Anti-ANG2-VEGF bispecific antibodies and uses thereof

Abstract

The present invention belongs to the field of biomedicine. Specifically, the present invention provides an anti-ANG2-VEGF bispecific antibody, a pharmaceutical composition comprising the bispecific antibody and its use. The present invention further provides a polynucleotide encoding the bispecific antibody and an expression vector and a method for producing the bispecific antibody.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention provides an anti-ANG2-VEGF bispecific antibody, a pharmaceutical composition comprising the bispecific antibody, and uses thereof. The present invention further provides a polynucleotide encoding the bispecific antibody and an expression vector, and a method for producing the bispecific antibody. Background Art

[0002] Vascular endothelial growth factor A (VEGF-A, also referred to as VEGF) is a key cytokine that promotes angiogenesis. The receptors of VEGF are VEGFR1 and VEGFR2, which are expressed in vascular endothelial cells. VEGFR2 is the main receptor tyrosine kinase receptor that mediates angiogenesis. VEGF activates VEGFR2 to promote mitosis of vascular endothelial cells and increase vascular permeability, thereby promoting cardiovascular sprouting. Therefore, targeting VEGF or VEGFR2 can effectively inhibit abnormal angiogenesis (Ferrara, N. (2010). Mol Biol Cell 21 (5): 687-690.).

[0003] Angiopoietin is part of the angiogenic growth factor family. ANG2 (also known as ANGPT2) belongs to the angiogenic family and is expressed by endothelial cells. It is usually stored in the Weibel-Paladebodies of endothelial cells. When encountering conditions that promote angiogenesis, such as hypoxia and inflammation, ANG2 is released from the Weibel-Paladebodies and competes with ANG1. As an antagonist of Tie2, it inhibits the stability of blood vessels, causes pericytes to detach from endothelial cells, increases endothelial cell permeability, and enables VEGF to better play its role in promoting angiogenesis (Saharinen, P., et al. (2017). Nat Rev Drug Discov 16(9): 635-661.). At the same time, many studies have shown that ANG2 overexpression plays a key role in promoting tumor lymph node metastasis, so ANG2 is an important target for pathological angiogenesis-related diseases and tumor treatment (Holash, J., et al. (1999). Science 284 (5422): 1994-1998; Gengenbacher, N., et al. (2021). Cancer Discov 11 (2): 424-445.). In 2004, for the first time, antibodies that neutralize the interaction between ANG2 and its receptor Tie2 were used to confirm that blocking ANG2 activity can effectively inhibit tumor angiogenesis and tumor growth (Oliner, J., et al. (2004). Cancer Cell 6 (5): 507-516.).

[0004] The complementary coordination and joint action of VEGF and ANG2 in abnormal angiogenesis have been confirmed in in vivo functional experiments. The combination of antibodies targeting ANG2 and VEGF, as well as bispecific antibodies, showed significant tumor inhibition effects in animal models, which were superior to the efficacy of monoclonal antibodies targeting any target (Hashizume, H., et al. (2010). Cancer Res 70(6):2213-2223. Kienast, Y., et al. (2013). Clin Cancer Res 19(24):6730-6740.). Therefore, simultaneous blocking of VEGF and ANG2 can more effectively inhibit abnormal angiogenesis and achieve the purpose of treating diseases related to angiogenesis.

[0005] Currently, several bispecific antibodies that simultaneously block VEGF and ANG2 signaling pathways and their uses are reported in the art and are in the clinical stage, including Vanucizumab (see, for example, WO2010040508A1), Faricimab (see, for example, WO2014009465A1) and BI836880 (see, for example, US9527925B2). However, there is still an urgent need to develop new and highly effective anti-ANG2-VEGF bispecific antibodies. Summary of the invention

[0006] In one aspect, the present invention provides a bispecific antibody comprising a first antigen-binding moiety that binds ANG2 and a second antigen-binding moiety that binds VEGF, wherein the first antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences are selected from any one of (1) to (3): (1) the HCDR1 sequence of SEQ ID NO:25; the HCDR2 sequence of SEQ ID NO:26; the HCDR3 sequence of SEQ ID NO:27; the LCDR1 sequence of SEQ ID NO:28; the LCDR2 sequence of SEQ ID NO:29; and the LCDR3 sequence of SEQ ID NO:30; (2) the HCDR1 sequence of SEQ ID NO:33; the HCDR2 sequence of SEQ ID NO:34; the HCDR3 sequence of SEQ ID NO:35; the LCDR1 sequence of SEQ ID NO:36; and the LCDR3 sequence of SEQ ID NO:37. LCDR2 sequence shown in SEQ ID NO:37; and LCDR3 sequence shown in SEQ ID NO:38; (3) HCDR1 sequence shown in SEQ ID NO:41; HCDR2 sequence shown in SEQ ID NO:42; HCDR3 sequence shown in SEQ ID NO:43; LCDR1 sequence shown in SEQ ID NO:44; LCDR2 sequence shown in SEQ ID NO:45; and LCDR3 sequence shown in SEQ ID NO:46.

[0007] In one embodiment, the heavy chain variable region and the light chain variable region are selected from any one of (1)-(3): (1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31; a light chain variable region comprising the amino acid sequence of SEQ ID NO:32; (2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:39; a light chain variable region comprising the amino acid sequence of SEQ ID NO:40; (3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47; a light chain variable region comprising the amino acid sequence of SEQ ID NO:48.

[0008] In one embodiment, the second antigen binding moiety comprises an immunoglobulin single variable domain (VHH) that binds VEGF. In a preferred embodiment, the immunoglobulin single variable domain comprises: a CDR1 sequence as shown in SEQ ID NO: 49, a CDR2 sequence as shown in SEQ ID NO: 50, and a CDR3 sequence as shown in SEQ ID NO: 51.

[0009] In one embodiment, said first antigen binding moiety and said second antigen binding moiety are connected via a linker.

[0010] In one embodiment, the bispecific antibody of the invention further comprises a heavy chain constant region of human IgG1 and a human kappa light chain constant region. In a preferred embodiment, the heavy chain constant region of human IgG1 comprises a mutation from leucine to alanine at positions 234 and 235 according to EU numbering.

[0011] The present invention also provides a polynucleotide encoding the bispecific antibody of the present invention.

[0012] The present invention also provides an expression vector comprising the polynucleotide of the present invention.

[0013] The present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention.

[0014] The present invention further provides a method for producing the bispecific antibody of the present invention, comprising:

[0015] (I) culturing the host cell of the present invention under appropriate conditions to express the bispecific antibody, and

[0016] (II) isolating the bispecific antibody from the host cell or its culture.

[0017] The present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier.

[0018] The present invention also provides use of the bispecific antibody or pharmaceutical composition of the present invention in preparing a medicament for treating the following diseases: (1) angiogenesis-related eye diseases; or (2) cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figures 1A-1E A schematic diagram of the structure of the anti-ANG2-VEGF bispecific antibody is shown: Figure 1A , BsAb1, BsAb13 and BsAb21; Figure 1B , BsAb2, BsAb14 and BsAb22; Figure 1C , BsAb3, BsAb15 and BsAb23; Figure 1D , BsAb4, BsAb16 and BsAb24; Figure 1E , BsAb29, BsAb30 and BsAb31.

[0020] Figure 2A-2E The binding activity of the anti-ANG2-VEGF bispecific antibody to the recombinant protein hVEGF-His is shown.

[0021] Figures 3A-3E The binding activity of the anti-ANG2-VEGF bispecific antibody to the recombinant protein hANG2-His is shown.

[0022] Figures 4A-4C The anti-ANG2-VEGF bispecific antibody was shown to neutralize VEGF activity.

[0023] Figure 5A-5B The blocking activity of anti-ANG2-VEGF bispecific antibody on ANG2 / Tie2 was shown.

[0024] Figures 6A-6C The anti-ANG2-VEGF bispecific antibody showed activity in inhibiting HUVEC cell proliferation.

[0025] Figures 7A-7C The anti-ANG2-VEGF bispecific antibody showed the effect of inhibiting the growth of subcutaneous transplanted human colon cancer cells: Fig. 7A The mean tumor volume of each group of mice over time is shown; Figure 7B The mean body weight of mice in each group over time is shown; Figure 7C The mean tumor weight of each group of mice at the end of the experiment is shown. DETAILED DESCRIPTION

[0026] definition

[0027] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0028] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of" are encompassed within the meaning of the expression "comprising."

[0029] As used herein, "bispecific antibody" refers to an antibody that specifically binds to two different antigens. The bispecific antibody of the present invention comprises an antigen-binding portion that specifically binds to two different antigens, ANG2 and VEGF.

[0030] As used herein, "antibody" refers to an immunoglobulin or a fragment thereof, which specifically binds to an antigenic epitope through at least one antigen binding site. The term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen binding fragments. Antibodies can be synthetic (e.g., produced by chemical coupling or biological coupling), enzymatically treated, or recombinantly produced. Antibodies include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b). Antibodies can be multivalent, i.e., contain multiple antigen binding sites. For example, antibodies can be divalent, trivalent, tetravalent, pentavalent, and hexavalent, which contain two, three, four, five, and six antigen binding sites, respectively.

[0031] As used herein, "antigen-binding fragment" refers to a portion of a full-length antibody that is less than the full length but contains at least a portion of the variable region of the full-length antibody (e.g., contains one or more CDRs and / or one or more antigen-binding sites), and thus retains at least a portion of the ability of the full-length antibody to specifically bind to an antigen. Examples of antigen-binding fragments include, but are not limited to, sdAb (e.g., the variable domain of a heavy chain antibody), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab') 2 , diabodies, Fd and Fd' fragments, and other fragments (e.g., fragments comprising modifications).

[0032] As used herein, a "full-length antibody" generally comprises four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Generally speaking, the heavy chain constant region of a full-length antibody may comprise CH1-hinge region-CH2-CH3 from N-terminus to C-terminus. In certain immunoglobulin types (e.g., IgM and IgE), the heavy chain constant region may comprise CH1-hinge region-CH2-CH3-CH4 from N-terminus to C-terminus.

[0033] The light chain variable region and the heavy chain variable region can each include three highly variable "complementarity determining regions (CDR)" and four relatively conservative "framework regions (FR)", and are connected in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus. Herein, the CDRs (CDRL or LCDR) of the light chain variable region can be referred to as LCDR1, LCDR2 and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region can be referred to as HCDR1, HCDR2 and HCDR3.

[0034] The CDR of an antibody can be defined by a variety of methods in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (see, e.g., Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on antibody sequence variability (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), AbM (Martin, ACR and J. Allen (2007) "Bioinformatics tools for antibody engineering," in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp.95-118), Contact (MacCallum, RM et al., (1996) J. Mol. Biol. 262:732-745), IMGT (Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined as defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection requested in the claims of the present invention is based on the sequences shown in the AbM definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the protection scope of the present invention.

[0035] Therefore, when referring to antibodies defined by specific CDR sequences defined herein, the scope of the antibodies also covers antibodies whose variable region sequences comprise the specific CDR sequences but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0036] As used herein, the terms "framework region" and "framework region" can be used interchangeably. As used herein, the terms "framework region", "framework region" or "FR" residues refer to those amino acid residues in the antibody variable region other than the CDR sequences defined above.

[0037] It is generally believed that the "Fv" fragment composed of a VH and a VL through non-covalent interaction is the smallest antigen-binding fragment containing an antigen-binding site. However, a single variable domain (single domain antibody) also has antigen-binding ability. VH and VL can be connected by a peptide linker to obtain a "single-chain Fv (scFv)". By introducing disulfide bonds into Fv or scFv, a "disulfide-stabilized Fv (dsFv)" or a "single-chain disulfide-stabilized Fv (scdsFv or dsscFv)" can be obtained respectively.

[0038] As used herein, "Fab" contains a complete antibody light chain (VL-CL) and an antibody heavy chain variable region and a heavy chain constant region (VH-CH1, also called Fd). A single-chain "Fab (scFab)" can be obtained by connecting CL and CH1 in "Fab" with a peptide linker. "F(ab') 2 "Essentially comprises two Fab fragments connected by a disulfide bond in the hinge region. "Fab'" is F(ab') 2 half of which can be obtained by reducing F(ab') 2 The disulfide bonds in the hinge region are obtained.

[0039] As used herein, "single domain antibody (sdAb)" or "nanobody" refers to an antibody comprising a single immunoglobulin variable domain (single variable domain) as a functional antigen binding fragment. Similar to the variable region of a full-length antibody, a single variable domain generally comprises CDR1, CDR2, and CDR3 that form an antigen binding site and a supporting framework region. A single variable domain can be, for example, a variable domain of a heavy-chain antibody (VHH), a shark's IgNAR variable domain, a human light chain antibody variable domain, and a heavy chain antibody variable domain.

[0040] As used herein, "percent (%) sequence identity" and "sequence identity" of amino acid sequences have a definition recognized in the art, which refers to the percentage of identity between two polypeptide sequences determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). It can be determined using methods known to those skilled in the art, such as using publicly available computer software such as BLAST, BLAST-2, Clustal Omega and FASTA software.

[0041] The polypeptide (e.g., CDR region, framework region, and constant region of an antibody) can be modified, for example, by substitution, addition, and / or deletion of one or more amino acids without changing the function of the polypeptide. The substitution is preferably a conservative substitution of amino acids. Suitable conservative substitutions are well known to those skilled in the art. In addition, antibodies can be modified using methods known in the art to change their properties, such as changing the type of antibody glycosylation modification, changing the ability to form interchain disulfide bonds, changing the effector function of the antibody (e.g., enhancing or reducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)), or providing active groups for the preparation of antibody conjugates. Such modified antibodies are also encompassed within the scope of the antibodies of the present invention.

[0042] "Affinity" or "binding affinity" is a measure of the strength of the non-covalent binding between an antibody and an antigen. The magnitude of "affinity" is usually reported as the equilibrium dissociation constant, K. D or EC 50 . K D It can be calculated by measuring the equilibrium association constant (ka) and the equilibrium dissociation constant (kd): D = kd / ka. Affinity can be determined using conventional techniques known in the art, such as biomembrane interferometry (e.g., ForteBio Octet or Gator detection systems can be used), surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS).

[0043] As used herein, the expression "isolated" means that a substance (eg, a polynucleotide or a polypeptide) is separated from its source or environment, ie, substantially does not contain any other components. The bispecific antibody of the present invention or the polynucleotide encoding the same may be isolated.

[0044] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives. Polynucleotides may include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0045] In this article, "vector" is a medium for introducing exogenous polynucleotides into host cells, and when the vector is transformed into an appropriate host cell, the exogenous polynucleotides are amplified or expressed. As used herein, the definition of vector covers plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. Viral vectors include but are not limited to retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes virus vectors, poxvirus vectors and baculovirus vectors, etc. As used herein, "expression vector" refers to a vector capable of expressing a polypeptide of interest. An expression vector can generally include a polynucleotide sequence encoding a polypeptide of interest and a regulatory sequence (such as a promoter and a ribosome binding site) operably connected thereto.

[0046] As used herein, "host cell" is a cell for receiving, maintaining, replicating or amplifying a vector. Host cells can also be used to express polynucleotides or polypeptides encoded by vectors. Host cells can be eukaryotic cells or prokaryotic cells. Prokaryotic cells are such as Escherichia coli (E. coli) or Bacillus subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus, insect cells (such as S2 Drosophila cells or Sf9) and animal cells (such as fibroblasts, CHO cells, COS cells, HeLa cells, NSO cells or HEK293 cells).

[0047] As used herein, the term "treatment" refers to the improvement of a disease / symptom, such as alleviating or eliminating the disease / symptom, preventing or slowing the occurrence, progression and / or worsening of the disease / symptom.

[0048] As used herein, "effective amount" means the amount of biological or medical response or desired therapeutic effect induced by an active substance (e.g., an antibody or pharmaceutical composition of the present invention) for a tissue, system, animal, mammal or human. Therefore, an "effective amount" can be the amount required to prevent, cure, improve, block or partially block a disease or symptom (e.g., cancer). Those skilled in the art can determine the effective amount based on factors such as the age, physical condition, sex, severity of symptoms, specific composition or route of administration of the subject.

[0049] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0050] As used herein, examples of mammals include, but are not limited to, humans, non-human primates, rats, mice, cows, horses, pigs, sheep, alpacas, dogs, cats, etc. As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject is a patient, such as a cancer patient.

[0051] Anti-ANG2-VEGF bispecific antibody

[0052] In one aspect, the present invention provides a bispecific antibody comprising a first antigen-binding moiety that binds ANG2 and a second antigen-binding moiety that binds VEGF, wherein the first antigen-binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences are selected from any one of (1) to (3): (1) the HCDR1 sequence of SEQ ID NO:25; the HCDR2 sequence of SEQ ID NO:26; the HCDR3 sequence of SEQ ID NO:27; the LCDR1 sequence of SEQ ID NO:28; the LCDR2 sequence of SEQ ID NO:29; and the LCDR3 sequence of SEQ ID NO:30; (2) the HCDR1 sequence of SEQ ID NO:33; the HCDR2 sequence of SEQ ID NO:34; the HCDR3 sequence of SEQ ID NO:35; the LCDR1 sequence of SEQ ID NO:36; and the LCDR3 sequence of SEQ ID NO:37. LCDR2 sequence shown in SEQ ID NO:37; and LCDR3 sequence shown in SEQ ID NO:38; (3) HCDR1 sequence shown in SEQ ID NO:41; HCDR2 sequence shown in SEQ ID NO:42; HCDR3 sequence shown in SEQ ID NO:43; LCDR1 sequence shown in SEQ ID NO:44; LCDR2 sequence shown in SEQ ID NO:45; and LCDR3 sequence shown in SEQ ID NO:46.

[0053] In some embodiments, the heavy chain variable region comprises an amino acid sequence of SEQ ID NO:31, SEQ ID NO:39 or SEQ ID NO:47. In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO:31, SEQ ID NO:39 or SEQ ID NO:47. In some embodiments, the heavy chain variable region polypeptide comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and / or deletions compared to SEQ ID NO:31, SEQ ID NO:39 or SEQ ID NO:47. Preferably, the amino acid substitutions, additions and / or deletions do not occur in the CDR regions.

[0054] In some embodiments, the light chain variable region comprises an amino acid sequence of SEQ ID NO:32, SEQ ID NO:40 or SEQ ID NO:48. In some embodiments, the light chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO:32, SEQ ID NO:40 or SEQ ID NO:48. In some embodiments, the light chain variable region comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and / or deletions compared to SEQ ID NO:32, SEQ ID NO:40 or SEQ ID NO:48. Preferably, the amino acid substitutions, additions and / or deletions do not occur in the CDR regions.

[0055] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:31, SEQ ID NO:39 or SEQ ID NO:47; the light chain variable region comprises the amino acid sequence of SEQ ID NO:32, SEQ ID NO:40 or SEQ ID NO:48.

[0056] In a specific embodiment, the heavy chain variable region and the light chain variable region are selected from any one of (1)-(3): (1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31; a light chain variable region comprising the amino acid sequence of SEQ ID NO:32; (2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:39; a light chain variable region comprising the amino acid sequence of SEQ ID NO:40; (3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47; a light chain variable region comprising the amino acid sequence of SEQ ID NO:48.

[0057] The first antigen binding portion and the second antigen binding portion can comprise any form of antigen binding fragment. In some embodiments, the first antigen binding portion is scFv, Fab, Fab', F(ab') 2 , Fv fragments or disulfide-stabilized Fv (dsFv). In one embodiment, the first antigen-binding portion is a fully human antibody or an antigen-binding portion thereof.

[0058] In some embodiments, the second antigen binding moiety comprises an immunoglobulin single variable domain that binds to VEGF. In one embodiment, the single variable domain is a VHH. In one embodiment, the single variable domain is a humanized VHH. In one embodiment, the immunoglobulin single variable domain comprises: a CDR1 sequence as shown in SEQ ID NO: 49, a CDR2 sequence as shown in SEQ ID NO: 50, and a CDR3 sequence as shown in SEQ ID NO: 51. In a specific embodiment, the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 11. In another specific embodiment, the immunoglobulin single variable domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 11.

[0059] In some embodiments, the first antigen binding moiety and the second antigen binding moiety are connected by a linker. The linker can be a peptide linker or a chemical bond, preferably a peptide linker (also referred to as a "connecting peptide"). The peptide linker can contain no more than 30 amino acids. Exemplary peptide linkers can include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S) or a combination thereof, such as GGAS, GGGS, GGGSG or (G 4 S) n, wherein n is an integer from 1 to 20. Preferably, n is an integer from 1 to 5. In a specific embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO:12.

[0060] In some embodiments, the bispecific antibody of the present invention further comprises an immunoglobulin constant region. The immunoglobulin constant region can be derived from the heavy chain constant region (CH) and the light chain constant region (CL) of immunoglobulins of any species.

[0061] The heavy chain constant region can be derived from any subtype (e.g., IgA, IgD, IgE, IgG, and IgM), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b) of an immunoglobulin heavy chain constant region or a combination thereof. In a preferred embodiment, the heavy chain constant region comprises at least an Fc region. In a preferred embodiment, the heavy chain constant region is a heavy chain constant region of human IgG1, an exemplary amino acid sequence of which is shown in SEQ ID NO: 1. In one embodiment, the bispecific antibody of the present invention further comprises an Fc region of human IgG1, an exemplary amino acid sequence of which is shown in SEQ ID NO: 3.

[0062] In some embodiments, the bispecific antibodies of the invention have reduced ADCC and CDC effector functions, for example by comprising amino acid substitutions (mutations) in the heavy chain constant region. In some embodiments, the bispecific antibodies of the invention may comprise a heavy chain constant region of human IgG1. In an exemplary embodiment, the heavy chain constant region of human IgG1 may comprise a mutation selected from the following: according to EU numbering, the leucine at position 234 (corresponding to position 117 of SEQ ID NO: 1) is mutated to alanine (L234A), the leucine at position 235 (corresponding to position 118 of SEQ ID NO: 1) is mutated to alanine (L235A), the aspartic acid at position 265 (corresponding to position 148 of SEQ ID NO: 1) is mutated to alanine (D265A), the asparagine at position 297 (corresponding to position 180 of SEQ ID NO: 1) is mutated to alanine (N297A) or glycine (N297G) or glutamine (N297Q), the lysine at position 322 (corresponding to position 205 of SEQ ID NO: 1) is mutated to alanine (K322A), the proline at position 329 (corresponding to position 212 of SEQ ID NO: 1) is mutated to alanine (P329A), and combinations thereof. In some embodiments, the bispecific antibody of the present invention may comprise a heavy chain constant region of human IgG4. In an exemplary embodiment, the heavy chain constant region of human IgG4 may comprise a mutation selected from the following: according to EU numbering, the serine at position 228 is mutated to proline (S228P), the phenylalanine at position 234 is mutated to alanine (F234A), the leucine at position 235 is mutated to alanine (L235A), and combinations thereof.

[0063] As used herein, "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to an antibody that binds to an antigen on a target cell (via its Fc region) that binds to an Fc receptor (FcR) on the surface of a cytotoxic cell (e.g., a natural killer (NK) cell, a neutrophil, and a macrophage), thereby targeting these cytotoxic cells to a target cell, and then killing the target cell using cytotoxins. The ADCC activity of an antibody can be assessed by methods known in the art (see, e.g., US5821337A).

[0064] As used herein, "complement dependent cytotoxicity (CDC)" refers to the activation of the complement system by an antibody bound to an antigen on a target cell, thereby causing lysis of the target cell. The CDC activity of an antibody can be assessed by methods known in the art (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996)).

[0065] The light chain constant region can be derived from a lambda light chain or a kappa light chain constant region. In a preferred embodiment, the light chain constant region is a human kappa light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 2.

[0066] In some embodiments, the bispecific antibodies of the present invention further comprise a heavy chain constant region (CH) and a light chain constant region (CL) of an immunoglobulin. In one embodiment, the heavy chain constant region is a heavy chain constant region of human IgG1 or human IgG4, and the light chain constant region is a human kappa light chain constant region. In one embodiment, the heavy chain constant region of human IgG1 comprises a mutation (L234A / L235A) of leucine to alanine at positions 234 and 235 according to EU numbering. In one embodiment, the heavy chain constant region of human IgG4 comprises a mutation (S228P / F234A / L235A) of serine to proline, phenylalanine to alanine and leucine to alanine at positions 228, 234 and 235 according to EU numbering.

[0067] In one embodiment, the VH and VL of the first antigen binding moiety are fused to the N-terminus of CH and CL, respectively, and the single variable domain (e.g., VHH) of the second antigen binding moiety is fused to the N-terminus of the VH, the N-terminus of the VL, the C-terminus of the CH and / or the C-terminus of the CL, optionally via a linker.

[0068] In some embodiments, the bispecific antibody of the present invention comprises a first polypeptide and a second polypeptide. In one embodiment, the first polypeptide comprises a heavy chain variable region (VH) and a heavy chain constant region (CH) of the first antigen binding moiety, the second polypeptide comprises a light chain variable region (VL) and a light chain constant region (CL) of the first antigen binding moiety, and the single variable domain (e.g., VHH) of the second antigen binding moiety is optionally fused to: (1) the N-terminus of the VH; (2) the N-terminus of the VL; (3) the N-termini of the VH and VL; (4) the C-terminus of the CH; (5) the C-terminus of the CL; (6) the C-termini of the CH and CL; (8) the N-terminus of the VH and the C-terminus of the CH; or (9) the N-terminus of the VL and the C-terminus of the CH.

[0069] In one embodiment, the first polypeptide and the second polypeptide are selected from any one of (1)-(5):

[0070] (1) The first polypeptide has a structure of formula (I), and the second polypeptide has a structure of formula (II)

[0071] VH-CH-Linker-VHH Formula (Ⅰ),

[0072] VL-CL formula (Ⅱ);

[0073] (2) The first polypeptide has a structure of formula (III), and the second polypeptide has a structure of formula (II)

[0074] VHH-Linker-VH-CH formula (III);

[0075] (3) The first polypeptide has a structure of formula (III), and the second polypeptide has a structure of formula (IV)

[0076] VHH-Linker-VL-CL formula (IV);

[0077] (4) The first polypeptide has a structure of formula (V), and the second polypeptide has a structure of formula (IV)

[0078] VH-CH formula (V);

[0079] (5) The first polypeptide has a structure of formula (V), and the second polypeptide has a structure of formula (VI)

[0080] VL-CL-Linker-VHH Formula (VI);

[0081] in

[0082] VH and VL are the heavy chain variable region and light chain variable region of the first antigen binding moiety, respectively;

[0083] VHH is a single variable domain of the second antigen binding moiety;

[0084] CH and CL are the heavy chain constant region and light chain constant region of immunoglobulin, respectively;

[0085] Linker is a connector.

[0086] In a specific embodiment, the first polypeptide and the second polypeptide are selected from any one of (1)-(15):

[0087] (1) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 6;

[0088] (2) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 6;

[0089] (3) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 15;

[0090] (4) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 16;

[0091] (5) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 17; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 8;

[0092] (6) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 8;

[0093] (7) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 19;

[0094] (8) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 20;

[0095] (9) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 10;

[0096] (10) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 10;

[0097] (11) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23;

[0098] (12) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 24;

[0099] (13) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 15;

[0100] (14) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 19;

[0101] (15) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 22; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23.

[0102] In one embodiment, the first polypeptide comprises a leucine to alanine mutation at the following amino acid positions: 238 and 239 (L238A / L239A) of SEQ ID NO:5 or SEQ ID NO:13; 242 and 243 (L242A / L243A) of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:17 or SEQ ID NO:21; 382 and 383 (L382A / L383A) of SEQ ID NO:14; or 386 and 387 (L386A / L387A) of SEQ ID NO:18 or SEQ ID NO:22.

[0103] In a specific embodiment, the first polypeptide and the second polypeptide are selected from any one of (1)-(15):

[0104] (1) a first polypeptide comprising an amino acid sequence having L238A and L239A mutations compared to SEQ ID NO: 13; a second polypeptide comprising an amino acid sequence of SEQ ID NO: 6;

[0105] (2) a first polypeptide comprising an amino acid sequence having L382A and L383A mutations compared to SEQ ID NO: 14; a second polypeptide comprising an amino acid sequence of SEQ ID NO: 6;

[0106] (3) a first polypeptide comprising an amino acid sequence having L238A and L239A mutations compared to SEQ ID NO: 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 15;

[0107] (4) a first polypeptide comprising an amino acid sequence having L238A and L239A mutations compared to SEQ ID NO: 5; a second polypeptide comprising an amino acid sequence of SEQ ID NO: 16;

[0108] (5) a first polypeptide comprising an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 17; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 8;

[0109] (6) a first polypeptide comprising an amino acid sequence having L386A and L387A mutations compared to SEQ ID NO: 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 8;

[0110] (7) a first polypeptide comprising an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 7; a second polypeptide comprising an amino acid sequence of SEQ ID NO: 19;

[0111] (8) a first polypeptide comprising an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 20;

[0112] (9) a first polypeptide comprising an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 21; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 10;

[0113] (10) a first polypeptide comprising an amino acid sequence having L386A and L387A mutations compared to SEQ ID NO: 22; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 10;

[0114] (11) a first polypeptide comprising an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 9; a second polypeptide comprising an amino acid sequence of SEQ ID NO: 23;

[0115] (12) a first polypeptide comprising an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 24;

[0116] (13) a first polypeptide comprising an amino acid sequence having L382A and L383A mutations compared to SEQ ID NO: 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 15;

[0117] (14) a first polypeptide comprising an amino acid sequence having L386A and L387A mutations compared to SEQ ID NO: 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 19;

[0118] (15) A first polypeptide comprising an amino acid sequence having L386A and L387A mutations compared to SEQ ID NO: 22; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23.

[0119] The bispecific antibodies of the present invention can be multivalent antibodies, such as bivalent, trivalent, tetravalent, pentavalent and hexavalent. In some embodiments, the bispecific antibodies of the present invention are tetravalent antibodies, which comprise two first antigen binding moieties and two second antigen binding moieties. In one embodiment, the bispecific antibodies of the present invention are hexavalent antibodies, which comprise two first antigen binding moieties and four second antigen binding moieties.

[0120] In one embodiment, the bispecific antibody of the present invention comprises two first polypeptides and two second polypeptides, wherein the first polypeptides are identical or different, and the second polypeptides are identical or different. In a specific embodiment, the bispecific antibody of the present invention comprises two identical first polypeptides and two identical second polypeptides. The first polypeptide and the second polypeptide are as described above.

[0121] In some embodiments, the bispecific antibodies of the invention specifically bind to ANG2 and VEGF. In some embodiments, the bispecific antibodies of the invention block the binding of ANG2 and VEGF to their respective receptors. In some embodiments, the bispecific antibodies of the invention specifically bind to ANG2, but do not bind or do not substantially bind to ANG1. As used herein, the expression "does not bind" or "does not substantially bind" means that the binding ability of the bispecific antibodies of the invention to ANG1 is significantly low relative to the binding ability to ANG2, for example as described in Example 4.

[0122] Antibodies or their antigen binding fragments can be prepared and produced using methods known in the art. Such methods may include, for example, preparing and separating the encoding nucleic acid of antibodies or antigen binding fragments from phage display libraries, yeast display libraries, immortalized B cells (e.g., mouse B cell hybridoma cells or EBV immortalized B cells). Methods for immunizing animals can also be used, such as immunizing animals (e.g., humanized mice) with antigens or DNA encoding antigens, and then separating the B cells expressing antibodies from the immunized animals. Polynucleotides encoding antibodies or their antigen binding fragments can also be prepared by separation or chemical synthesis from immune animals or human bodies, and then expression vectors expressing antibodies or antigen binding fragments are constructed using polynucleotides.

[0123] Polynucleotides, vectors and host cells

[0124] In another aspect, the present invention provides a polynucleotide encoding the bispecific antibody of the present invention.

[0125] The polynucleotides of the present invention can be obtained using methods known in the art. For example, the polynucleotides of the present invention can be isolated from human body, phage display library, yeast display library, immune animal, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells) or chemical synthesis. The polynucleotides can be codon optimized for the host cell used for expression.

[0126] In yet another aspect, the present invention also provides an expression vector comprising the polynucleotide of the present invention. The expression vector may further comprise additional polynucleotide sequences, such as transcriptional regulatory sequences and antibiotic resistance genes.

[0127] The present invention also provides a host cell comprising a polynucleotide or expression vector of the present invention. Various methods known in the art can be used to introduce the polynucleotide or expression vector of the present invention into a suitable host cell. Such methods include, but are not limited to, viral transduction, liposome transfection, electroporation, and calcium phosphate transfection, etc. In a preferred embodiment, the host cell is used to express the bispecific antibody of the present invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK293 cells, Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for expressing antibodies.

[0128] The present invention also provides a method for producing the bispecific antibody of the present invention, comprising:

[0129] (I) culturing the host cell of the present invention under appropriate conditions to express the bispecific antibody of the present invention, and

[0130] (II) isolating the bispecific antibody from the host cell or its culture.

[0131] Pharmaceutical composition

[0132] The present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier.

[0133] Pharmaceutically acceptable carriers may include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitan For compositions comprising antibodies or antibody conjugates, suitable carriers may be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delay agents, chelating agents and emulsifiers. For compositions comprising antibodies or antibody conjugates, suitable carriers may be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer) or combinations thereof.

[0134] The pharmaceutical compositions provided herein can be in a variety of dosage forms, including but not limited to solid, semisolid, liquid, powder or lyophilized forms. Preferably, the pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion).

[0135] The pharmaceutical compositions provided herein can be administered by a variety of routes. The route of administration includes, but is not limited to, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular or intracavitary), topical (e.g., intratumoral), epidural or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual or topical). The method of administration can be, for example, injection or infusion.

[0136] As a general guide, the dosage range of the bispecific antibodies of the present invention can be about 0.0001 to 100 mg / kg, more generally 0.01 to 20 mg / kg of the subject's body weight. For example, the dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight or 20 mg / kg body weight, or in the range of 1-20 mg / kg. Exemplary treatment regimens require administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or a slightly shorter initial dosing interval and a longer later dosing interval.

[0137] treat

[0138] Without wishing to be bound by any theory, the bispecific antibodies of the present invention block the ANG2 and VEGF signaling pathways by binding to ANG2 and VEGF, including blocking angiogenesis associated with the ANG2 and VEGF signaling pathways. As used herein, "angiogenesis" refers to the formation of new blood vessels. Studies have shown that angiogenesis is associated with a variety of diseases, such as cancer and angiogenesis-related eye diseases.

[0139] In a general aspect, a bispecific antibody or pharmaceutical composition of the present invention is provided for use in cancer and angiogenesis-related eye diseases. The present invention also provides the use of the bispecific antibody or pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer and angiogenesis-related eye diseases. The present invention also provides a method for treating cancer and angiogenesis-related eye diseases in a subject, comprising administering an effective amount of the bispecific antibody or pharmaceutical composition of the present invention to the subject.

[0140] Angiogenesis-related eye diseases can be eye diseases associated with choroidal and retinal vascular diseases, including but not limited to choroidal neovascular diseases, retinal neovascular diseases, and diseases associated with vascular leakage. In one embodiment, angiogenesis-related eye diseases are macular degeneration (e.g., dry or wet age-related macular degeneration (AMD)), retinal vein occlusion, retinopathy, retinopathy of prematurity (ROP), diabetic retinopathy, neovascular glaucoma, pathological myopia, macular edema, retinal edema, diabetic macular edema (DME), or choroidal neovascular disease.

[0141] As used herein, the term "cancer" or "tumor" refers to or describes a physiological condition in an individual that is generally characterized by unregulated cell growth. Preferably, the cancer is a solid tumor associated with angiogenesis. Cancer can include primary cancers and metastatic cancers. Non-limiting examples of cancer include lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, skin cancer, head and neck cancer, melanoma, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, prostate cancer, uterine cancer, Hodgkin's lymphoma, esophageal cancer, anal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, kidney cancer (e.g., renal cell carcinoma, renal pelvic cancer, and adrenal cancer), soft tissue sarcoma, bladder cancer, central nervous system (CNS) tumors, mesothelioma, glioma, meningioma, and pituitary adenoma. In a preferred embodiment, the cancer is colorectal cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, or hepatocellular carcinoma.

[0142] Reagent test kit

[0143] The present invention also provides a kit comprising an anti-ANG2-VEGF bispecific antibody or pharmaceutical composition of the present invention. In one embodiment, the kit further comprises instructions for use of the bispecific antibody or pharmaceutical composition of the present invention. The kit may also comprise a suitable container, such as an ampoule. In some embodiments, the kit further comprises a device for administration. The kit may also comprise a label indicating the intended use and / or method of use of the contents of the kit. The term "label" includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

[0144] Beneficial Effects

[0145] The anti-ANG2-VEGF bispecific antibodies of the present invention achieve at least one of the following beneficial effects:

[0146] (1) specifically binds to ANG2 but does not bind or substantially binds to ANG1;

[0147] (2) specifically binds to and neutralizes VEGF;

[0148] (3) blocking the binding of ANG2 to Tie2;

[0149] (4) inhibiting angiogenesis associated with ANG2 and VEGF activity; and

[0150] (5) Inhibit tumor growth.

[0151] Example

[0152] The invention generally described herein will be more easily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. The experimental methods in the following examples where specific conditions are not indicated are carried out according to conventional methods and conditions, or according to the commercial instructions.

[0153] Example 1 Preparation of raw materials

[0154] 1.1 Antigen preparation

[0155] The coding sequence of human IgG1 Fc segment (SEQ ID NO: 3) or His tag was connected to the 3' end of the coding sequence of antigen VEGF (hVEGF, UniProt ID: P15692-4, SEQ ID NO: 52) and antigen Ang2 (hANG2, UniProt ID: O15123-1, SEQ ID NO: 53) by PCR amplification, and then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination to prepare the expression vectors of recombinant proteins hVEGF-Fc, hANG2-Fc, hANG2-His and hVEGF-His. The recombinant protein expression vectors were transformed into Escherichia coli DH5α, cultured at 37°C overnight, and then the plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) and the plasmids were extracted by Expi293 TM The expression system kit (ThermoFisher, A14635) was used for expression. The transient transfection method was described in the kit manual.

[0156] After 7 days of transfection, the cell expression supernatant was centrifuged at high speed for 10 min. hANG2-Fc and hVEGF-Fc expression supernatants were affinity purified with COLUMN XK16 / 20 (purchased from Cytiva), and then the target protein was eluted with 100 mM sodium acetate (pH 3.0), followed by neutralization with 1 M Tris-HCl; hANG2-His or hVEGF-His expression supernatant was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with gradient concentrations of imidazole. The eluted proteins were exchanged into PBS buffer through ultrafiltration concentration tubes (Millipore, UFC901096). After SDS-PAGE identification and activity identification, they were frozen at -80°C for use.

[0157] 1.2 Preparation of positive control antibody

[0158] The anti-VEGF positive control antibodies used were Bevacizumab (synthesized according to the antibody sequence information provided in patent US9527925B2) and P30-10-26 (VHH VEGF-huFc, amino acid sequence as shown in SEQ ID NO:4); the anti-ANG2 positive control antibody used was Nesvacumab (synthesized according to the sequence disclosed in patent application US20170355762A1) and the fully human antibody 17 of IgG1 type independently developed by the applicant (the heavy chain amino acid sequence is shown in SEQ ID NO:5, and the light chain amino acid sequence is shown in SEQ ID NO:6), 78A14 (the heavy chain amino acid sequence is shown in SEQ ID NO:7, and the light chain amino acid sequence is shown in SEQ ID NO:8) and 78A74-7 (the heavy chain amino acid sequence is shown in SEQ ID NO:9, and the light chain amino acid sequence is shown in SEQ ID NO:10); the anti-ANG2-VEGF positive control antibody used was Faricimab (synthesized according to the antibody sequence information provided in patent WO2016073157A1, purchased from Taizhou Baiying Biotechnology Co., Ltd.).

[0159] Except for Faricimab, the other control antibodies mentioned above were expressed using the ExpiCHO transient expression system (Gibco, A29133). The resulting supernatant was filtered through a 0.22 μm filter membrane and purified using a Protein A / G affinity chromatography column affinity method. The target protein was then eluted with 100 mM sodium acetate (pH 3.0), then neutralized with 1 M Tris-HCl, and finally replaced with PBS buffer using an ultrafiltration concentration tube (Millipore, UFC901096). The purified antibodies were identified by SDS-PAGE and activity assay and then stored in aliquots at -80°C.

[0160] 1.3 Cell line construction

[0161] A HEK293 cell line overexpressing human Tie2 (hereinafter referred to as huTie2-HEK293 cell line) and a HEK293 cell line overexpressing VEGFR2 and NFAT luciferase reporter gene (hereinafter referred to as VEGFR2 / NFAT-HEK293 cell line) were constructed. The cell line construction methods are as follows:

[0162] 1.3.1 Construction of huTie2-HEK293 cell line

[0163] A DNA fragment encoding human Tie2 protein (NCBI Gene ID: 7010) was synthesized by gene synthesis technology and cloned into the expression vector pLVX-Puro (Clontech, 632164) to obtain Tie2 expression plasmid. HEK293 cells ( CRL-1573 TMThe Tie2 expression plasmid was introduced into HEK293 cells using Invitrogen's electroporation kit (Cat. No.: MPK10096) and electroporator (Cat. No.: MP922947). Monoclonal cell lines were screened in a culture medium containing puromycin at a final concentration of 2 μg / mL, and huTie2-HEK293 cell lines were obtained by FACS identification.

[0164] 1.3.2 Construction of VEGFR2 / NFAT-HEK293 cell line

[0165] First, the NFAT-HEK293 cell line was constructed: the pGL4.30 plasmid (Promega, #E8481) containing the luciferase gene (whose transcription and expression are driven by the NFAT response element (NFAT-RE)) was electroporated by an Invitrogen, Neon TM Transfection System, MP922947) and electroporated into HEK293 cells ( CRL-1573 TM ), and then the monoclonal cell line was screened and identified by using Hygromycin B (source culture, S160J7) at a final concentration of 500 μg / mL. After the NFAT-HEK293 cell line was identified as qualified, the plasmid expressing VEGFR2 (gene sequence see NCBI Gene ID: 3791, synthesized by Universal Bio) was electroporated into the NFAT-HEK293 cell line by the method of Example 1.3.1, and the VEGFR2 / NFAT-HEK293 cell line was obtained by FACS identification.

[0166] Example 2 Construction of anti-ANG2-VEGF bispecific antibody

[0167] This example describes the construction of an anti-ANG2-VEGF bispecific antibody (BsAb), wherein the first antigen-binding moiety uses the heavy chain variable region (VH) and light chain variable region (VL) of the anti-ANG2 fully human antibody 17, 78A14 or 78A74-7 (HCDR1-3 and LCDR1-3 as well as the amino acid sequences of VH and VL as defined by AbM (SEQ ID NO: ) are shown in Table 1A), and the second antigen-binding moiety uses the VHH of the humanized anti-VEGF single domain antibody P30-10-26 (the amino acid sequences of CDR1, CDR2 and CDR as defined by AbM are shown in SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, respectively; the amino acid sequence of VHH is shown in SEQ ID NO: 11). The bispecific antibody of the present invention further comprises a human IgG1 heavy chain constant region (SEQ ID NO: 1) or a human IgG1 heavy chain constant region mutant (abbreviated as LALA; leucine at positions 117 and 118 of SEQ ID NO: 1 are mutated to alanine) and a human Kappa light chain constant region (SEQ ID NO: 2), which are fused to the C-termini of VH and VL of the first antigen binding portion, respectively; and a connecting peptide (SEQ ID NO: 12). Figures 1A-1E The schematic diagram of the structure of the candidate bispecific antibody is shown. The candidate bispecific antibody contains two identical first polypeptides and two identical second polypeptides, and the amino acid sequences are shown in Table 1B.

[0168] The expression vectors of the first polypeptide and the second polypeptide were prepared as follows: the target fragments were amplified by PCR, the fragments were connected by overlapping extension PCR, and then the fragments were constructed by homologous recombination to obtain the expression vectors of the first polypeptide and the second polypeptide. The expression vectors of the first polypeptide and the second polypeptide were transformed into Escherichia coli DH5α, and then the plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain the expression plasmids of the first polypeptide chain and the second polypeptide chain without endotoxin for eukaryotic expression.

[0169] Table 1A

[0170] Antibody Name HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 VH V L 17 25 26 27 28 29 30 31 32 78A14 33 34 35 36 37 38 39 40 78A74-7 41 42 43 44 45 46 47 48

[0171] Table 1B

[0172]

[0173] Example 3 Expression and purification of anti-ANG2-VEGF bispecific antibody

[0174] The anti-ANG2-VEGF bispecific antibody of Example 2 was expressed using the ExpiCHO transient expression system (ThermoFisher, A29133). The specific method was as follows: On the day of transfection, the cell density was confirmed to be 7×10 6 Up to 1×10 7 The cell viability was >98%. At this time, the cells were adjusted to a final concentration of 6×10 cells / mL using fresh ExpiCHO expression medium pre-warmed at 37°C. 6 cells / mL. Use OptiPRO pre-cooled at 4°C TM The target plasmid was diluted in SFM (1 μg of plasmid was added to 1 mL of the culture medium) and OptiPRO TM Dilute ExpiFectamine in SFM TM CHO, and then mix the two in equal volumes and gently pipette to mix to prepare ExpiFectamine TM The CHO / plasmid DNA mixture was incubated at room temperature for 1-5 min, then slowly added to the prepared cell suspension while gently shaking, and finally placed in a cell culture shaker at 37°C and 8% CO. 2 18-22h after transfection, add ExpiCHO TM Enhancer and ExpiCHO TM Feed the flask and place it in a shaker at 32°C and 5% CO 2 On the 5th day after transfection, add the same volume of ExpiCHO TM Feed, slowly add and gently mix the cell suspension. 7-15 days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10 minutes, and the resulting supernatant was affinity purified with COLUMN XK16 / 20 (purchased from Cytiva), and then the target protein was eluted with 100mM sodium acetate (pH3.0), followed by neutralization with 1M Tris-HCl, and finally the resulting protein was replaced into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096). The purified antibody was identified by SDS-PAGE and SEC-HPLC to have a purity greater than 90%, and was stored in aliquots at -80°C.

[0175] Example 4 Antigen Binding Activity Analysis of Anti-ANG2-VEGF Bispecific Antibodies

[0176] 4.1 ELISA method to detect the binding activity of anti-ANG2-VEGF bispecific antibody to recombinant protein hVEGF-His

[0177] The recombinant protein hVEGF-His was coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, different concentrations of the antibody to be tested were added and incubated for 1 hour. After that, after washing three times with PBST, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 hour. After the incubation was completed, the plate was washed six times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the OD450 value was read using an enzyme reader (Molecular Devices, SpecterMax 190). Using PRISM TM The data were analyzed by GraphPad Software (San Diego, CA), and the EC was calculated. 50 value.

[0178] ELISA binding assay results Figure 2A-2E As shown, the candidate bispecific antibody exhibited binding activity to the recombinant protein hVEGF-His comparable to that of antibodies P30-10-26 and Faricimab.

[0179] 4.2 ELISA method to detect the binding activity of candidate bispecific antibodies to recombinant protein hANG2-His

[0180] The recombinant protein hANG2-His was coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, different concentrations of the antibody to be tested were added and incubated for 1 hour. After that, after washing three times with PBST, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 hour. After the incubation was completed, the plate was washed six times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the OD450 value was read using an enzyme reader (Molecular Devices, SpecterMax 190). Using PRISM TM The data were analyzed by GraphPad Software (San Diego, CA), and the EC was calculated. 50 value.

[0181] ELISA binding assay results Figures 3A-3EAs shown, the candidate bispecific antibodies exhibited binding activity to the recombinant protein hANG2-His comparable to antibodies 17, 78A14, and 78A74-7, and were significantly better than the positive bispecific antibody Faricimab.

[0182] 4.3 ELISA-based detection of binding activity of anti-ANG2 fully human antibodies to hANG1-Fc

[0183] 30 μL / well of 2 μg / mL hANG1-Fc (fusion protein of hANG1 (Uniprot ID: Q15389-1) and human IgG1 Fc segment (SEQ ID NO: 3); see Example 1.1 for preparation method) was coated on a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed 3 times with PBST and blocked with 5% skim milk for 2 h. After washing the plate 3 times with PBST, a gradient dilution of the antibody to be tested was added and incubated for 1 h. After that, anti-human Kappa HRP (Millipore, AP502P) was added and incubated for 1 h after washing 3 times with PBST. After incubation, the plate was washed six times with PBST, TMB was added for color development, 2M HCl was added to terminate the reaction according to the color development results, and the OD450 value was read by an enzyme reader.

[0184] The results showed that antibodies 17, 78A14 and 78A74-7 did not bind to hANG1.

[0185] Example 5 Detection of VEGF neutralization activity of anti-ANG2-VEGF bispecific antibody

[0186] The VEGFR2 / NFAT-HEK293 cell line constructed in Example 1 was used to screen candidate bispecific antibodies capable of neutralizing VEGF activity. In this cell line culture system, VEGF protein activates the transcriptional activity of the intracellular transcription factor NFAT through the VEGF-VEGFR2 signal axis, thereby initiating the transcription and expression of the luciferase reporter gene; when the candidate bispecific antibody is added, the candidate bispecific antibody neutralizes VEGF and blocks its binding to VEGFR2, thereby blocking the expression of the luciferase reporter gene.

[0187] The specific method is as follows: VEGFR2 / NFAT-HEK293 cell line was revived, cells that had been passaged 2-4 times and were in good growth condition were used for the experiment, the cells were resuspended after trypsin digestion, centrifuged at room temperature and 300g to remove the supernatant, and then the cells were resuspended in DMEM medium, and the cell density was adjusted to 4×10 after counting. 5cells / mL, add 100 μL per well to a new 96-well plate and place it in a 37°C cell culture incubator. At the same time, use DMEM medium to gradient dilute the antibody to be tested, add 60 ng / mL recombinant protein hVEGF-Fc, mix and incubate at room temperature for 30 minutes. Subsequently, the co-incubated antibody / hVEGF-Fc mixture is added to the 96-well plate and cultured in a 37°C incubator for 18 hours. After the incubation, add 30 μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) to each well, shake for 2 minutes, detect the fluorescence value of the 96-well plate and calculate the IC 50 .

[0188] The results are as follows Figures 4A-4C As shown in Tables 2A-2C, the activity of candidate bispecific antibodies BsAb3, BsAb14, BsAb30, BsAb31, BsAb22 and BsAb29 in neutralizing VEGF is comparable to that of P30-10-26; the effect of candidate bispecific antibodies BsAb14-LALA and BsAb22-LALA in neutralizing VEGF is comparable to that of P30-10-26 and Faricimab.

[0189] Table 2A

[0190] Antibody Name <![CDATA[IC for neutralizing VEGF activity 50 (nM)]]> BsAb3 0.194 BsAb14 0.213 BsAb22 0.418 P30-10-26 0.292

[0191] Table 2B

[0192] Antibody Name <![CDATA[IC for neutralizing VEGF activity 50 (nM)]]> BsAb29 1.130 BsAb30 0.608 BsAb31 0.754 P30-10-26 0.878

[0193] Table 2C

[0194] Antibody Name <![CDATA[IC that neutralizes VEGF activity 50 (nM)]]> BsAb14-LALA 0.469 BsAb22-LALA 0.401 Faricimab 0.582 P30-10-26 0.482

[0195] Example 6 Detection of the activity of anti-ANG2-VEGF bispecific antibody in blocking the binding of ANG2 to Tie2 based on FACS method

[0196] The binding activity of candidate antibodies to block ANG2 and cell surface expressed receptor Tie2 was evaluated based on the FACS method. The specific method is as follows: the antibody to be tested, which was serially diluted in FACS buffer (1×PBS+2% FBS), was added to a 96-well round bottom plate at 100 μL per well. Then, 2 μg / mL biotin-labeled hANG2-Fc protein (biotin-hANG2-Fc) in 100 μL FACS buffer was added to the 96-well plate, and then incubated at 4°C for 1 hour.

[0197] huTie2-HEK293 cells that had been passaged 2-4 and were in good growth condition were used for the experiment. 6Cells in FACS buffer of 100 μL / well were added to a new 96-well round-bottom plate, centrifuged at 4°C / 300g, and the supernatant was removed; then the pre-incubated antibody / biotin-hANG2-Fc mixture was added to the cells, 180 μL per well, and then incubated at 4°C for 30 min; then the cells were centrifuged at 4°C / 300g and the supernatant was removed, then 200 μL / well of FACS buffer was added and the cells were resuspended, centrifuged at 4°C / 300g and the supernatant was removed; this step was repeated twice. PE-labeled Streptavidin (Invitrogen, 12-4317-87; 1:200 diluted in FACS buffer) was added to the well at 200 μL / well and the cells were gently pipetted to resuspend, and then the cells were placed at 4°C in the dark for 30 min. After the incubation, the cells were centrifuged at 4°C, 300g to remove the supernatant, and the cells were resuspended in FACS buffer, and this step was repeated twice. Finally, the amount of biotin-hANG2-Fc bound to the cells was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102) (expressed as the mean fluorescence intensity (MFI) of PE).

[0198] The results are as follows Figure 5A-5B and as shown in Tables 3A-3B. Among them, Figure 5A and Table 3A shows that the blocking effects of candidate bispecific antibodies BsAb14, BsAb30, BsAb31, and BsAb22 are comparable to antibodies 78A14 and 78A74-7; Figure 5B Table 3B shows that the blocking effects of candidate bispecific antibodies BsAb14-LALA and BsAb22-LALA are comparable to those of antibodies 78A14 and 78A74-7, and significantly better than Faricimab.

[0199] Table 3A

[0200] Antibody Name <![CDATA[Blocking IC 50 Value (nM)]]> BsAb14 0.415 BsAb22 0.350 BsAb30 0.362 BsAb31 0.299 78A14 0.390 78A74-7 0.226

[0201] Table 3B

[0202] Antibody Name <![CDATA[Blocking IC 50 Value (nM)]]> BsAb14-LALA 0.411 BsAb22-LALA 0.320 78A14 0.464 78A74-7 0.440 Faricimab 17.76

[0203] Example 7 Detection of the efficacy of anti-ANG2-VEGF bispecific antibody in inhibiting HUVEC proliferation

[0204] In this example, the inhibitory effect of anti-ANG2-VEGF bispecific antibodies BsAb14 and BsAb22 on VEGF-induced endothelial cell proliferation was verified. The specific implementation method is as follows: human umbilical vein endothelial cell line (HUVEC) (purchased from Beina Biotechnology) was revived, and cells that were passaged 2-4 times and in good growth state were used for the experiment. The cells were resuspended after trypsin digestion, centrifuged at room temperature and 300g to remove the supernatant, and then the cells were resuspended in EBM-2 medium with 0.5% FBS, and the cell density was adjusted to 5×10 after counting. 4 cells / mL, 50 μL per well was added to a new 96-well cell culture plate, and cultured in a 37°C cell culture incubator overnight. The next day, the antibody to be tested was gradiently diluted using EBM-2 medium, and 400 ng / mL recombinant protein hVEGF-Fc was added, mixed and incubated at room temperature for 30 minutes. Subsequently, the co-incubated antibody / hVEGF-Fc mixture was added to the 96-well cell plate and cultured in a 37°C incubator for 72 hours. After the culture was completed, 20 μL of MTS (Progema, G3581) was added to each well, and the OD492 value of the 96-well plate was detected after shaking for 2 minutes. The proliferation inhibition rate was calculated by the respective OD492, and the proliferation inhibition rate (%) = [1-(OD492 样品 -OD492 仅细胞 ) / (OD492 VEGF -OD492 仅细胞 )]×100%.

[0205] The results are as follows Figures 6A-6C As shown, the candidate bispecific antibodies BsAb14 and BsAb22 significantly inhibited VEGF-induced HUVEC proliferation, and their activity was comparable to that of P30-10-26; at higher antibody concentrations, for example, when the antibody concentration reached 100 nM, the activity of P30-10-26 in inhibiting VEGF-induced HUVEC proliferation was better than that of Bevacizumab.

[0206] Example 8 Detection of the efficacy of anti-ANG2-VEGF bispecific antibody in inhibiting Colo205 tumor cell animal model

[0207] The tumor inhibition effects of anti-ANG2-VEGF bispecific antibodies BsAb3, BsAb14 and BsAb22 in the human colon cancer cell Colo205 transplanted tumor model were verified and compared with antibodies Nesvacumab, Bevacizumab and P30-10-26. 6 Colo205 cells (purchased from Beina Biotechnology) were subcutaneously injected into 6-8 week old male nude mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). The experimental mice were raised under standard conditions. 3Around 2:00 p.m., the mice were divided into groups and cages and given medication. There were 8 tumor-bearing nude mice in each group, for a total of 7 groups, including 3 candidate bispecific antibody groups (BsAb3 3.4952 mg / kg, BsAb14 3.5224 mg / kg, and BsAb22 3.528 mg / kg; equimolar doses), 3 positive control antibody groups (Nesvacumab 3 mg / kg and Bevacizumab 3 mg / kg, equimolar doses with the bispecific antibodies; P30-10-26 3 mg / kg, 2.3 times the molar dose of the bispecific antibodies) and a PBS negative control group. The medication was given by intraperitoneal injection, and the dose was as follows: Figures 7A-7C As shown, the drug was administered once every 3-4 days, and the tumor volume (V = (L × W) was measured twice a week. 2 ) / 2, where L is the longest tumor diameter and W is the shortest tumor diameter (mm), and the drug was administered 9 times in total. Tumors were taken 10 days after the end of the drug administration and the tumor weight was measured. The changes in the average tumor volume, tumor weight and body weight of each group of mice were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 7A-7C and Table 4.

[0208] like Figure 7B As shown in the figure, there was no significant difference in the body weight of mice in each group, indicating that the mice tolerated the antibody well. Fig. 7A , 7C As shown in Table 4, the tumors in the mice in the PBS group grew fastest. Compared with the PBS group, all antibodies had significant tumor inhibition effects. At equimolar doses, the tumor inhibition effects of the candidate bispecific antibodies BsAb3, BsAb14, and BsAb22 were superior to those of Nesvacumab and Bevacizumab. Surprisingly, the tumor inhibition effects of BsAb14 and BsAb22 were comparable to those of the anti-VEGF antibody P30-10-26, which had a molar number 2.3 times that of the anti-VEGF antibody, showing excellent anti-tumor activity.

[0209] Table 4

[0210]

[0211] Those skilled in the art will further recognize that the present invention may be implemented in other specific forms without departing from its spirit or central features. Since the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. On the contrary, the scope and content of the present invention should be indicated with reference to the appended claims.

[0212] Sequence Listing

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[0221] Sequence Listing <110> Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. <120> Anti-ANG2-VEGF bispecific antibodies and uses thereof <130> I2021TC6223CS <160> 53 <170> PatentIn version 3.5 <210> 1 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> hIgG1 heavy chain constant region <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> hIgG1 CL (Kappa) <400> 2 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 3 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 Fc <400> 3 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 4 <211> 361 <212> PRT <213> Artificial Sequence <220> <223> P30-10-26 VHH-huFc <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 130 135 140 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 145 150 155 160 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 165 170 175 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 180 185 190 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 195 200 205 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 210 215 220 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 225 230 235 240 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 245 250 255 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 260 265 270 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 275 280 285 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 290 295 300 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 305 310 315 320 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 325 330 335 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 340 345 350 Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 5 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> 17 / BsAb3 / BsAb4 heavy chain <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Thr Ile Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser Val Tyr 65 70 75 80 Leu Leu Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 6 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> 17 / BsAb1 / BsAb2 light chain <400> 6 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Glu Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 7 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> 78A14 / BsAb15 / BsAb16 heavy chain <400> 7 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Val Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 8 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> 78A14 / BsAb13 / BsAb14 light chain <400> 8 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Pro Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Val Ser Gln His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 9 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 / BsAb23 / BsAb24 heavy chain <400> 9 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 10 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 / BsAb21 / BsAb22 light chain <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Met Ser Asn Trp Pro Pro 85 90 95 Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 11 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> P30-10-26 VHH <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker peptide <400> 12 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 13 <211> 595 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of BsAb1 <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Thr Ile Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser Val Tyr 65 70 75 80 Leu Leu Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 465 470 475 480 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp 485 490 495 Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 500 505 510 Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp 515 520 525 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 530 535 540 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 545 550 555 560 Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His 565 570 575 Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 580 585 590 Val Ser Ser 595 <210> 14 <211> 595 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of BsAb2 / BsAb29 <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 165 170 175 Thr Ile Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Leu Val 180 185 190 Ala Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn Tyr Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser Val Tyr 210 215 220 Leu Leu Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Arg Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser Trp Gly 245 250 255 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 260 265 270 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 275 280 285 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 290 295 300 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 305 310 315 320 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 325 330 335 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 340 345 350 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 355 360 365 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 370 375 380 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 385 390 395 400 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 405 410 415 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 420 425 430 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 435 440 445 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 450 455 460 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 465 470 475 480 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 485 490 495 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 500 505 510 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 515 520 525 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 530 535 540 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 545 550 555 560 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 565 570 575 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 580 585 590 Pro Gly Lys 595 <210> 15 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> Light chain of BsAb3 / BsAb29 <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 145 150 155 160 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 165 170 175 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 180 185 190 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 195 200 205 Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 210 215 220 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Glu Ser Tyr Pro Leu 225 230 235 240 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 245 250 255 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 260 265 270 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 275 280 285 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 290 295 300 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 305 310 315 320 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 325 330 335 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 340 345 350 Phe Asn Arg Gly Glu Cys 355 <210> 16 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> BsAb4 light chain <400> 16 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Glu Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 225 230 235 240 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 245 250 255 Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 260 265 270 Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn 275 280 285 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 290 295 300 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 305 310 315 320 Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu 325 330 335 Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr 340 345 350 Leu Val Thr Val Ser Ser 355 <210> 17 <211> 599 <212> PRT <213> Artificial Sequence <220> <223> BsAb13 heavy chain <400> 17 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Val Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 465 470 475 480 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 485 490 495 Phe Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly 500 505 510 Lys Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr 515 520 525 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 530 535 540 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 545 550 555 560 Thr Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser 565 570 575 Leu Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly 580 585 590 Thr Leu Val Thr Val Ser Ser 595 <210> 18 <211> 599 <212> PRT <213> Artificial Sequence <220> <223> BsAb14 / BsAb30 heavy chain <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Val Tyr 165 170 175 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 180 185 190 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 210 215 220 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met 245 250 255 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 260 265 270 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 275 280 285 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 290 295 300 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 305 310 315 320 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 325 330 335 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 340 345 350 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 355 360 365 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 370 375 380 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 385 390 395 400 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 405 410 415 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 420 425 430 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 435 440 445 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 450 455 460 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 465 470 475 480 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 485 490 495 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 500 505 510 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 515 520 525 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 530 535 540 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 545 550 555 560 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 565 570 575 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 580 585 590 Leu Ser Leu Ser Pro Gly Lys 595 <210> 19 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> BsAb15 / BsAb30 light chain <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 145 150 155 160 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 165 170 175 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 180 185 190 Tyr Gly Pro Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 195 200 205 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 210 215 220 Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Val Ser Gln His 225 230 235 240 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 245 250 255 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 260 265 270 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 275 280 285 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 290 295 300 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 305 310 315 320 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 325 330 335 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 340 345 350 Phe Asn Arg Gly Glu Cys 355 <210> 20 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> BsAb16 light chain <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Pro Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Val Ser Gln His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 225 230 235 240 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 245 250 255 Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 260 265 270 Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn 275 280 285 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 290 295 300 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 305 310 315 320 Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu 325 330 335 Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr 340 345 350 Leu Val Thr Val Ser Ser 355 <210> 21 <211> 599 <212> PRT <213> Artificial Sequence <220> <223> BsAb21 heavy chain <400> 21 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 465 470 475 480 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 485 490 495 Phe Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly 500 505 510 Lys Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr 515 520 525 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 530 535 540 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 545 550 555 560 Thr Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser 565 570 575 Leu Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly 580 585 590 Thr Leu Val Thr Val Ser Ser 595 <210> 22 <211> 599 <212> PRT <213> Artificial Sequence <220> <223> BsAb22 / BsAb31 heavy chain <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 165 170 175 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 180 185 190 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 210 215 220 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met 245 250 255 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 260 265 270 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 275 280 285 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 290 295 300 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 305 310 315 320 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 325 330 335 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 340 345 350 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 355 360 365 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 370 375 380 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 385 390 395 400 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 405 410 415 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 420 425 430 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 435 440 445 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 450 455 460 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 465 470 475 480 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 485 490 495 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 500 505 510 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 515 520 525 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 530 535 540 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 545 550 555 560 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 565 570 575 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 580 585 590 Leu Ser Leu Ser Pro Gly Lys 595 <210> 23 <211> 360 <212> PRT <213> Artificial Sequence <220> <223> BsAb23 / BsAb31 light chain <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly 100 105 110 Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 145 150 155 160 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 165 170 175 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 180 185 190 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 195 200 205 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 210 215 220 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Met Ser Asn Trp Pro Pro 225 230 235 240 Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 245 250 255 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 260 265 270 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 275 280 285 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 290 295 300 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 305 310 315 320 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 325 330 335 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 340 345 350 Lys Ser Phe Asn Arg Gly Glu Cys 355 360 <210> 24 <211> 360 <212> PRT <213> Artificial Sequence <220> <223> BsAb24 light chain <400> 24 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Met Ser Asn Trp Pro Pro 85 90 95 Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly 210 215 220 Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly 225 230 235 240 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 245 250 255 Gly Phe Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro 260 265 270 Gly Lys Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu 275 280 285 Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 290 295 300 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 305 310 315 320 Asp Thr Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile 325 330 335 Ser Leu Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln 340 345 350 Gly Thr Leu Val Thr Val Ser Ser 355 360 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 17 HCDR1 <400> 25 Gly Phe Thr Phe Asn Lys Tyr Thr Ile Asn 1 5 10 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 17 HCDR2 <400> 26 Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn 1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 17 HCDR3 <400> 27 Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser 1 5 10 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 17 LCDR1 <400> 28 Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala 1 5 10 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 17 LCDR2 <400> 29 Ala Ala Ser Thr Leu Gln Ser 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 17 LCDR3 <400> 30 Gln Gln Phe Glu Ser Tyr Pro Leu Thr 1 5 <210> 31 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 17 VH <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Thr Ile Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser Val Tyr 65 70 75 80 Leu Leu Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 32 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 17 VL <400> 32 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Glu Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 33 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 78A14 HCDR1 <400> 33 Gly Phe Thr Phe Asn Val Tyr Gly Met His 1 5 10 <210> 34 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 78A14 HCDR2 <400> 34 Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr 1 5 10 <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 78A14 HCDR3 <400> 35 Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 36 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A14 LCDR1 <400> 36 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A14 LCDR2 <400> 37 Gly Pro Ser Asn Arg Ala Thr 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 78A14 LCDR3 <400> 38 Gln His Tyr Gly Val Ser Gln His Thr 1 5 <210> 39 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 78A14 VH <400> 39 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Val Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 40 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 78A14 VL <400> 40 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Pro Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Val Ser Gln His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 41 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 HCDR1 <400> 41 Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn 1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 HCDR2 <400> 42 Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr 1 5 10 <210> 43 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 HCDR3 <400> 43 Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 44 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 LCDR1 <400> 44 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 45 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 LCDR2 <400> 45 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 46 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 LCDR3 <400> 46 Gln Leu Met Ser Asn Trp Pro Pro Glu Trp Thr 1 5 10 <210> 47 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 VH <400> 47 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 48 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 VL <400> 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Met Ser Asn Trp Pro Pro 85 90 95 Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> P30-10-26 CDR1 <400> 49 Gly Phe Gly Leu Asp Tyr Tyr Ala Ile Gly 1 5 10 <210> 50 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> P30-10-26 CDR2 <400> 50 Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn 1 5 10 <210> 51 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> P30-10-26 CDR3 <400> 51 Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly Leu Tyr 1 5 10 15 Glu Tyr Asp Tyr 20 <210> 52 <211> 191 <212> PRT <213> Homo sapiens <400> 52 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly 130 135 140 Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr 145 150 155 160 Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln 165 170 175 Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 180 185 190 <210> 53 <211> 496 <212> PRT <213> Homo sapiens <400> 53 Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala 1 5 10 15 Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 20 25 30 Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro 35 40 45 Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala 50 55 60 Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu 65 70 75 80 Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys 85 90 95 Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile 100 105 110 Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly 115 120 125 Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp 130 135 140 Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu 145 150 155 160 Leu Glu His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp 165 170 175 Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu 180 185 190 Lys Lys Val Leu Ala Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser 195 200 205 Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn 210 215 220 Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn 225 230 235 240 Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val Asn 245 250 255 Asn Leu Leu Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr 260 265 270 Val Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe 275 280 285 Lys Ser Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn 290 295 300 Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly 305 310 315 320 Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln 325 330 335 Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu 340 345 350 Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg 355 360 365 Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr 370 375 380 Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg 385 390 395 400 Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile 405 410 415 Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys 420 425 430 Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp Phe Asp 435 440 445 Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln 450 455 460 Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser 465 470 475 480 Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe 485 490 495

Claims

1. A bispecific antibody comprising a first antigen binding moiety that binds ANG2 and a second antigen binding moiety that binds VEGF, wherein the first antigen binding moiety comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences are selected from any one of (1) to (3): (1) HCDR1 sequence shown in SEQ ID NO:25; HCDR2 sequence shown in SEQ ID NO:26; HCDR3 sequence shown in SEQ ID NO:27; LCDR1 sequence shown in SEQ ID NO:28; LCDR2 sequence shown in SEQ ID NO:29; and the LCDR3 sequence shown in SEQ ID NO:30; (2) HCDR1 sequence shown in SEQ ID NO:33; HCDR2 sequence shown in SEQ ID NO:34; HCDR3 sequence shown in SEQ ID NO:35; LCDR1 sequence shown in SEQ ID NO:36; LCDR2 sequence shown in SEQ ID NO:37; and the LCDR3 sequence shown in SEQ ID NO:38; (3) HCDR1 sequence shown in SEQ ID NO:41; HCDR2 sequence shown in SEQ ID NO:42; HCDR3 sequence shown in SEQ ID NO:43; LCDR1 sequence shown in SEQ ID NO:44; LCDR2 sequence shown in SEQ ID NO:45; and the LCDR3 sequence shown in SEQ ID NO:46; and The second antigen binding portion comprises an immunoglobulin single variable domain (VHH) that binds to VEGF, wherein the immunoglobulin single variable domain comprises: a CDR1 sequence shown in SEQ ID NO:49, a CDR2 sequence shown in SEQ ID NO:50, and a CDR3 sequence shown in SEQ ID NO:

51.

2. The bispecific antibody of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:31, SEQ ID NO:39 or SEQ ID NO:

47.

3. The bispecific antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:32, SEQ ID NO:40 or SEQ ID NO:

48.

4. The bispecific antibody of claim 1, wherein the heavy chain variable region and the light chain variable region are selected from any one of (1) to (3). (1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 32; (2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39; a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40; (3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

48. The bispecific antibody of claim 1 , wherein the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO:

11.

6. The bispecific antibody of any one of claims 1 to 5, wherein the first antigen binding moiety and the second antigen binding moiety are connected via a linker. 7 . The bispecific antibody of claim 1 , further comprising a heavy chain constant region (CH) and a light chain constant region (CL) of an immunoglobulin. The bispecific antibody of claim 7 , wherein the heavy chain constant region is a heavy chain constant region of human IgG1, and the light chain constant region is a human kappa light chain constant region. 9 . The bispecific antibody of claim 8 , wherein the heavy chain constant region of human IgG1 comprises a leucine to alanine mutation at positions 234 and 235 according to EU numbering.

10. The bispecific antibody of claim 7, comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the heavy chain variable region (VH) and the heavy chain constant region (CH), the second polypeptide comprises the light chain variable region (VL) and the light chain constant region (CL), and the single variable domain of the second antigen binding portion is optionally fused via a linker to: (1) the N-terminus of the VH; (2) the N-terminus of the VL; (3) the N-termini of the VH and VL; (4) the C-terminus of the CH; (5) the C-terminus of the CL; (6) the C-termini of the CH and CL; (8) the N-terminus of the VH and the C-terminus of the CH; or (9) the N-terminus of the VL and the C-terminus of the CH.

11. The bispecific antibody of claim 10, wherein the first polypeptide and the second polypeptide are selected from any one of (1) to (12): (1) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having L238A and L239A mutations compared to SEQ ID NO: 13; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 6; (2) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having L382A and L383A mutations compared to SEQ ID NO: 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 6; (3) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having L238A and L239A mutations compared to SEQ ID NO: 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 15; (4) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 17; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 8; (5) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having L386A and L387A mutations compared to SEQ ID NO: 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 8; (6) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 19; (7) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 21; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 10; (8) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having L386A and L387A mutations compared to SEQ ID NO: 22; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 10; (9) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO: 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23; (10) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having L382A and L383A mutations compared to SEQ ID NO: 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 15; (11) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having L386A and L387A mutations compared to SEQ ID NO: 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 19; (12) A first polypeptide comprising the amino acid sequence of SEQ ID NO:22 or an amino acid sequence having L386A and L387A mutations compared to SEQ ID NO:22; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:

23. 12 . The bispecific antibody of claim 10 , comprising two of the first polypeptides and two of the second polypeptides, wherein the first polypeptides are identical or different, and the second polypeptides are identical or different.

13. A polynucleotide encoding the bispecific antibody of any one of claims 1 to 12.

14. An expression vector comprising the polynucleotide of claim 13.

15. A host cell comprising the polynucleotide of claim 13 or the expression vector of claim 14.

16. A method for producing the bispecific antibody of any one of claims 1 to 12, comprising: (I) culturing the host cell of claim 15 under suitable conditions to express the bispecific antibody, and (II) isolating the bispecific antibody from the host cell or its culture. 17 . A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

18. Use of the bispecific antibody of any one of claims 1 to 12 or the pharmaceutical composition of claim 17 in the preparation of a medicament for treating angiogenesis-related eye diseases, wherein the angiogenesis-related eye diseases are macular degeneration, retinal vein occlusion, retinopathy, retinopathy of prematurity, diabetic retinopathy, neovascular glaucoma, pathological myopia, macular edema, retinal edema, diabetic macular edema, or choroidal neovascular disease.

19. Use of the bispecific antibody of any one of claims 1 to 12 or the pharmaceutical composition of claim 17 in the preparation of a medicament for treating cancer, wherein the cancer is lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, prostate cancer, uterine cancer, Hodgkin lymphoma, esophageal cancer, anal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue sarcoma, bladder cancer, central nervous system (CNS) tumors, mesothelioma, glioma, meningioma or pituitary adenoma.

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