anti-VEGF scFv were selected for better activity and stability. The number of G4S linkers to connect the anti-EGFR arm and the anti-VEGF arm was optimized for better stability and developability.

[0259] Example 8: TAVO412E Binding to Fcγ Receptors and C1q TAVO412E was designed to have enhanced ADCC by including the following Fc manipulations F243L, R292P, Y300L, V305I, and P396L. Fc-mediated effector functions of antibodies, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), have been shown to be crucial for the therapeutic efficacy of most clinically approved anti-cancer antibodies. Most of these effector functions are induced through the constant (Fc) region of the antibody, allowing the antibody to interact with complement proteins and specialized Fc-receptors. Alternative assays for such activity can be demonstrated in the binding of TAVO412E to CD16A, CD32A, CD64, and C1q. The binding activity of TAVO412 to recombinant CD16A, CD32A, CD64, and purified human C1q was assessed using ELISA (TAVO412-009). TAVO412 has mutations that enhance Fc effector function. To a large extent, the antitumor effect of TAVO412 depends on Fc effector function through binding to complement component 1q (C1q), Fc gamma receptor IIIa (FcgRIIIa or CD16A), and / or Fc gamma receptor I (Fc gamma RI or CD64). To measure binding to human CD16a, CD32a, or CD64, biotinylated recombinant proteins were added to plates precoated with streptavidin. Test antibodies were serially diluted and added to the plates. After 1 hour of incubation, binding was detected by adding HRP-conjugated goat anti-human Fc antibody. The color reaction results were measured at 450 nm. For binding to C1q, ELISA plates were coated with serially diluted test antibodies, followed by addition of human C1q. Binding was detected using sheep anti-human C1q-HRP-labeled antibody, and color reaction was performed. Binding curves for CD16a, CD32a, C1q, and CD64 are shown in Figure 6. TAVO412E bound to CD16a (EC50 value 0.46 nM) (Figure 6A), CD32a (EC50 value 2.91 nM) (Figure 6B), CD64 (EC50 value 0.16 nM) (Figure 6C), and C1q (EC50 value 0.16 nM) (Figure 6D). Overall, TAVO412E bound better to CD16a, CD32A, and C1q than the human IgG1 isotype.

[0260] Example 9: Inhibition of EGF binding to EGFR in H292 cells FIG. 7A shows the assay format of the FACS-based assay used to characterize ligand blockade of H292 cells. Anti-cMET×anti-EGFR multispecific antibodies were added to compete with 0.2 mg / mL EGF for binding to the cells. EGF was detected using AF488 nm labeled rabbit anti-EGF antibody. FIG. 7B shows that gMFI was measured to determine the level of EGF binding in the presence of competing mAbs. In this assay, competing cMET antibodies did not block EGF binding to the H292 cell line. EGFR antibodies were able to block EGF binding to EGFR. FIG. 8 shows the inhibition of EGF binding to EGFR in HCC827 cells. FIG. 8A shows the assay format of the FACS-based assay used to characterize ligand blockade of HCC827 cells. Anti-cMET×anti-EGFR multispecific antibodies were added to compete with 1 mg / mL EGF for binding to the cells. EGF was detected using AF488 nm labeled rabbit anti-EGF antibody. FIG. 8B shows that gMFI was measured to determine the level of EGF binding in the presence of competing mAbs. In this assay, the competing cMET antibody did not block EGF binding to the HCC827 cell line. The EGFR antibody blocked EGF from binding to EGFR on the HCC827 cell line. In this EGFR-EGF inhibition experiment, the EC50 values ​​in ng / mL for the EGFR x cMet hits were: 7D VH6 x TV4 - 0.63 nM; 7D VH6 x EV1 - 0.63 nM; 7D VH4 x TV4 - 0.93 nM; 7D VH4 x EV1 - 0.81 nM; cetuximab x gp120 - 0.60 nM; cetuximab - 0.33 nM; 7D VH4-Fc - 0.18 nM; and 7D VH6-Fc - 0.23 nM.

[0261] Example 10: Inhibition of EGFR phosphorylation in H1975 cells using Western blotting As shown in Figure 9, cMETxEGFR BsAb can inhibit EGFR phosphorylation in NCI-H1975 cells using Western blot. 5 Cells / well were seeded in 12-well plates. NCI-H1975 cells have the L858R T790M EGFR and cMET WT genotype. After 18 hours of starvation at 37°C in non-FBS medium, 33.3 nM antibody was added for 1 hour, followed by 500 ng / mL EGF ligand treatment for 30 minutes. Cells were harvested and lysed with cell extraction buffer supplemented with phosphatase and protease inhibitors. In FIG. 9A, the top panel has western blot lanes corresponding to (1) medium only; (2) EGF only; (3) 7D VH4-Fc; (4) gp120; (5) 7D VH6-Fc; (6) EV1 (SEQ ID NO:22 and SEQ ID NO:24); (7) TV4; (8) 7D VH4×EV1; (9) 7D VH4×TV4; (10) 7D VH4×EV1; (11) 7D VH6×TV4; (12) cetuximab×gp120. Integration values ​​were normalized to b-actin levels in each lane. All four candidate BsAbs were able to inhibit EGFR phosphorylation, with one armed cetuximab×gp120 showing similar inhibitory effects. In Figure 9B, the top panel has western blot lanes corresponding to (1) medium only; (2) EGF only; (3) 7D VH4 x EV1; (4) 7D VH4 x TV1; (5) 7D VH6 x EV1; (6) 7D VH6 x TV4; (7) cetuximab x gp120; (8) gp120; (9) 7D VH4 x gp120; (10) 7D VH6 x gp120. Integration values ​​were normalized to b-actin levels in each lane. All four of our candidate BsAbs were able to inhibit EGFR phosphorylation, with one armed cetuximab x gp120 showing similar inhibitory effects.

[0262] In FIG. 9C, H292 and HCC827 cells were cultured at 2×10 5Cells were seeded in 12-well plates at a concentration of 1:1. HCC827 cells have deletions E746 and A750 in EGFR and WT cMET. H292 cells have WT EGFR and WT cMET. After starvation with no FBS medium and incubation at 37°C for 18 hours, 33.3 nM antibody was added and incubated for 1 hour, followed by 500 ng / mL EGF ligand treatment for 30 minutes. Cells were harvested and lysed with cell extraction buffer containing phosphatase and protease inhibitors. In Figure 9C, results for H292 cells with western blot lanes correspond to (1) media only, (2) EGF only, (3) 7D VH4 x EV1, (4) 7D VH4 x TV1, (5) 7D VH6 x EV1, (6) 7D VH6 x TV4, (7) cetuximab x gp120, (8) gp120, (9) 7D VH4 x gp120, (10) 7D VH6 x gp120. In Figure 9D, the results for HCC827 cells with western blot lanes correspond to (1) medium only, (2) EGF only, (3) 7D VH4 x EV1, (4) 7D VH4 x TV1, (5) 7D VH6 x EV1, (6) 7D VH6 x TV4, (7) cetuximab x gp120, (8) gp120, (9) 7D VH4 x gp120, (10) 7D VH6 x gp120. The integrals were normalized to b-actin levels in each lane. All four candidate BsAbs could inhibit EGFR phosphorylation, and showed similar inhibitory effects with one armed cetuximab x gp120 in H292 cells, but not in HCC827 cells.

[0263] Example 11: Demonstration of the usefulness of TAVO412E in non-small cell lung cancer cell line HCC827 FIG. 10 demonstrated TAVO412E cell binding, blocking of EGF from binding to EGFR on HCC827 cells, and blocking of HGF from binding to cMET on HCC827 cells. In cell binding experiments, HCC827 cells were seeded at 50,000 cells / well in 96-well plates. Serial dilutions of antibodies were added and incubated for 1 hour at 4° C. in the dark. After washing, Alexa Fluor 647 Fcγ fragment-specific goat anti-human IgG was used for detection at 638 nm excitation and 660 nm emission on a Beckman flow cytometer. In blocking of EGF from binding to EGFR in HCC827 cell experiments, cells were harvested and seeded at 50,000 cells / well in 96-well plates. Serial dilutions of antibodies were added and incubated for 1 hour at 4° C. in the dark. After washing, cells were incubated with EGF for 1 hour at 4° C. in the dark. Rabbit polyclonal anti-human EGF and Alexa Fluor 488 anti-rabbit IgG1 were used for detection on a Beckman flow cytometer at 488 nm excitation and 525 nm emission. In blocking HGF binding to cMET in HCC827 experiments, cells were harvested and plated at 50,000 cells / well in a 96-well plate. Serial antibody dilutions were added and incubated for 0.5 hours at 4°C in the dark. 0.7 μg / mL biotinylated HGF was added and washed and incubated for 0.5 hours at 4°C in the dark. Streptavidin-AF488 was used to detect biotinylated HGF on a Beckman flow cytometer at 488 nm excitation and 525 nm emission. In Figure 10A-C, the y-axis represents gMFI reflecting the level of binding to HCC827 cells, and the x-axis represents the concentration of the test reagent. Figure 10A shows that TAVO412E has an EC50 value of 1.04nM for binding to HCC827 cells. Isotype mAb does not bind to HCC827 cells. Figure 10B shows that TAVO412E has an IC50 value of 2.56nM for blocking EGF binding to EGFR on HCC827 cells. Isotype mAb does not block EGF binding to EGFR on HCC827 cells.Figure 10C shows that TAVO412E had an IC50 value of 0.28 nM for blocking HGF binding to cMET on HCC827 cells. Isotype mAb did not block HGF binding to cMET on HCC827 cells.

[0264] Example 12: TAVO412E inhibition of EGFR and cMET phosphorylation in H292 and HCC827 cells In this assay, either H292 or HCC827 cells were seeded in 96-well plates at a density of 40,000 cells / well and incubated overnight. Cells were starved for 24 hours in serum-free medium. Serial antibody dilutions were added to the plates and incubated at 37°C for 1 hour, then ligands (HGF or HGF+EGF) were added and incubated at 37°C for 15 minutes. Cells were lysed with lysis buffer containing phosphoinhibitors and cell lysates were transferred to 384-well plates and incubated with HTRF antibodies for 4 hours at RT. Plates were read at 320 / 615, 320 / 665 on a Decan Spark plate reader. Phosphorylation ratio percentages (%) were determined for each drug / concentration and dose-response curves were generated. In Figure 11A-D, the y-axis is shown as the value of percent EGFR phosphorylation shown with the control mAb and the x-axis is the concentration of the test substance. FIG. 11A shows that TAVO412E inhibited EGFR phosphorylation in H292 cells in the presence of EGF with an IC50 value of 0.79 nM. Isotype mAb did not inhibit EGFR phosphorylation. FIG. 11B shows that TAVO412E inhibited EGFR phosphorylation in H292 cells in the presence of EGF and HGF with an IC50 value of 0.78 nM. Isotype mAb did not inhibit EGFR phosphorylation. FIG. 11C shows that TAVO412E inhibited cMET phosphorylation in HCC827 cells in the presence of HGF with an IC50 value of 1.41 nM. Isotype mAb did not inhibit cMET phosphorylation. FIG. 11D shows that TAVO412E inhibited cMET phosphorylation in HCC827 cells in the presence of HGF and EGF with an IC50 value of 1.99 nM. Isotype mAbs did not inhibit cMET phosphorylation.

[0265] Example 13: TAVO412E inhibition of HCC827 cell proliferation HCC827 cells were seeded at 10,000 cells / well in 96-well plates and incubated overnight. The cells were then starved with serum-free medium followed by 24-hour culture. The next day, serial antibody dilutions were added to the plates. After 3 days of incubation, PrestoBlue reagent was added for cell viability detection at 560 nm and 590 nm using a Tecan Spark microplate reader. Viability was calculated as (fluorescence of test antibody medium control) / (fluorescence of untreated cell control-fluorescence of medium control). Figure 12A shows that TAVO412E inhibited the proliferation of HCC827 cells with an IC50 value of 1.76 nM. Isotype mAbs did not inhibit the proliferation of HCC827 cells. Figure 12B shows that TAVO412E inhibited the proliferation of HCC827 cells in the presence of EGF and HGF with an IC50 value of 1.39 nM. Isotype mAb did not inhibit the proliferation of HCC827 cells.

[0266] Example 14: In vitro inhibition of cMET phosphorylation in H1975, HCC827, and H292 cells using Western blot In Figure 13, cells are 2 × 10 5Cells / well were seeded in 12-well plates. After starvation in non-FBS medium for 18 hours at 37°C, cells were incubated with 33.3 nM antibody for 1 hour, followed by 500 ng / mL HGF ligand treatment for 30 minutes. Cells were harvested and lysed with cell extraction buffer supplemented with phosphatase and protease inhibitors. Figure 13A shows the results for HCI-H1975, Figure 13B shows the results for HCC827, and Figure 13C shows the results for H292 cells. Western blot lanes corresponded to (1) medium alone, (2) HGF alone, (3) 7D VH4 x EV1, (4) 7D VH4 x TV1, (5) 7D VH6 x EV1, (6) 7D VH6 x TV4, (7) cetuximab x gp120, (8) gp120, (9) 7D VH4 x gp120, and (10) 7D VH6 x gp120. The four BsAbs showed significantly more inhibitory effects than their parental Abs alone in HCC827, H292, and NCI-H1975 cells.

[0267] Example 15: In vitro Fc effector function of TAVO412E on HCC827 cells For reporter assays as shown in Figures 14A and 14B, target cells (2 x 10 per well) were 4 cells) and Jurkat-CD16A-V158 or Jurkat-CD32A-H131 reporter cells (2 × 10 per well 5 HCC827 cells) were harvested and co-cultured in a 96-well plate at an E:T ratio of 10:1. Serially diluted test antibodies were dispensed into the plate and incubated at 37°C for 6 hours. After incubation, cell supernatants were transferred to white-walled plates and Bio-Lite reagent was added to each well. Luminescence was measured using a Decan Spark®. To measure ADCC activity in HCC827 cells, target cells were cultured at 1 × 10 in a round-bottom 96-well plate as shown in Figure 14C. 4Cells / well were seeded and incubated with serial dilutions of test antibodies at 37°C for 15 min first, then frozen PBMCs were harvested and added to the plate at an E:T ratio of 50:1. The plate was centrifuged to ensure contact between effector and target cells and incubated at 37°C for 4 h. After centrifugation, cell supernatant was transferred to a new flat-bottom plate. Cell lysis was tested using an LDH kit. Absorbance was read at 492 nm and 650 nm using a Decan Spark®. ADCC% was calculated as (experimental release-spontaneous release) / (maximum release-spontaneous release). As shown in Figure 14D, to measure ADCP-macrophage killing activity in NSCLC cancer cell lines, monocytes were isolated from PBMCs and induced to differentiate into macrophages with cytokines of 25 ng / mL MCSF and 50 ng / mL IFNγ. Target cells were harvested and stained with CSFE. Differentiated macrophages (1×10 5 cells / well) and labeled target cells (5 x 10 4Cells / well) were co-cultured in 96-well plates at E:T=2:1. After 24 h of incubation, cells were harvested and stained with Alexa647-labeled CD14 and CD11b antibodies for 30 min. After washing, cells were measured at 638 nm and 660 nm on a Beckman flow cytometer. Percent killing was determined using a formula such as ((average %FITC+AF647 of [lowest mAb] for each antibody)-%FITC+AF647-sample) / (average %FITC+AF647 of [lowest mAb] for each antibody). For the CDC experiment shown in Figure 14E, cells were harvested and seeded in 96-well plates at optimized cell density in basal medium. Cells were incubated with a series of antibody dilutions and incubated at RT for 1 h. Rabbit serum was dispensed into the plate and cultured at 37°C for 1 h. After incubation, cell supernatants were transferred to new plates and cell lysis was detected using an LDH kit. Absorbance values ​​were read at 492 nm and 650 nm on a Decan Spark®. Percent lysis was calculated by dividing the absorbance value of the sample by that of the control. Dose-response curves were generated by GraphPad Prism 9.3.1. Figure 14A shows that TAVO412E had ADCC reporter activity on HCC827 cells with an IC50 value of 0.2 nM. Isotype mAb had no ADCP reporter activity on HCC827 cells. Figure 14B shows that TAVO412E had ADCP reporter activity on HCC827 cells with an IC50 value of about 1 nM. Isotype mAb had no ADCP reporter activity on HCC827 cells. Figure 14C shows that TAVO412E had ADCC killing activity on HCC827 cells with an IC50 value of 0.12 nM. Isotype mAb had no ADCP reporter activity of HCC827 cells. Figure 14D shows that TAVO412E induced ADCP killing activity on HCC827 cells with IC50 value of 0.16nM. Isotype mAb had no ADCP reporter activity of HCC827 cells. Figure 14E shows that TAVO412E had CDC killing activity on HCC827 cells with IC50 value of 3.76nM. Isotype mAb had no ADCP reporter activity of HCC827 cells.

[0268] Example 16: In vivo antitumor activity of TAVO412E against non-small cell lung cancer cell line H1975 The following guidelines were used in xenograft model experiments. All protocols and amendments or procedures involving the care and use of animals were reviewed and approved by the GenePharma Institutional Animal Care and Use Committee (IACUC) prior to conducting the studies. Tumor cells were implanted subcutaneously into female Balb / c nude mice, and tumors were grown to a mean tumor volume of 100–200 mm. 3 Treatment was initiated when the tumor volume reached 100 mg / kg / day. Test antibodies were injected intraperitoneally with a twice weekly dosing regimen. Tumor growth and body weight were measured twice weekly until the end point, and tumor volume was determined as length x width 2 x 0.5. Tumor growth inhibition (TGI) was calculated as TGI% = 1-T / C, where T and C were the mean tumor volumes of the treatment and control groups on the last day, respectively.

[0269] The following protocol was used for in vivo receptor degradation experiments. Mice bearing established tumors were treated with two doses of vehicle control. 24 hours after the 2nd dose, tumors were harvested and flash frozen in liquid nitrogen. Tumors were lysed in ice-cold RIPA buffer containing protease and phosphatase inhibitor cocktail using a homogenizer. Lysates were cleared by centrifugation and protein concentrations were determined by BCA protein assay. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% BSA blocking buffer for 1 hour at room temperature and incubated with appropriate primary antibodies overnight at 4°C. ECL detection was performed by incubating the membrane and ECL reagent. Images were acquired using e-BLOT WB IMAGER. Western blot images were analyzed with image J software. Mean total protein (GAPDH) versus loading control was graphed and statistical analysis was performed using GraphPad Prism version 9.3.1.

[0270] Figure 15A shows H1975 tumor growth inhibition at day 13 of 42% at 1 mg / kg, 76% at 3 mg / kg, and 94% at 10 mg / kg. TAVO412E had dose-dependent tumor growth inhibition in H1975 cells. Figure 15B shows that TAVO412E induces degradation of EGFR in tumors in an in vivo H1975 xenograft model, as well as a decrease in EGFR phosphorylation. Figure 15C shows that TAVO412E induced degradation of cMET in tumors in an in vivo H1975 xenograft model, as well as a decrease in cMET phosphorylation. Figure 15D shows a bar graph representation of the results for control isotype mAb and TAVO412E in Figures 15B and C. TAVO412E reduced the levels of total and phosphorylated forms of cMET and EGFR in an in vivo H1975 xenograft model experiment.

[0271] Example 17: In vivo antitumor activity of TAVO412E against non-small cell lung cancer cell line HCC827 Xenografting and receptor degradation protocols are outlined in Example 16. Figure 16A shows HCC827 tumor growth inhibition of 45% at 1 mg / kg, 79% at 3 mg / kg, and 94% at 10 mg / kg on day 13. TAVO412E showed dose-dependent tumor growth suppression in HCC827 cells. Figure 16B shows that TAVO412E induces degradation of EGFR and cMET in tumors in an in vivo HCC827 xenograft model experiment. Figure 16C shows a bar graph representation of the results for control isotype mAb and TAVO412E in Figure 16B. TAVO412E reduced the levels of all forms of cMET and EGFR in an in vivo HCC827 xenograft model experiment.

[0272] Example 18: In vitro antitumor activity of TAVO412E against triple-negative breast cancer cell line MDA-MB-468 Figure 17A shows the binding of TAVO412E to MDA-MB-468, with an EC50 value for binding of 1.11 nM. TAVO412E had dose-dependent tumor growth inhibition in H1975 cells. Figure 17B shows that TAVO412E inhibited human EGFR phosphorylation in MDA-MB-468 cells in the presence of human EGF, with an IC50 value of 9.08 nM. Isotype mAb did not inhibit human EGFR phosphorylation. Figure 17C shows that TAVO412E inhibited human EGFR phosphorylation in MDA-MB-468 cells in the presence of human EGF and human HGF, with an IC50 value of 8.50 nM. Isotype mAb did not inhibit EGFR phosphorylation.

[0273] Example 19: In vitro antitumor activity of TAVO412E against triple-negative breast cancer cell line MDA-MB-231 For ADCC experiments, frozen PBMCs were harvested and cultured overnight. The next day, target cells were seeded in round-bottom 96-well plates and first incubated with serial dilutions of test antibodies at 37°C for 15 min, then PBMCs were added at an E:T ratio of 50:1. The plates were centrifuged to ensure contact between effector and target cells and incubated at 37°C for 4 h. After centrifugation, cell supernatants were transferred to new flat-bottom plates. Cell lysis was tested using an LDH kit. Absorbance was read at 492 nm and 650 nm using a Decan Spark®. ADCC% was calculated as (experimental release-spontaneous release) / (maximum release-spontaneous release). For ADCP experiments, monocytes were isolated from PBMCs and differentiated into macrophages by treatment with MCSF and IFNγ cytokines. Target cells were harvested and stained with CSFE. Differentiated macrophages and labeled target cells were co-cultured at a 2:1 E:T ratio, and serial dilutions of test antibodies were added and incubated. After 24 hours of incubation, cells were harvested and stained with Alexa647-labeled CD14 and CD11b antibodies for 30 minutes. After washing, cells were measured at 638 nm and 660 nm on a Beckman flow cytometer. Percent killing was determined using a formula such as ((mean %FITC+AF647 of [lowest mAb] for each antibody)-%FITC+AF647-sample) / (mean %FITC+AF647 of [lowest mAb] for each antibody). The results of three independent measurements were then collected, graphed, and processed with GraphPad Prism 9.3.1. For CDC experiments, cells were harvested and seeded in 96-well plates at optimized cell density in basal medium. Serial antibody dilutions were added and incubated for 1 hour at RT. Rabbit serum was dispensed into the plates and incubated at 37°C for 1-4 hours. After incubation, cell supernatants were transferred to new plates and cell lysis was tested using an LDH kit. Absorbance values ​​were read at 492 nm and 650 nm on a Decan Spark®. Percent lysis was calculated by dividing the absorbance value of the sample by that of the control. Dose-response curves were generated using GraphPad Prism 9.3.1.

[0274] Figure 18A shows TAVO412E bound to MDA-MB-231 with an EC50 value for binding of 0.37 nM. Figure 18B shows TAVO412E had ADCP reporter activity on MDA-MB-231 cells with an EC50 value of 0.087 nM. Isotype mAbs had no ADCP reporter assay response. Figure 18C shows TAVO412E had ADCP killing of MDA-MB-231 cells with an EC50 value of 0.156 nM. Isotype mAbs had no ADCP killing response. Figure 18D shows TAVO412E had ADCC reporter activity on MDA-MB-231 cells with an EC50 value of 0.18 nM. Isotype mAbs had no ADCP reporter assay response. Figure 18E shows that TAVO412E had ADCC killing of MDA-MB-231 cells with an EC50 value of 0.13 nM. Isotype mAb had no ADCC killing response. Figure 18F shows that TAVO412E had CDC killing of MDA-MB-231 cells with an EC50 value of 1.22 nM. Isotype mAb had no CDC killing response.

[0275] Example 20: In vivo antitumor activity of TAVO412E against triple-negative breast cancer cell line MDA-MB-231 Xenograft and receptor degradation protocols are outlined in Example 16. Figure 19A shows 62% MDA-MB-231 tumor growth inhibition at 10 mg / kg dose at day 20. Figure 19B shows TAVO412E induces degradation of EGFR and cMET in tumors in an in vivo MDA-MB-231 xenograft model experiment. Figure 19C shows a bar graph representation of the results for control isotype mAb and TAVO412E in Figure 19B. TAVO412E reduced levels of cMET and all forms of EGFR in an in vivo MDA-MB-231 xenograft model experiment.

[0276] Example 21: In vitro utility of TAVO412E in gastric cancer cell lines SNU-5 and MKN-45 as demonstrated by cell binding, blocking HGF binding to cMET on MKN45 cells, and growth inhibition of SNU-5 cells For HGF blockade in MKN45 experiments, cells were harvested and seeded in 96-well plates at 50,000 cells / well. Serial dilutions of HGF and 0.1 mg / mL of test antibody were added sequentially and incubated for 1 hour at 4°C in the dark. After washing, test antibodies were detected on a Beckman flow cytometer using Alexa Fluor 488 anti-rabbit IgG1 at excitation 488 nm and emission 525 nm. For proliferation inhibition studies, 3k SNU-5 cells / well without starvation were placed in 96-well plates. Cells were treated with test articles for 6 days and cell proliferation / viability was measured using Alamar Blue. Figure 20A shows that TAVO412E had an EC50 value of 1.78 nM for binding to MKN45 cells. Isotype mAbs did not bind to MKN45 cells. Figure 20B shows that TAVO412E had an IC50 value of 0.28nM for blocking HGF binding to cMET on MKN45 cells. Isotype mAb did not block HGF binding to cMET on MKN45 cells. Figure 20C shows that TAVO412E had an EC50 value of 1.99nM for binding to SNU-5 cells. Isotype mAb bound to SNU-5 cells. Figure 20D shows that TAVO412E had an IC50 value of 2.66nM for inhibiting the proliferation of SNU-5 cells. Isotype mAb did not inhibit the proliferation of SNU-5 cells.

[0277] Example 22: In vitro antitumor activity of TAVO412E against gastric cancer cell line SNU-5 The experimental protocol was described in FIG. 18. FIG. 21A shows that TAVO412E had ADCC reporter activity on SNU-5 cells with an EC50 value of 0.18 nM. The isotype mAb had no ADCC reporter assay response. FIG. 21B shows that TAVO412E had ADCP reporter activity on SNU-5 cells with an EC50 value of 0.20 nM. The isotype mAb had no ADCP reporter assay response. FIG. 21C shows that TAVO412E had CDC killing of SNU-5 cells with an EC50 value of 1.19 nM. The isotype mAb had no CDC killing response.

[0278] Example 23: In vivo antitumor activity of TAVO412E against gastric cancer cell line MKN45 Xenograft and receptor degradation protocols are outlined in Example 16. Figure 22A shows 70% MKN-45 tumor growth inhibition at a dose of 3 mg / kg on day 21. Figure 22B shows that TAVO412E induced degradation of EGFR and cMET in tumors in an in vivo MKN45 xenograft model experiment. Figure 22C shows a bar graph representation of the results for control isotype mAb and TAVO412E in Figure 22B. TAVO412E reduced levels of cMET and all forms of EGFR in tumors excised from an in vivo MKN45 xenograft model experiment.

[0279] Example 24: In vitro utility of TAVO412E in pancreatic ductal adenocarcinoma cell line BxPC-3 as demonstrated by cell binding, ADCC reporter assay, and ADCP reporter assay Figure 23A shows that TAVO412E had an EC50 value of 0.90nM for binding to BxPC-3 cells. Isotype mAb did not bind to BxPC-3 cells. Figure 23B shows that TAVO412E had an EC50 value of 0.20nM for ADCC reporter assay on BxPC-3 cells. Isotype mAb did not have ADCC reporter assay activation on BxPC-3 cells. Figure 23C shows that TAVO412E had an EC50 value of 0.65nM for ADCP reporter assay on BxPC-3 cells. Isotype mAb did not show ADCP reporter assay activation on BxPC-3 cells.

[0280] Example 25: In vitro inhibition of EGFR and cMET phosphorylation in BxPC-3 cells This experiment was performed similarly to that described for FIG. 11. In FIG. 24A-D, the y-axis is shown as the value of percent receptor phosphorylation shown with the control mAb, and the x-axis is the concentration of the test substance. FIG. 24A shows that TAVO412E inhibited EGFR phosphorylation in BxPC-3 cells in the presence of recombinant human EGF with an IC50 value of 3.45 nM. Isotype mAbs did not inhibit EGFR phosphorylation. FIG. 24B shows that TAVO412E inhibited cMET phosphorylation in BxPC-3 cells in the presence of recombinant human HGF with an IC50 value of 1.18 nM. Isotype mAbs did not inhibit cMET phosphorylation. FIG. 24C shows that TAVO412E inhibited EGFR phosphorylation in BxPC-3 cells in the presence of recombinant human EGF and recombinant human HGF with an IC50 value of 1.13 nM. Isotype mAbs did not inhibit EGFR phosphorylation. Figure 24D shows that TAVO412E inhibits cMET phosphorylation in BxPC-3 cells in the presence of recombinant human EGF and recombinant human HGF with an IC50 value of 0.44 nM. Isotype mAbs did not inhibit cMET phosphorylation.

[0281] Example 26: In vitro antitumor activity of TAVO412E against pancreatic ductal adenocarcinoma cell line BxPC-3 Xenograft and receptor degradation protocols are outlined in Example 16. Figure 25A shows TAVO412 treatment results in 80% BxPC-3 tumor growth inhibition at 10 mg / kg dose on day 34. Figure 25B shows TAVO412E induced degradation of EGFR and cMET in tumors in an in vivo BxPC-3 xenograft model experiment. Figure 25C shows a bar graph representation of the results for control isotype mAb and TAVO412E in Figure 25B. TAVO412E reduced levels of cMET and all forms of EGFR in tumors excised from an in vivo BxPC-3 xenograft model experiment.

[0282] Example 27: Antitumor activity of TAVO412E against liver cancer cell line HCC9810 in vitro, triple-negative breast cancer cell line HCC70 in vivo, and head and neck cancer cell line FaDu in vivo Xenograft and receptor decomposition protocols are outlined in Example 16. Figure 26A shows that TAVO412E had ADCC activity against the HCC9810 cell line with an EC50 value of 0.098 nM. Figure 26B shows that TAVO412 treatment resulted in 26% HCC-70 tumor growth inhibition at day 21 at a dose of 10 mg / kg. Figure 26C shows that TAVO412 treatment resulted in 95% FaDu tumor growth inhibition at day 21 at a dose of 10 mg / kg.

[0283] Example 28: In vitro anticancer activity in the head and neck esophageal mesoesophageal squamous cell carcinoma cell line KYSE-150 as demonstrated by cell binding, ADCC reporter assay, and ADCC bactericidal assay Figure 27A shows that TAVO412E had an EC50 value for binding to KYSE-150 cells of 0.39nM. Isotype mAb did not bind to KYSE-150 cells. Figure 27B shows that TAVO412E had an EC50 value for ADCC reporter assay on KYSE-150 cells of 0.15nM. Isotype mAb did not show ADCC reporter assay activation on KYSE-150 cells. Figure 27C shows that TAVO412E had an EC50 value for ADCC killing assay on KYSE-150 cells of 0.038nM. Isotype mAb did not have ADCC killing response on KYSE-150 cells.

[0284] Example 29: TAVO412 Antitumor In Vitro Activity in Mesothelioma Cancer Cell Line NCI-H226 as Shown by Cell Binding, ADCC Reporter Assay, and ADCC Killing Assay Figure 28A shows that TAVO412E had an EC50 value of 0.78nM for binding to NCI-H226 cells. Isotype mAb did not bind to NCI-H226 cells. Figure 28B shows that TAVO412E had an EC50 value of 0.17nM for ADCC reporter assay on NCI-H226 cells. Isotype mAb did not have ADCC reporter assay activation on NCI-H226 cells. Figure 28C shows that TAVO412E had an EC50 value of 0.025nM for ADCC killing assay on NCI-H226 cells. Isotype mAb did not show ADCC killing response on NCI-H226 cells.

[0285] Example 30: In vitro antitumor activity of TAVO412 in colon cancer cell line HT-29 as demonstrated by cell binding, ADCC reporter assay, and ADCC killing assay Figure 29A shows that TAVO412E had an EC50 value of 0.23nM for binding to HT-29 cells. Isotype mAb did not bind to HT-29 cells. Figure 28B shows that TAVO412E had an EC50 value of 0.078nM for ADCC reporter assay on HT-29 cells. Isotype mAb did not have ADCC reporter assay activation on HT-29 cells. Figure 27C shows that TAVO412E had an EC50 value of 0.023nM for ADCC killing assay on HT-29 cells. Isotype mAb did not show ADCC killing response on HT-29 cells.

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Claims

1. 1. A multispecific antibody comprising an EGFR arm capable of binding to EGFR and a cMet arm capable of binding to cMET, the EGFR arm comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 95, 96, and 97; SEQ ID NOs: 95, 96, and 98; SEQ ID NOs: 95, 96, and 105; SEQ ID NOs: 102, 100, and 101; and SEQ ID NOs: 102, 103, and 104, respectively; or The EGFR arm comprises a light chain sequence and a heavy chain sequence selected from SEQ ID NOs: 1-4; a single domain heavy chain sequence selected from SEQ ID NOs: 5-12; or a tandem single domain heavy chain sequence selected from SEQ ID NOs: 13-18; and the cMET arm comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 106, 107, and 133; SEQ ID NOs: 111, 112, and 113; SEQ ID NOs: 111, 114, and 113; SEQ ID NOs: 99, 118, and 119; SEQ ID NOs: 99, 120, and 119; SEQ ID NOs: 99, 121, and 119; SEQ ID NOs: 99, 122, and 119; and wherein LCDR1, LCDR2, and LCDR3 are selected from SEQ ID NOs: 108, 109, and 110; SEQ ID NOs: 115, 116, and 117; and SEQ ID NOs: 123, 124, and 125, respectively; or A multispecific antibody, wherein the cMET arms comprise a light chain sequence and a heavy chain sequence selected from SEQ ID NOs: 23-40, or at least one single domain heavy chain domain selected from SEQ ID NOs: 41-44.

2. 2. The multispecific antibody of claim 1, wherein the EGFR arm comprises at least one single-domain heavy chain domain selected from SEQ ID NOs: 5-12.

3. 2. The multispecific antibody of claim 1, wherein the EGFR arms comprise tandem single domain heavy chains selected from SEQ ID NOs: 13-18.

4. 2. The multispecific antibody of claim 1 , wherein the anti-EGFR arms comprise heavy chain SEQ ID NO: 1 and light chain SEQ ID NO: 2; heavy chain SEQ ID NO: 3 and light chain SEQ ID NO: 4; a single domain heavy chain selected from SEQ ID NOs: 5 to 12; or tandem single domain heavy chains selected from SEQ ID NOs: 13 to 18.

5. The multispecific antibody of any one of claims 1 to 4, wherein the cMET arms comprise light chain and heavy chain sequences selected from SEQ ID NOs: 23 to 40.

6. The multispecific antibody of any one of claims 1 to 4, wherein the cMET arm comprises at least one single-domain heavy chain domain selected from SEQ ID NOs: 41 to 44.

7. The anti-cMET arm is composed of heavy chain SEQ ID NO:23 and light chain SEQ ID NO:25; heavy chain SEQ ID NO:23 and light chain SEQ ID NO:26; heavy chain SEQ ID NO:24 and light chain SEQ ID NO:25; heavy chain SEQ ID NO:24 and light chain SEQ ID NO:26; heavy chain SEQ ID NO:27 and light chain SEQ ID NO:30; heavy chain SEQ ID NO:27 and light chain SEQ ID NO:31; heavy chain SEQ ID NO:27 and light chain SEQ ID NO:32; heavy chain SEQ ID NO:28 and light chain SEQ ID NO:30; heavy chain SEQ ID NO:28 and light chain SEQ ID NO:31; heavy chain SEQ ID NO:28 and light chain SEQ ID NO:32; chain SEQ ID NO:29 and light chain SEQ ID NO:30; heavy chain SEQ ID NO:29 and light chain SEQ ID NO:31; heavy chain SEQ ID NO:29 and light chain SEQ ID NO:32; heavy chain SEQ ID NO:33 and light chain SEQ ID NO:38 5. The multispecific antibody of any one of claims 1 to 4, comprising heavy chain SEQ ID NO:33 and light chain SEQ ID NO:39; heavy chain SEQ ID NO:33 and light chain SEQ ID NO:40; heavy chain SEQ ID NO:34 and light chain SEQ ID NO:38; heavy chain SEQ ID NO:34 and light chain SEQ ID NO:39; heavy chain SEQ ID NO:34 and light chain SEQ ID NO:40; heavy chain SEQ ID NO:35 and light chain SEQ ID NO:38; heavy chain SEQ ID NO:35 and light chain SEQ ID NO:39; heavy chain SEQ ID NO:35 and light chain SEQ ID NO:40; heavy chain SEQ ID NO:36 and light chain SEQ ID NO:38; heavy chain SEQ ID NO:36 and light chain SEQ ID NO:39; heavy chain SEQ ID NO:36 and light chain SEQ ID NO:40; heavy chain SEQ ID NO:37 and light chain SEQ ID NO:39; or heavy chain SEQ ID NO:37 and light chain SEQ ID NO:

40.

8. 5. The multispecific antibody of any one of claims 1 to 4, further comprising an anti-VEGF antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from SEQ ID NOs: 129, 130, and 131; and SEQ ID NOs: 129, 132, and 131, respectively; and LCDR1, LCDR2, and LCDR3 are SEQ ID NOs: 126, 127, and 128, respectively.

9. 5. The multispecific antibody of any one of claims 1 to 4, wherein the anti-VEGF antibody or antigen-binding fragment comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 73 to 76.

10. The multispecific antibody of any one of claims 1 to 4, further comprising an anti-PD-L1 antibody or antigen-binding fragment thereof comprising an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 71 to 72.

11. The multispecific antibody of any one of claims 1 to 4, wherein the multispecific antibody comprises a shielding domain selected from SEQ ID NOs: 45 to 51.

12. The multispecific antibody of any one of claims 1 to 4, wherein the multispecific antibody comprises a shielding domain selected from SEQ ID NOs: 52 to 61.

13. The multispecific antibody of claim 11, wherein the multispecific antibody comprises a protease-recognition peptide sequence selected from SEQ ID NOs: 62 to 69.

14. 2. The multispecific antibody of claim 1, wherein the multispecific antibody comprises an amino acid sequence selected from SEQ ID NOs: 83 to 94.

15. 2. The multispecific antibody of claim 1, wherein the multispecific antibody comprises the amino acid sequences shown in SEQ ID NO: 90, SEQ ID NO: 84, and SEQ ID NO: 85, respectively.

16. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

17. A nucleic acid sequence encoding the multispecific antibody of any one of claims 1 to 4.

18. A recombinant expression vector comprising the nucleic acid sequence of claim 17.

19. A recombinant expression transformant comprising the recombinant expression vector according to claim 18.

20. A method for preparing the multispecific antibody of any one of claims 1 to 4, comprising culturing the recombinant expression transformant of claim 19 and obtaining the antibody or antigen-binding fragment, or the multispecific antibody from the culture, optionally using controlled Fab arm exchange.

21. 10. Use of a multispecific antibody according to any one of claims 1 to 4 for the manufacture of a medicament for treating or preventing cancer in a subject.

22. Use of the pharmaceutical composition of claim 16 for the manufacture of a medicament for treating or preventing cancer in a subject.

23. 22. The method for treating or preventing cancer according to claim 21, wherein the cancer is lung cancer, breast cancer, pancreatic cancer, colon cancer, head and neck cancer, esophageal cancer, or gastric cancer.

24. 22. The use according to claim 21, wherein the cancer is a relapsed EGFR-positive cancer and / or a cMET-positive cancer.

25. 5. The multispecific antibody of any one of claims 1 to 4 for the manufacture of a medicament for treating or preventing a disease mediated by EGFR, VEGF or PD-L1, and cMET in a subject.

26. The pharmaceutical composition of claim 16 for producing a medicament for treating or preventing a disease mediated by EGFR, VEGF or PD-L1, and cMET in a subject.

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