Anti-ANG2 antibodies and their uses

Abstract

The present invention relates to the field of biomedicine. Specifically, the present invention provides anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof, particularly fully human anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof. The present invention also provides polynucleotides encoding such anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof, as well as expression vectors and host cells for expressing such anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof. The present invention further provides methods for producing such anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof, and uses thereof.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention provides anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof, particularly fully human anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof. Background Art

[0002] Angiopoietin is part of the angiogenic growth factor family. Angiopoietin-2 (ANG2 or ANGPT2), a member of the angiopoietin family, is a secreted glycoprotein whose overexpression promotes angiogenesis. The direct receptor for ANG2 is TIE2, which binds to the angiopoietins ANG1, ANG2, and ANG4. As an orphan receptor, TIE1 cannot directly bind to ANG to be activated. However, upon ANG-TIE2 binding, a complex of TIE1 and TIE2 forms and becomes activated. The signaling pathways following ANG-TIE ligand binding can regulate vascular permeability, inflammation, and pathological vascular remodeling in tumors.

[0003] ANG proteins stimulate the translocation and localization of TIE receptors expressed on endothelial cells to cell-cell and cell-matrix junctions. ANG1 and ANG2 have opposing functions in regulating angiogenesis. ANG1, expressed by mesenchymal cells, acts as an agonist to activate TIE2, promoting endothelial cell survival, tight junction formation, and vascular stability. ANG2, on the other hand, is expressed by endothelial cells and normally stored in Weibel–Palade bodies (WPBs). Under conditions that promote angiogenesis, such as hypoxia and inflammation, ANG2 is released from WPBs and competes with ANG1, acting as a TIE2 antagonist to inhibit vascular stability and, together with VEGF and other proteins, promote angiogenesis. Furthermore, numerous studies have demonstrated that overexpression of ANG2 plays a key role in promoting tumor lymph node metastasis. Therefore, ANG2 is an important target for pathological angiogenesis-related diseases and tumor therapy.

[0004] In 2004, the first study demonstrated that blocking ANG2 activity could effectively inhibit tumor angiogenesis and growth by neutralizing the interaction between ANG2 and its receptor, TIE2 (Oliner, J., et al. (2004) Cancer Cell 6(5):507-516). Therefore, the development of monoclonal antibodies targeting ANG2 has great application value in the treatment of solid tumors and ocular diseases related to pathological angiogenesis.

[0005] Anti-ANG2 antibodies are available in the prior art (see, for example, WO2015179166A1 and WO2011014469A1), including nesvacumab, a fully human antibody targeting ANG2 that is currently under clinical development. There is still a desire in the art to develop new and improved fully human anti-ANG2 antibodies. Summary of the Invention

[0006] In one aspect, the present invention provides a heavy chain variable region polypeptide that specifically binds to ANG2, comprising HCDR1, HCDR2, and HCDR3 sequences, wherein

[0007] (a) The HCDR1 sequence is shown in SEQ ID NO: 110

[0008] GFTFX1X2YX3MX4 (SEQ ID NO:110)

[0009] Wherein X1 is S or N; X2 is S or V; X3 is S or G; X4 is N or H;

[0010] (b) the HCDR2 sequence is shown as VISYDGSNKY (SEQ ID NO: 5);

[0011] (c) The HCDR3 sequence is shown in SEQ ID NO: 111

[0012] X5TLDGYTAGYYYGMDV (SEQ ID NO:111)

[0013] Where X5 is A or E.

[0014] In another aspect, the present invention provides an anti-ANG2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region polypeptide and a light chain variable region polypeptide, wherein the heavy chain variable region polypeptide comprises HCDR1, HCDR2 and HCDR3 sequences, and the light chain variable region polypeptide comprises LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2 and HCDR3 sequences are as shown above, and the LCDR1, LCDR2 and LCDR3 sequences are selected from any one of (1) to (21):

[0015] (1) the LCDR1 sequence set forth in SEQ ID NO:7; the LCDR2 sequence set forth in SEQ ID NO:8; and the LCDR3 sequence set forth in SEQ ID NO:68; (2) the LCDR1 sequence set forth in SEQ ID NO:82; the LCDR2 sequence set forth in SEQ ID NO:8; and the LCDR3 sequence set forth in SEQ ID NO:83; (3) the LCDR1 sequence set forth in SEQ ID NO:7; the LCDR2 sequence set forth in SEQ ID NO:8; and the LCDR3 sequence set forth in SEQ ID NO:84; (4) the LCDR1 sequence set forth in SEQ ID NO:7; the LCDR2 sequence set forth in SEQ ID NO:8; and the LCDR3 sequence set forth in SEQ ID NO:85; (5) the LCDR1 sequence set forth in SEQ ID NO:7; the LCDR2 sequence set forth in SEQ ID NO:8; and the LCDR3 sequence set forth in SEQ ID NO:86; (6) the LCDR1 sequence set forth in SEQ ID NO:7; the LCDR2 sequence set forth in SEQ ID NO:8; and the LCDR3 sequence set forth in SEQ ID NO:80; (7) the LCDR1 sequence set forth in SEQ ID NO:7; the LCDR2 sequence set forth in SEQ ID NO:8; and the LCDR3 sequence set forth in SEQ ID NO:81. NO:8; and the LCDR3 sequence shown in SEQ ID NO:39; (8) the LCDR1 sequence shown in SEQ ID NO:17; the LCDR2 sequence shown in SEQ ID NO:18; and the LCDR3 sequence shown in SEQ ID NO:19; (9) the LCDR1 sequence shown in SEQ ID NO:25; the LCDR2 sequence shown in SEQ ID NO:18; and the LCDR3 sequence shown in SEQ ID NO:59; (10) the LCDR1 sequence shown in SEQ ID NO:25; the LCDR2 sequence shown in SEQ ID NO:18; and the LCDR3 sequence shown in SEQ ID NO:36; (11) the LCDR1 sequence shown in SEQ ID NO:25; the LCDR2 sequence shown in SEQ ID NO:28; and the LCDR3 sequence shown in SEQ ID NO:29; (12) the LCDR1 sequence shown in SEQ ID NO:7; the LCDR2 sequence shown in SEQ ID NO:13; and the LCDR3 sequence shown in SEQ ID NO:14; (13) the LCDR1 sequence shown in SEQ ID NO:25; the LCDR2 sequence shown in SEQ ID NO:18; and the LCDR3 sequence shown in SEQ ID NO:59; LCDR2 sequence shown in SEQ ID NO:18; and LCDR3 sequence shown in SEQ ID NO:26; (14) LCDR1 sequence shown in SEQ ID NO:7; LCDR2 sequence shown in SEQ ID NO:8; and LCDR3 sequence shown in SEQ ID NO:9; (15) LCDR1 sequence shown in SEQ ID NO:63;The LCDR2 sequence shown in SEQ ID NO:64; and the LCDR3 sequence shown in SEQ ID NO:65; (16) the LCDR1 sequence shown in SEQ ID NO:25; the LCDR2 sequence shown in SEQ ID NO:32; and the LCDR3 sequence shown in SEQ ID NO:33; (17) the LCDR1 sequence shown in SEQ ID NO:42; the LCDR2 sequence shown in SEQ ID NO:43; and the LCDR3 sequence shown in SEQ ID NO:44; (18) the LCDR1 sequence shown in SEQ ID NO:50; the LCDR2 sequence shown in SEQ ID NO:51; and the LCDR3 sequence shown in SEQ ID NO:52; (19) the LCDR1 sequence shown in SEQ ID NO:46; the LCDR2 sequence shown in SEQ ID NO:47; and the LCDR3 sequence shown in SEQ ID NO:48; (20) the LCDR1 sequence shown in SEQ ID NO:21; the LCDR2 sequence shown in SEQ ID NO:22; and the LCDR3 sequence shown in SEQ ID NO:23; (21) the LCDR1 sequence shown in SEQ ID NO:50; the LCDR2 sequence shown in SEQ ID NO:51; and the LCDR3 sequence shown in SEQ ID NO:52; The LCDR1 sequence shown in SEQ ID NO: 54; the LCDR2 sequence shown in SEQ ID NO: 55; and the LCDR3 sequence shown in SEQ ID NO: 56.

[0016] In another aspect, the present invention provides an anti-ANG2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region polypeptide and a light chain variable region polypeptide, wherein the heavy chain variable region polypeptide comprises a HCDR1, HCDR2, and HCDR3 sequence, and the light chain variable region polypeptide comprises a LCDR1, LCDR2, and LCDR3 sequence, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequence are selected from any one of (1) to (3): (1) the HCDR1 sequence of SEQ ID NO: 87; the HCDR2 sequence of SEQ ID NO: 88; the HCDR3 sequence of SEQ ID NO: 89; the LCDR1 sequence of SEQ ID NO: 90; the LCDR2 sequence of SEQ ID NO: 91; and the LCDR3 sequence of SEQ ID NO: 92; (2) the HCDR1 sequence of SEQ ID NO: 95; the HCDR2 sequence of SEQ ID NO: 96; the HCDR3 sequence of SEQ ID NO: 97; the LCDR1 sequence of SEQ ID NO: 98; LCDR2 sequence shown in SEQ ID NO:99; and LCDR3 sequence shown in SEQ ID NO:100; (3) HCDR1 sequence shown in SEQ ID NO:103; HCDR2 sequence shown in SEQ ID NO:104; HCDR3 sequence shown in SEQ ID NO:105; LCDR1 sequence shown in SEQ ID NO:106; LCDR2 sequence shown in SEQ ID NO:91; and LCDR3 sequence shown in SEQ ID NO:107.

[0017] The present invention further provides a multispecific antibody comprising a first antigen-binding portion that specifically binds to ANG2 and a second antigen-binding portion that specifically binds to a second antigen, wherein the first antigen-binding portion comprises a heavy chain variable region polypeptide, antibody, or antigen-binding fragment thereof of the present invention.

[0018] The present invention also provides a polynucleotide encoding the heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, or multispecific antibody of the present invention.

[0019] The present invention also provides an expression vector comprising the polynucleotide of the present invention.

[0020] The present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention.

[0021] The present invention also provides a pharmaceutical composition comprising the heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, or multispecific antibody of the present invention, and a pharmaceutically acceptable carrier.

[0022] The present invention also relates to the use of the heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, multispecific antibody or pharmaceutical composition of the present invention in the preparation of a medicament for treating the following diseases: (1) angiogenesis-related eye disease; or (2) cancer. In one embodiment, the angiogenesis-related eye disease is 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. In another embodiment, 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's lymphoma, esophageal cancer, anal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue sarcoma, bladder cancer, central nervous system (CNS) tumor, mesothelioma, glioma, meningioma or pituitary adenoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A-1C The binding activity of candidate monoclonal supernatants to the antigen ANG2 determined by ELISA is shown.

[0024] Figures 2A-2C The blocking activity of the candidate monoclonal supernatants against the binding of ANG2 to the receptor TIE2 was shown based on an ELISA assay.

[0025] Figures 3A-3B The binding activity of candidate antibodies to the antigen ANG2 determined based on the ELISA method is shown.

[0026] Figure 4 The binding activity of the candidate antibodies to the antigen ANG1 determined based on the ELISA method is shown.

[0027] Figures 5A-5B The blocking activity of candidate antibodies to block the binding between ANG2 and receptor TIE2 based on ELISA assay is shown.

[0028] Figure 6 The blocking activity of candidate antibodies to block the binding of ANG2 and hTIE2-HEK293 cells based on FACS assay is shown.

[0029] Figure 7 The inhibitory activity of the candidate antibodies against ANG2-mediated TIE2 phosphorylation was shown.

[0030] Figures 8A-8C The inhibitory effect of the candidate antibodies on tumor growth in a mouse subcutaneous xenograft tumor model is shown; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0031] Figure 9 The in vivo plasma concentration-time curves of the candidate antibodies in Balb / C mice are shown. DETAILED DESCRIPTION

[0032] definition

[0033] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0034] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended and mean 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."

[0035] As used herein, "antibody" refers to an immunoglobulin or its fragment, which specifically binds to an antigenic epitope by 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 provided herein 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).

[0036] The molecules, antibodies or product numbers used herein are only used to distinguish or identify molecules or products, and are not intended to indicate that such identification is a feature of the molecules or products of the present invention. It will be understood by those skilled in the art that, for example, other molecules, antibodies or products may also use such identification for the purpose of distinguishing or identifying, but do not refer to the same or equivalent molecules, antibodies or products. Similarly, the similar numbering or identification used in the embodiments is also only for illustrative convenience, and the molecules, antibodies or products of the present invention are limited by the features described in the appended claims.

[0037] As used herein, "antigen-binding fragment" refers to a portion of a full-length antibody that is less than full-length but comprises at least a portion of the variable region of the full-length antibody (e.g., comprising one or more CDRs and / or one or more antigen-binding sites), and thus retains at least a portion of the full-length antibody's ability 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).

[0038] 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).

[0039] The light chain variable region and the heavy chain variable region can each include three highly variable "complementarity determining regions (CDRs)" and four relatively conserved "framework regions (FRs)", and are connected from the N-terminus to the C-terminus in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. 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.

[0040] In the present invention, the amino acid sequences of CDRs are all shown according to the AbM definition rules (the sequences in the claims of the present invention are also shown according to the AbM definition rules). However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (see, for example, 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, for example, 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 ColdSpring 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 as defined by any of the above-mentioned known schemes described herein.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 scope of protection of the present invention.

[0041] Thus, when referring to antibodies defined by specific CDR sequences defined herein, the scope of said antibodies also encompasses antibodies whose variable region sequences comprise said 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).

[0042] As used herein, the terms "framework region" and "framework region" are 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 excluding the CDR sequences as defined above.

[0043] An "Fv" fragment, consisting of a single VH and a single VL through non-covalent interactions, is generally considered the smallest antigen-binding fragment containing an antigen-binding site. However, a single variable domain (single-domain antibody) also possesses antigen-binding ability. A "single-chain Fv (scFv)" can be obtained by linking the VH and VL via a peptide linker. By introducing disulfide bonds into an Fv or scFv, a "disulfide-stabilized Fv (dsFv)" or "single-chain disulfide-stabilized Fv (scdsFv or dsscFv)" can be obtained, respectively.

[0044] As used herein, "Fab" comprises a complete antibody light chain (VL-CL) and an antibody heavy chain variable region and a heavy chain constant region (VH-CH1, also referred to as Fd). A single-chain "Fab (scFab)" can be obtained by linking the CL and CH1 in "Fab" with a peptide linker. "F(ab')2" essentially comprises two Fab fragments linked by a disulfide bond in the hinge region. "Fab'" is half of F(ab')2, which can be obtained by reducing the disulfide bond in the hinge region of F(ab')2.

[0045] As used herein, the terms "fully human antibody," "completely human antibody," and "human antibody" are used interchangeably and refer to an antibody produced by a human or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to an antibody produced by a human. The definition of a fully human antibody encompasses intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide. Fully human antibodies have low immunogenicity in humans. In some embodiments, the anti-ANG2 antibody or antigen-binding fragment thereof of the present invention is a fully human antibody.

[0046] As used herein, an "affinity matured" antibody comprises one or more modifications (e.g., substitutions of amino acid residues) in one or more CDRs such that the affinity matured antibody has improved affinity for the antigen compared to a parent antibody that does not comprise such modifications. Methods for affinity maturation of antibodies are known in the art, see, e.g., Marks et al., Bio / Technology 10:779-783 (1992); Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Scier et al., Gene 169:147-155 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0047] As used herein, "percent (%) sequence identity" or "sequence identity" of amino acid sequences has an art-recognized definition and refers to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). This can be determined using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0048] The polypeptide (e.g., the 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, 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.

[0049] "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 The equilibrium association constant (ka) and the equilibrium dissociation constant (kd) can be calculated: D = kd / ka. Affinity can be determined using conventional techniques known in the art, such as biomembrane interferometry (using, for example, the ForteBio Octet or Gator detection systems), surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS).

[0050] In this article, the "specific binding" between an antibody and an antigen refers to the binding between the antibody and the antigen with a high affinity. D The value may be at least about 10 -7 M to at least about 10 -10 M or less, such as at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 In some embodiments, the anti-ANG2 antibodies or antigen-binding fragments thereof of the invention have a K of 5 nM, 2 nM, 1 nM or less. D Value combined with ANG2.

[0051] As used herein, the term "isolated" refers to a substance (e.g., a polynucleotide or polypeptide) that is separated from its source or environment, i.e., substantially free of any other components. The anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments of the present invention, or polynucleotides encoding the same, may be isolated.

[0052] 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).

[0053] In this article, "vector" is a medium for introducing exogenous polynucleotides into a host cell, 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 encompasses 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, pox virus 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.

[0054] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, or amplify a vector. A host cell can also be used to express a polynucleotide or a polypeptide encoded by a vector. The host cell can be a eukaryotic cell or a prokaryotic cell. Prokaryotic cells include Escherichia coli (E. coli) or 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).

[0055] As used herein, the term "treating" refers to improving a disease / symptom, such as reducing or eliminating the disease / symptom, preventing or slowing down the occurrence, progression and / or worsening of the disease / symptom.

[0056] As used herein, an "effective amount" refers to the amount of an active substance (e.g., an antibody or pharmaceutical composition of the present invention) that induces a biological or medical response or desired therapeutic effect on a tissue, system, animal, mammal, or human. Thus, an "effective amount" can be the amount required to prevent, cure, improve, block, or partially block a disease or symptom (e.g., cancer). One skilled in the art can determine an effective amount based on factors such as the subject's age, physical condition, sex, severity of symptoms, specific composition, or route of administration.

[0057] 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, eg, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0058] 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.

[0059] Anti-ANG2 polypeptides, antibodies or antigen-binding fragments thereof

[0060] In one aspect, the present invention provides a heavy chain variable region polypeptide that specifically binds to ANG2, comprising HCDR1, HCDR2, and HCDR3 sequences, wherein

[0061] (a) The HCDR1 sequence is shown in SEQ ID NO: 110

[0062] GFTFX1X2YX3MX4 (SEQ ID NO:110)

[0063] Wherein X1 is S or N; X2 is S or V; X3 is S or G; X4 is N or H;

[0064] (b) the HCDR2 sequence is shown as VISYDGSNKY (SEQ ID NO: 5);

[0065] (c) The HCDR3 sequence is shown in SEQ ID NO: 111

[0066] X5TLDGYTAGYYYGMDV (SEQ ID NO:111)

[0067] Where X5 is A or E.

[0068] In a specific embodiment, the HCDR1, HCDR2 and HCDR3 sequences are selected from any one of (1)-(4):

[0069] (1) the HCDR1 sequence shown in SEQ ID NO: 62; the HCDR2 sequence shown in SEQ ID NO: 5; and the HCDR3 sequence shown in SEQ ID NO: 6;

[0070] (2) the HCDR1 sequence shown in SEQ ID NO: 62; the HCDR2 sequence shown in SEQ ID NO: 5; and the HCDR3 sequence shown in SEQ ID NO: 81;

[0071] (3) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; and the HCDR3 sequence shown in SEQ ID NO: 6;

[0072] (4) The HCDR1 sequence shown in SEQ ID NO: 12; the HCDR2 sequence shown in SEQ ID NO: 5; and the HCDR3 sequence shown in SEQ ID NO: 6.

[0073] In another embodiment, the anti-ANG2 antibody or antigen-binding fragment thereof further comprises a light chain variable region polypeptide comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences are selected from any one of (1) to (21):

[0074] (1) LCDR1 sequence shown in SEQ ID NO:7; LCDR2 sequence shown in SEQ ID NO:8; and LCDR3 sequence shown in SEQ ID NO:68;

[0075] (2) LCDR1 sequence shown in SEQ ID NO:82; LCDR2 sequence shown in SEQ ID NO:8; and LCDR3 sequence shown in SEQ ID NO:83;

[0076] (3) LCDR1 sequence shown in SEQ ID NO:7; LCDR2 sequence shown in SEQ ID NO:8; and LCDR3 sequence shown in SEQ ID NO:84;

[0077] (4) LCDR1 sequence shown in SEQ ID NO:7; LCDR2 sequence shown in SEQ ID NO:8; and LCDR3 sequence shown in SEQ ID NO:85;

[0078] (5) LCDR1 sequence shown in SEQ ID NO:7; LCDR2 sequence shown in SEQ ID NO:8; and LCDR3 sequence shown in SEQ ID NO:86;

[0079] (6) LCDR1 sequence shown in SEQ ID NO:7; LCDR2 sequence shown in SEQ ID NO:8; and LCDR3 sequence shown in SEQ ID NO:80;

[0080] (7) LCDR1 sequence shown in SEQ ID NO:7; LCDR2 sequence shown in SEQ ID NO:8; and LCDR3 sequence shown in SEQ ID NO:39;

[0081] (8) LCDR1 sequence shown in SEQ ID NO: 17; LCDR2 sequence shown in SEQ ID NO: 18; and LCDR3 sequence shown in SEQ ID NO: 19;

[0082] (9) LCDR1 sequence shown in SEQ ID NO: 25; LCDR2 sequence shown in SEQ ID NO: 18; and LCDR3 sequence shown in SEQ ID NO: 59;

[0083] (10) LCDR1 sequence shown in SEQ ID NO: 25; LCDR2 sequence shown in SEQ ID NO: 18; and LCDR3 sequence shown in SEQ ID NO: 36;

[0084] (11) LCDR1 sequence shown in SEQ ID NO: 25; LCDR2 sequence shown in SEQ ID NO: 28; and LCDR3 sequence shown in SEQ ID NO: 29;

[0085] (12) LCDR1 sequence shown in SEQ ID NO:7; LCDR2 sequence shown in SEQ ID NO:13; and LCDR3 sequence shown in SEQ ID NO:14;

[0086] (13) LCDR1 sequence shown in SEQ ID NO: 25; LCDR2 sequence shown in SEQ ID NO: 18; and LCDR3 sequence shown in SEQ ID NO: 26;

[0087] (14) LCDR1 sequence shown in SEQ ID NO: 7; LCDR2 sequence shown in SEQ ID NO: 8; and LCDR3 sequence shown in SEQ ID NO: 9;

[0088] (15) LCDR1 sequence shown in SEQ ID NO: 63; LCDR2 sequence shown in SEQ ID NO: 64; and LCDR3 sequence shown in SEQ ID NO: 65;

[0089] (16) LCDR1 sequence shown in SEQ ID NO: 25; LCDR2 sequence shown in SEQ ID NO: 32; and LCDR3 sequence shown in SEQ ID NO: 33;

[0090] (17) LCDR1 sequence shown in SEQ ID NO:42; LCDR2 sequence shown in SEQ ID NO:43; and LCDR3 sequence shown in SEQ ID NO:44;

[0091] (18) LCDR1 sequence shown in SEQ ID NO: 50; LCDR2 sequence shown in SEQ ID NO: 51; and LCDR3 sequence shown in SEQ ID NO: 52;

[0092] (19) LCDR1 sequence shown in SEQ ID NO:46; LCDR2 sequence shown in SEQ ID NO:47; and LCDR3 sequence shown in SEQ ID NO:48;

[0093] (20) LCDR1 sequence shown in SEQ ID NO: 21; LCDR2 sequence shown in SEQ ID NO: 22; and LCDR3 sequence shown in SEQ ID NO: 23;

[0094] (21) LCDR1 sequence shown in SEQ ID NO:54; LCDR2 sequence shown in SEQ ID NO:55; and LCDR3 sequence shown in SEQ ID NO:56.

[0095] In a further embodiment, the anti-ANG2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region polypeptide and a light chain variable region polypeptide, wherein the heavy chain variable region polypeptide comprises a HCDR1, HCDR2, and HCDR3 sequence, and the light chain variable region polypeptide comprises a LCDR1, LCDR2, and LCDR3 sequence, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences are selected from any one of (1) to (21):

[0096] (1) the HCDR1 sequence shown in SEQ ID NO: 62; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 7; the LCDR2 sequence shown in SEQ ID NO: 8; and the LCDR3 sequence shown in SEQ ID NO: 68;

[0097] (2) the HCDR1 sequence shown in SEQ ID NO:62; the HCDR2 sequence shown in SEQ ID NO:5; the HCDR3 sequence shown in SEQ ID NO:6; the LCDR1 sequence shown in SEQ ID NO:7; the LCDR2 sequence shown in SEQ ID NO:8; and the LCDR3 sequence shown in SEQ ID NO:80;

[0098] (3) the HCDR1 sequence shown in SEQ ID NO:62; the HCDR2 sequence shown in SEQ ID NO:5; the HCDR3 sequence shown in SEQ ID NO:6; the LCDR1 sequence shown in SEQ ID NO:7; the LCDR2 sequence shown in SEQ ID NO:8; and the LCDR3 sequence shown in SEQ ID NO:84;

[0099] (4) the HCDR1 sequence shown in SEQ ID NO:62; the HCDR2 sequence shown in SEQ ID NO:5; the HCDR3 sequence shown in SEQ ID NO:6; the LCDR1 sequence shown in SEQ ID NO:7; the LCDR2 sequence shown in SEQ ID NO:8; and the LCDR3 sequence shown in SEQ ID NO:85;

[0100] (5) the HCDR1 sequence shown in SEQ ID NO: 62; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 7; the LCDR2 sequence shown in SEQ ID NO: 8; and the LCDR3 sequence shown in SEQ ID NO: 86;

[0101] (6) the HCDR1 sequence shown in SEQ ID NO: 62; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 63; the LCDR2 sequence shown in SEQ ID NO: 64; and the LCDR3 sequence shown in SEQ ID NO: 65;

[0102] (7) the HCDR1 sequence shown in SEQ ID NO:62; the HCDR2 sequence shown in SEQ ID NO:5; the HCDR3 sequence shown in SEQ ID NO:81; the LCDR1 sequence shown in SEQ ID NO:82; the LCDR2 sequence shown in SEQ ID NO:8; and the LCDR3 sequence shown in SEQ ID NO:83;

[0103] (8) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 7; the LCDR2 sequence shown in SEQ ID NO: 8; and the LCDR3 sequence shown in SEQ ID NO: 9;

[0104] (9) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 17; the LCDR2 sequence shown in SEQ ID NO: 18; and the LCDR3 sequence shown in SEQ ID NO: 19;

[0105] (10) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 21; the LCDR2 sequence shown in SEQ ID NO: 22; and the LCDR3 sequence shown in SEQ ID NO: 23;

[0106] (11) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 25; the LCDR2 sequence shown in SEQ ID NO: 18; and the LCDR3 sequence shown in SEQ ID NO: 26;

[0107] (12) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 25; the LCDR2 sequence shown in SEQ ID NO: 28; and the LCDR3 sequence shown in SEQ ID NO: 29;

[0108] (13) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 25; the LCDR2 sequence shown in SEQ ID NO: 32; and the LCDR3 sequence shown in SEQ ID NO: 33;

[0109] (14) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 25; the LCDR2 sequence shown in SEQ ID NO: 18; and the LCDR3 sequence shown in SEQ ID NO: 36;

[0110] (15) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 7; the LCDR2 sequence shown in SEQ ID NO: 8; and the LCDR3 sequence shown in SEQ ID NO: 39;

[0111] (16) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 42; the LCDR2 sequence shown in SEQ ID NO: 43; and the LCDR3 sequence shown in SEQ ID NO: 44;

[0112] (17) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 46; the LCDR2 sequence shown in SEQ ID NO: 47; and the LCDR3 sequence shown in SEQ ID NO: 48;

[0113] (18) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 50; the LCDR2 sequence shown in SEQ ID NO: 51; and the LCDR3 sequence shown in SEQ ID NO: 52;

[0114] (19) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 54; the LCDR2 sequence shown in SEQ ID NO: 55; and the LCDR3 sequence shown in SEQ ID NO: 56;

[0115] (20) the HCDR1 sequence shown in SEQ ID NO: 4; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 25; the LCDR2 sequence shown in SEQ ID NO: 18; and the LCDR3 sequence shown in SEQ ID NO: 59;

[0116] (21) The HCDR1 sequence shown in SEQ ID NO: 12; the HCDR2 sequence shown in SEQ ID NO: 5; the HCDR3 sequence shown in SEQ ID NO: 6; the LCDR1 sequence shown in SEQ ID NO: 7; the LCDR2 sequence shown in SEQ ID NO: 13; and the LCDR3 sequence shown in SEQ ID NO: 14.

[0117] In some embodiments, the heavy chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 10, SEQ ID NO: 40, SEQ ID NO: 75, SEQ ID NO: 15, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 57, SEQ ID NO: 60, or SEQ ID NO: 66. In some embodiments, the heavy chain variable region polypeptide comprises an amino acid sequence that has 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 to SEQ ID NO: 69, SEQ ID NO: 10, SEQ ID NO: 40, SEQ ID NO: 75, SEQ ID NO: 15, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 57, SEQ ID NO: 60, or SEQ ID NO: 66. 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: 69, SEQ ID NO: 10, SEQ ID NO: 40, SEQ ID NO: 75, SEQ ID NO: 15, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 57, SEQ ID NO: 60, or SEQ ID NO: 66. Preferably, the amino acid substitutions, additions, and / or deletions do not occur in the CDR regions.

[0118] In some embodiments, the light chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:76, SEQ ID NO:16, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:58, SEQ ID NO:61, or SEQ ID NO:67. In some embodiments, the light chain variable region polypeptide comprises an amino acid sequence that is 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% identical to SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:76, SEQ ID NO:16, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:58, SEQ ID NO:61, or SEQ ID NO:67.In some embodiments, the light 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: 70, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 76, SEQ ID NO: 16, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 58, SEQ ID NO: 61, or SEQ ID NO: 67. Preferably, the amino acid substitutions, additions, and / or deletions do not occur in the CDR regions.

[0119] In some embodiments, the heavy chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO:69, SEQ ID NO:10, SEQ ID NO:40, SEQ ID NO:75, SEQ ID NO:15, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:57, SEQ ID NO:60, or SEQ ID NO:66; and the light chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:76, SEQ ID NO:16, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:58, SEQ ID NO:61, or SEQ ID NO: The amino acid sequence of NO:67.

[0120] In a specific embodiment, the heavy chain variable region polypeptide and the light chain variable region polypeptide are selected from any one of (1) to (21):

[0121] (1) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 69; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 70;

[0122] (2) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 69; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 74;

[0123] (3) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 69; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 77;

[0124] (4) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 69; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 78;

[0125] (5) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 69; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 79;

[0126] (6) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 10; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 11;

[0127] (7) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 10; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 20;

[0128] (8) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 10; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 24;

[0129] (9) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 10; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 27;

[0130] (10) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 40; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 41;

[0131] (11) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 40; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 45;

[0132] (12) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 40; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 49;

[0133] (13) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 40; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 53;

[0134] (14) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 75; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 76;

[0135] (15) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 15; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 16;

[0136] (16) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 30; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 31;

[0137] (17) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 34; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 35;

[0138] (18) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 37; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 38;

[0139] (19) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 57; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 58;

[0140] (20) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 60; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 61;

[0141] (21) A heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 66; and a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 67.

[0142] In another aspect, the present invention further provides an anti-ANG2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region polypeptide and a light chain variable region polypeptide, wherein the heavy chain variable region polypeptide comprises HCDR1, HCDR2, and HCDR3 sequences, and the light chain variable region polypeptide comprises LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences are selected from any one of (1) to (3):

[0143] (1) the HCDR1 sequence shown in SEQ ID NO: 87; the HCDR2 sequence shown in SEQ ID NO: 88; the HCDR3 sequence shown in SEQ ID NO: 89; the LCDR1 sequence shown in SEQ ID NO: 90; the LCDR2 sequence shown in SEQ ID NO: 91; and the LCDR3 sequence shown in SEQ ID NO: 92;

[0144] (2) the HCDR1 sequence shown in SEQ ID NO:95; the HCDR2 sequence shown in SEQ ID NO:96; the HCDR3 sequence shown in SEQ ID NO:97; the LCDR1 sequence shown in SEQ ID NO:98; the LCDR2 sequence shown in SEQ ID NO:99; and the LCDR3 sequence shown in SEQ ID NO:100;

[0145] (3) HCDR1 sequence shown in SEQ ID NO: 103; HCDR2 sequence shown in SEQ ID NO: 104; HCDR3 sequence shown in SEQ ID NO: 105; LCDR1 sequence shown in SEQ ID NO: 106; LCDR2 sequence shown in SEQ ID NO: 91; and LCDR3 sequence shown in SEQ ID NO: 107.

[0146] In some embodiments, the heavy chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO: 93, SEQ ID NO: 101, or SEQ ID NO: 108. In some embodiments, the heavy chain variable region polypeptide 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 to SEQ ID NO: 93, SEQ ID NO: 101, or SEQ ID NO: 108. 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: 93, SEQ ID NO: 101, or SEQ ID NO: 108. Preferably, the amino acid substitutions, additions, and / or deletions do not occur in the CDR regions.

[0147] In some embodiments, the light chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO: 94, SEQ ID NO: 102, or SEQ ID NO: 109. In some embodiments, the light chain variable region polypeptide comprises an amino acid sequence that has 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 to SEQ ID NO: 94, SEQ ID NO: 102, or SEQ ID NO: 109. In some embodiments, the light chain variable region polypeptide comprises an amino acid sequence that has 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: 94, SEQ ID NO: 102, or SEQ ID NO: 109. Preferably, the amino acid substitutions, additions, and / or deletions do not occur in the CDR regions.

[0148] In some embodiments, the heavy chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO:93, SEQ ID NO:101 or SEQ ID NO:108; and the light chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO:94, SEQ ID NO:102 or SEQ ID NO:109.

[0149] In a specific embodiment, the heavy chain variable region polypeptide and the light chain variable region polypeptide are selected from any one of (1) to (3):

[0150] (1) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 93; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 94;

[0151] (2) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 101; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 102;

[0152] (3) a heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 108; and a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 109.

[0153] In some embodiments, the anti-ANG2 antibody or antigen-binding fragment thereof of the present invention is a scFv, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv (dsFv), or a diabody.

[0154] In some embodiments, the anti-ANG2 antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region and / or a light chain constant region.

[0155] The heavy chain constant region and the light chain constant region can each independently be derived from the heavy chain constant region and the light chain constant region of the immunoglobulin of any species. The heavy chain constant region can be derived from the heavy chain constant region of the immunoglobulin of 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), or a combination thereof. The light chain constant region can be derived from λ (Lambda) light chain or κ (Kappa) light chain constant region.

[0156] Appropriate immunoglobulin constant regions (e.g., CH1 and light chain constant regions, hinge region-CH2-CH3, CH1-hinge region-CH2-CH3 and light chain constant regions or Fc region), as well as types (e.g., IgG, such as IgG1, IgG2, IgG3 and IgG4) can be selected and, optionally, modified to obtain an antibody with the desired properties.

[0157] In some embodiments, the heavy chain constant region is a heavy chain constant region (e.g., Fc region or CH1-hinge-CH2-CH3) of a human IgG (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). In one embodiment, the heavy chain constant region is a heavy chain constant region of a human IgG1 (an exemplary amino acid sequence is shown in SEQ ID NO: 71). In one embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 71.

[0158] In a preferred embodiment, the light chain constant region is a human kappa light chain constant region (an exemplary amino acid sequence is shown in SEQ ID NO: 72) or a human lambda light chain constant region (an exemplary amino acid sequence is shown in SEQ ID NO: 73). In one embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 73.

[0159] In some embodiments, the anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof of the present 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 polypeptides, antibodies, or antigen-binding fragments thereof of the present invention to ANG1 is significantly lower than the binding ability to ANG2. For example, Figure 4 As shown, the molecules of the present invention exhibit weak or no binding to ANG1.

[0160] Antibodies or their antigen-binding fragments can be prepared and produced using methods known in the art. Such methods can 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 hybridomas or EBV immortalized B cells). It is also possible to use methods for immunizing animals, such as immunizing animals (e.g., humanized mice) with antigens or DNA encoding antigens, and then separating the B cells expressing the antibodies from the animals after immunization. It is also possible to separate or prepare polynucleotides encoding antibodies or their antigen-binding fragments from immune animals or human bodies using chemical synthesis methods, and then utilize polynucleotides to construct expression vectors expressing the antibodies or antigen-binding fragments.

[0161] Multispecific antibodies

[0162] In another aspect, the present invention provides a multispecific antibody comprising a first antigen-binding moiety that specifically binds to ANG2 and a second antigen-binding moiety that specifically binds to a second antigen, wherein the first antigen-binding moiety comprises an anti-ANG2 heavy chain variable region polypeptide, antibody, or antigen-binding fragment thereof of the present invention.

[0163] As used herein, the term "multispecific antibody" refers to an antibody that can specifically bind to two or more (e.g., 2, 3, 4, 5, or 6) different antigenic epitopes. A multispecific antibody can, for example, be a bispecific, trispecific, or tetraspecific antibody, which can specifically bind to 2, 3, or 4 antigenic epitopes, respectively. As used herein, the term "epitope" or "antigenic determinant" refers to a region in an antigen that specifically binds to the antigen binding site of an antibody. An antigenic epitope is typically composed of chemically active surface groups (such as amino acids or sugar side chains) of an antigen and typically has specific three-dimensional structural properties and specific charge properties. A multispecific antibody can be a multivalent (e.g., 2, 3, 4 valence) antibody, i.e., it has multiple antigen binding sites. A multispecific antibody can, for example, be a chimeric antibody, a humanized antibody, a fully human antibody, scFab, F(ab')2, or a diabody.

[0164] Methods for constructing multispecific antibodies using an antibody or antigen-binding fragment of interest are well known to those skilled in the art (see, e.g., WO 93 / 08829; Suresh et al., (1986) Methods in Enzymology, 121:210; and Traunecker et al., (1991) EMBO, 10:3655-3659).

[0165] As used herein, "first antigen-binding moiety" and "second antigen-binding moiety" refer to amino acid sequences that contain an antigen-binding site and are capable of binding to an antigen epitope, and their definitions fall within the meaning of an antibody or antigen-binding fragment. The first antigen-binding moiety and the second antigen-binding moiety can be any form of antibody or antigen-binding fragment, including but not limited to Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', and F(ab')2.

[0166] The first antigen binding portion and the second antigen binding portion can optionally be connected by a linker. In some embodiments, the first antigen binding portion and the second antigen binding portion are not connected by a linker. In other embodiments, the first antigen binding portion and the second antigen binding portion are connected by a linker, such as a peptide linker or a chemical bond. Preferably, the first antigen binding portion and the second antigen binding portion are connected by a peptide linker. 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 (G4S) n , where n is an integer from 1 to 20.

[0167] Polynucleotides, vectors and host cells

[0168] In another aspect, the present invention provides a polynucleotide encoding the anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, or multispecific antibody of the present invention.

[0169] 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, EBV-mediated immortalized B cells) or chemically synthesized. The polynucleotides can be codon-optimized for the host cell used for expression.

[0170] In 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.

[0171] The present invention also provides a host cell comprising a polynucleotide or expression vector of the present invention. The polynucleotide or expression vector of the present invention can be introduced into a suitable host cell using various methods known in the art. Such methods include, but are not limited to, viral transduction, lipofectamine transfection, electroporation, and calcium phosphate transfection. In preferred embodiments, the host cell is used to express the anti-ANG2 heavy chain variable region polypeptide, antibody, or antigen-binding fragment thereof, or multispecific antibody of the present invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, and mammalian cells). Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells, Chinese Hamster Ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

[0172] The present invention also provides a method for producing the anti-ANG2 heavy chain variable region polypeptide, antibody, or antigen-binding fragment thereof, or multispecific antibody of the present invention, comprising:

[0173] (I) culturing the host cell of the present invention under suitable conditions to express the anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, or multispecific antibody of the present invention, and

[0174] (II) isolating the heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, or multispecific antibody from the host cell or its culture.

[0175] Pharmaceutical composition

[0176] The present invention also provides a pharmaceutical composition comprising the anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, or multispecific antibody of the present invention, and a pharmaceutically acceptable carrier.

[0177] 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, α-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitan,

[0014] In some embodiments, the carrier may be a surfactant (e.g., a glycerol ...

[0178] 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).

[0179] The pharmaceutical compositions provided herein can be administered by a variety of routes. Routes of administration include, but are 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). Methods of administration can be, for example, injection or infusion.

[0180] As a general guide, the anti-ANG2 antibodies or antigen-binding fragments thereof of the present invention may be administered at a dosage ranging from about 0.0001 to 100 mg / kg, more typically 0.01 to 20 mg / kg of the subject's body weight. For example, the dosage may be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, or 20 mg / kg, or within the range of 1-20 mg / kg. Exemplary treatment regimens entail dosing once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, once every three to six months, or initially at a shorter interval followed by a longer interval.

[0181] treat

[0182] The anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof, or multispecific antibodies of the present invention bind to human ANG2, blocking the binding of human ANG2 to the human TIE2 receptor and inhibiting TIE2 phosphorylation, thereby inhibiting angiogenesis associated with ANG2 activity. As used herein, "angiogenesis" refers to the formation of new blood vessels. Studies have shown that angiogenesis is associated with various diseases, such as cancer and angiogenesis-related eye diseases.

[0183] The anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof, multispecific antibodies, or pharmaceutical compositions of the present invention can be used to treat cancer or angiogenesis-related eye diseases. The present invention also provides use of the anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof, multispecific antibodies, or pharmaceutical compositions of the present invention in the preparation of a medicament for treating cancer or angiogenesis-related eye diseases. The present invention also provides a method for treating cancer or angiogenesis-related eye diseases in a subject, comprising administering to the subject an effective amount of the anti-ANG2 heavy chain variable region polypeptides, antibodies, or antigen-binding fragments thereof, multispecific antibodies, or pharmaceutical compositions of the present invention.

[0184] 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.

[0185] As used herein, the term "cancer" or "tumor" refers to or describes a physiological condition in an individual that is typically 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 pelvis 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.

[0186] For the treatment of cancer, the anti-ANG2 heavy chain variable region polypeptides, antibodies or antigen-binding fragments thereof, multispecific antibodies, or pharmaceutical compositions of the present invention can be used in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, immune checkpoint inhibitors, and angiogenesis inhibitors. The example of chemotherapeutic agent includes but is not limited to:Cyclophosphamide, ifosfamide, melphalan, busulfan, nitrogen mustard, chlorambucil, cyclohexane, carmustine (BCNU), lomustine (CCNU), cisplatin (DDP), carboplatin (CBP), oxaliplatin (OXA), methotrexate (MTX), 6-mercaptopurine (6-MP), 5-fluorouracil (5-FU), cytarabine, gemcitabine, vincristine, vindesine, camptothecin, irinotecan, topotecan, rubitecan, etoposide, teniposide, paclitaxel, taxane, docetaxel, liposome paclitaxel, actinomycin D, idarubicin, doxorubicin, epirubicin, mitomycin, bleomycin and doxorubicin.Immune checkpoint inhibitors include but are not limited to antibodies targeting PD-1, PD-L1, CTLA4, LAG-3 or TIM-3. Angiogenesis inhibitors include, but are not limited to, various receptor tyrosine kinase inhibitors, anti-VEGF antibodies (eg, Bevacizumab), anti-VEGFR antibodies (eg, Ramucirumab), small molecule inhibitors of VEGFR, and VEGF inhibitory fusion proteins (eg, aflibercept).

[0187] Reagent test kit

[0188] The present invention also provides a kit comprising an anti-ANG2 heavy chain variable region polypeptide, antibody, or antigen-binding fragment thereof, multispecific antibody, or pharmaceutical composition of the present invention, and instructions for use. The kit may also comprise a suitable container, such as an ampoule. In some embodiments, the kit also comprises a device for administration. The kit may also comprise a label indicating the intended use and / or method of use of the kit contents. The term "label" includes any written or recorded material provided on or with the kit, or otherwise accompanying the kit.

[0189] Beneficial effects

[0190] The anti-ANG2 heavy chain variable region polypeptide, antibody, or antigen-binding fragment thereof, or multispecific antibody of the present invention can achieve at least the following beneficial effects:

[0191] (1) specifically binds to ANG2 but does not bind or substantially binds to ANG1;

[0192] (2) blocking the binding of ANG2 to TIE2 and inhibiting TIE2 phosphorylation;

[0193] (3) inhibiting angiogenesis associated with ANG2 activity; and / or

[0194] (4) Inhibit tumor growth.

[0195] The anti-ANG2 antibody or antigen-binding fragment thereof of the present invention may be a fully human antibody and thus have low immunogenicity.

[0196] Example

[0197] The invention generally described herein will be more readily 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, for which no specific conditions are specified, were performed according to conventional methods and conditions, or according to commercial specifications.

[0198] Example 1 Preparation of raw materials

[0199] 1.1 Preparation of antigenic proteins ANG2 and ANG1 and receptor protein TIE2

[0200] The coding sequences of the extracellular domain of human ANG2 (hANG2, Uniprot ID: O15123-1), monkey ANG2 (cANG2, Uniprot ID: A0A2K5VNX6), mouse ANG2 sequence (mANG2, Uniprot ID: O35608-1), human ANG1 (hANG1, Uniprot ID: Q15389-1), and human TIE2 (hTIE2 ECD, Uniprot ID: Q02763-1) were synthesized by General Biotech Co., Ltd. The C-terminus of the gene sequence was connected to the human IgG1 Fc segment (SEQ ID NO: 1) gene sequence and His tag by PCR amplification, and then constructed into the eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen) by homologous recombination. The constructed recombinant protein expression vector was transformed into Escherichia coli DH5α and cultured overnight at 37°C. The plasmid was then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) and expressed using the Expi293 transient expression system (ThermoFisher, A14635). For transient transfection methods, see Expi293. TM ExpressionSystem USER GUIDE.

[0201] Seven days after transfection, the cell expression supernatant was centrifuged at 15,000 g for 10 minutes. The resulting Fc-tagged protein expression supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403). The target protein was eluted with 100 mM sodium acetate (pH 3.0) and then neutralized with 1 M Tris-HCl. The His-tagged protein expression supernatant was affinity purified using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), followed by elution with a gradient of imidazole concentrations. The eluted proteins were individually filtered through ultrafiltration concentrators (Millipore, UFC901096) into PBS buffer and, after SDS-PAGE and activity assay, stored frozen at -80°C until use. The proteins are referred to as hANG2-Fc, hANG2-His, cANG2-His, mANG2-Fc, hANG1-Fc, and hTIE2 ECD-Fc.

[0202] 1.2 Preparation of positive control antibody

[0203] The positive control antibody used in this application was the anti-ANG2 antibody nesvacumab, synthesized according to the sequence disclosed in US2011027286A1. Plasmids containing the nesvacumab heavy chain (SEQ ID NO: 2) and nesvacumab light chain (SEQ ID NO: 3) genes were constructed using molecular cloning methods. The remaining steps were similar to those in Example 1.1.

[0204] 1.3 Preparation of hTIE2-HEK293 cell line

[0205] The DNA sequence of full-length human TIE2 (NCBI Gene ID: 7010) was constructed into pLVX-puro plasmid (Clontech, Cat# 632164). The resulting plasmid was then electroporated into HEK293 cells ( CRL-1573 TM ). Through pressure selection with 2 μg / mL puromycin, the resulting single-cell clones were identified using the antibody nesvacumab (conventional FACS method), successfully obtaining a HEK293 cell line overexpressing human TIE2, also referred to herein as the "hTIE2-HEK293 cell line."

[0206] Example 2 Construction and Screening of Natural Human Antibody Phage Display Library

[0207] In this example, an antibody gene phage display library was constructed, and the library was screened using the antigen protein ANG2 (including hANG2-Fc and hANG2-His) prepared in Example 1.1 as a screening antigen to obtain multiple antibody molecules that specifically bind to ANG2.

[0208] 2.1 Construction of a human antibody gene library

[0209] Peripheral blood mononuclear cells (PBMCs) from normal human blood were separated using Ficoll-Paque density gradient separation medium (purchased from GE, catalog number: 17144003S). Total RNA was extracted from the isolated PBMCs using conventional methods and reverse transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa, catalog number: 6210A). Based on the sequence similarity of the heavy and light chain germline genes, degenerate primers were designed at the front end of the V region and the back end of the first constant region of the heavy and light chains, respectively (Li Xiaolin, Construction and Preliminary Screening of a Large-Capacity Non-Immune Human Fab Phage Antibody Library, Master's Thesis, Peking Union Medical College, June 2007). After PCR, the heavy and light chain variable region gene fragments of the antibody were obtained. A fragment containing the variable regions of the antibody light and heavy chains was amplified by fusion PCR. The PCR product and the phage display vector were digested, recovered, and ligated. The ligated product was recovered using a recovery kit (Omega, catalog number: D6492-02). Finally, the transformed E. coli SS320 (Lucigen, MC1061 F) was transformed using an electroporator (Bio-Rad, MicroPulser). The transformed E. coli SS320 bacterial solution was plated on ampicillin-resistant 2-YT solid plates. By serial dilution plating, the library capacity was determined to be 3×10 11 cfu, i.e. 3 × 10 11 An antibody gene library containing 100 antibody genes (for library capacity calculation, refer to Example 2.2 in CN112250763B ). VSCM13 helper phage (purchased from Stratagene) was used to package the library to obtain an antibody gene phage display library (for preparation of the antibody gene phage display library, refer to Example 2.3 in CN112250763B ).

[0210] 2.2 Screening of Antibody Gene Phage Display Library

[0211] 2.2.1 Screening of Antibody Gene Phage Display Library by Magnetic Bead Method

[0212] Magnetic bead screening involves biotin-labeling hANG2-Fc, then binding it to streptavidin-coupled magnetic beads. A panning process involves incubating the antigen-bound magnetic beads with a phage display library of antibody genes, followed by washing and elution. Typically, three to four rounds of panning are performed, allowing for the substantial enrichment of antigen-specific monoclonal antibodies. In this example, biotin-labeled hANG2-Fc was used for phage display library screening. After three rounds of panning, initial screening for monoclonal antibodies targeting ANG2 was performed. For detailed procedures, refer to Example 2.4.1 of CN112250763B.

[0213] 2.2.2 Screening of Antibody Gene Phage Display Library by Immunotube Method

[0214] Both the immunotube and magnetic bead methods aim to enrich for antigen-specific antibodies, serving as complementary and validated experimental methods. The principle of immunotube screening involves coating hANG2-Fc on the highly adsorbable surface of an immunotube. A panning process involving incubation with the antigenic protein adsorbed to the surface of the immunotube, followed by washing and elution, is then performed. After two to four rounds of panning, antigen-specific monoclonal antibodies are ultimately enriched. In this example, initial screening for monoclonal antibodies against ANG2 was performed after three rounds of panning. For detailed procedures, refer to Example 2.4.2 of CN112250763B.

[0215] 2.3 Selection of monoclones

[0216] The phage pool eluted in each round was tested by ELISA to evaluate the enrichment effect, and 10 clones were randomly selected from the phage pool in each round of screening for sequence analysis. The enrichment effect and the repeatability ratio of the measured sequences were comprehensively analyzed to select the appropriate round for single clone selection.

[0217] ELISA monoclonal screening used hANG2-His. Positive antibodies that bound hANG2-His were retested using cANG2-His and mANG2-Fc. ELISA data for exemplary monoclonal antibodies (OD450 values for binding to human, monkey, and mouse ANG2 proteins) are shown in Table 1. Candidate fully human antibodies were named by clone number. The amino acid sequences of their variable regions are shown in Table 2. The complementarity-determining region sequences were determined using the AbM method for defining CDRs.

[0218] Table 1 OD450 of the exemplary monoclonal antibodies screened for binding to antigens (ELISA)

[0219]

[0220] Table 2 Variable region amino acid sequences of candidate antibodies

[0221]

[0222] Example 3 Preliminary identification of antigen binding and blocking activities of candidate monoclonal supernatants

[0223] 3.1 Specific binding of candidate monoclonal supernatants to hANG2-Fc

[0224] A 96-well ELISA plate was coated with hANG2-Fc (2 μg / mL, 30 μL / well) 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 three times with PBST, serially diluted supernatants of candidate monoclonal antibodies (obtained by overnight expression of plasmids containing the monoclonal Fab fragment in E. coli SS320) were added and incubated for 1 hour. After washing three times with PBST, a mixture of anti-human Kappa HRP and anti-human Lambda HRP secondary antibodies (Millipore, AP502P and AP506P) was added and incubated for 1 hour. After incubation, the plate was washed six times with PBST and developed with TMB. The reaction was terminated with 2M HCl based on the color development result, and the OD450 value was read using a microplate reader.

[0225] Test results are shown in Figures 1A-1C : The supernatants of candidate monoclonal antibodies all specifically bound to hANG2-Fc with comparable binding abilities.

[0226] 3.2 Blocking activity of candidate monoclonal supernatants in blocking the binding between ANG2 and receptor TIE2.

[0227] The plates were coated with hTIE2 ECD-Fc (4 μg / mL, 30 μL / well) overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 5% skim milk for 2 hours. The supernatant of the candidate monoclonal clones was then serially diluted and premixed with biotinylated hANG2-Fc (4 μg / mL) for 0.5 hours. After blocking and washing, the plates were added to a 96-well ELISA plate and incubated for 1 hour. The plates were then washed three times with PBST, and NeutrAvidin-HRP (Therofisher, 31001) was added and incubated for 1 hour. Following incubation, the plates were washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was terminated with 2 M HCl, and the OD450 value was read using a microplate reader (Molecular Devices, SpecterMax 190).

[0228] The results are shown in Figures 2A-2C : The supernatants of the candidate monoclonal antibodies all have good ability to block the binding between ANG2 and receptor TIE2, and the abilities are comparable.

[0229] Example 4 Construction, expression and purification of candidate antibodies

[0230] 4.1 Plasmid construction

[0231] The VH in the Fab sequence of the screened monoclonal clones 7, 78A46, 78A74, 78A14, 78A51, 78A5, 17, and 35 was connected to the coding sequence of the constant region of human IgG1 (SEQ ID NO: 71) to construct the heavy chain coding sequence of the fully human antibody. The VL in the Fab sequence was connected to the coding sequence of the Kappa type (SEQ ID NO: 72) of the human light chain constant region (CL) or the Lambda type (SEQ ID NO: 73) of human CL to construct the light chain coding sequence of the fully human antibody. The coding sequences of the antibody heavy chain and light chain were respectively inserted into the eukaryotic expression vector plasmid pcDNA3.4-TOPO (Invitrogen), transformed into Escherichia coli DH5α, and cultured at 37°C overnight. Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain an endotoxin-free antibody plasmid for eukaryotic expression.

[0232] 4.2 Antibody Expression and Purification

[0233] The candidate antibodies were expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133) as follows: on the day of transfection, the cell density was confirmed to be 7 × 10 6 to 1×10 7 The cell viability was >98%. Fresh ExpiCHO expression medium pre-warmed at 37°C was used to adjust the cells to a final concentration of 6×10 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 minutes, then slowly added to the prepared cell suspension while gently shaking. Finally, the mixture was placed in a cell culture shaker and cultured at 37°C and 8% CO2.

[0234] 18-22 hours after transfection, add ExpiCHO to the culture medium TM Enhancer and ExpiCHO TM Feed, shake flasks were placed in a 32 ° C shaker and 5% CO2 conditions to continue to culture. On the 5th day after transfection, the same volume of ExpiCHO TMFeed, slowly add and gently mix the cell suspension. 7 days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10 minutes. The resulting supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403), and the target protein was eluted with 100mM sodium acetate (pH 3.0), followed by neutralization with 1M Tris-HCl. Finally, the resulting protein was exchanged into PBS buffer using an ultrafiltration concentrator (Millipore, UFC901096).

[0235] Example 5 Determination of antigen binding activity and blocking activity of candidate antibodies based on ELISA method

[0236] In this example, the binding activity of eight candidate antibodies, 7, 78A46, 78A74, 78A14, 78A51, 78A5, 17, and 35, to hANG2-His and hANG1-Fc was tested by ELISA. The activity of the candidate antibodies in blocking the binding of ANG2 to the receptor TIE2 was also tested by ELISA.

[0237] 5.1 ELISA-based detection of candidate antibody binding activity to hANG2-His

[0238] A 96-well ELISA plate was coated with hANG2-His (2 μg / mL, 30 μL / well) 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 three times with PBST, serial dilutions of the candidate antibody and the positive control antibody nesvacumab were added and incubated for 1 hour. After washing three times with PBST, an anti-human Fc HRP secondary antibody (Jackson Immuno Research, 109-035-008) was added and incubated for 1 hour. After incubation, the plate was washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was terminated by adding 2 M HCl, and the OD450 value was read using a microplate reader (Molecular Devices, SpecterMax 190).

[0239] The results are shown in Figures 3A-3B And Table 3: All candidate antibodies have high antigen binding activity to hANG2-His and are significantly better than the positive control antibody nesvacumab.

[0240] 5.2 ELISA-based detection of candidate antibody binding activity to hANG1-Fc

[0241] A 96-well ELISA plate was coated with hANG1-Fc (2 μg / mL, 30 μL / well) 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 three times with PBST, serial dilutions of the candidate antibodies and the positive control antibody nesvacumab were added and incubated for 1 hour. After washing three times with PBST, a mixture of anti-human kappa HRP and anti-human lambda HRP secondary antibodies (Millipore, AP502P and AP506P) were added and incubated for 1 hour. After incubation, the plate was washed six times with PBST and developed with TMB. The reaction was terminated with 2 M HCl based on the color development result, and the OD450 value was read using a microplate reader.

[0242] The results are shown in Figure 4 : Except for antibody 78A5, which weakly bound to hANG1-Fc, the remaining seven antibodies and the positive control antibody did not bind to hANG1.

[0243] 5.3 ELISA-based detection of the blocking activity of candidate antibodies against the binding of ANG2 to the receptor TIE2

[0244] The plates were coated with hTIE2 ECD-Fc (4 μg / mL, 30 μL / well) overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 5% skim milk for 2 hours. The candidate antibody or the positive control antibody, nesvacumab, was then serially diluted and premixed with biotinylated hANG2-Fc (4 μg / mL) for 0.5 hours. After blocking and washing, the plates were added to a 96-well ELISA plate and incubated for 1 hour. The plates were then washed three times with PBST, and the secondary antibody, NeutrAvidin-HRP (Therofisher, 31001), was added and incubated for 1 hour. Following incubation, the plates were washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). The reaction was terminated with 2 M HCl based on the color development result, and the OD450 value was read using a microplate reader (Molecular Devices, SpecterMax 190).

[0245] The results are shown in Figures 5A-5B And Table 3: The candidate antibodies all have good ability to block the binding between ANG2 and receptor TIE2, and all candidate antibodies show blocking activity that is better than the control antibody nesvacumab.

[0246] Table 3 Binding activity and blocking activity of candidate antibodies based on ELISA method

[0247] Antibody name <![CDATA[Combined active EC 50 (μg / mL)]]> <![CDATA[Blocking activity IC 50 (μg / mL)]]> nesvacumab 0.442 1.578 78A46 0.049 0.367 78A74 0.097 0.626 78A51 0.136 0.566 7 0.128 0.611 78A14 0.122 0.631 78A5 0.166 0.648 17 0.120 0.547 35 0.173 0.676

[0248] Example 6 Detection of blocking activity of candidate antibodies based on FACS method

[0249] In this example, the activity of candidate antibodies in blocking the binding between ANG2 and the cell surface-expressed receptor TIE2 was evaluated based on the FACS method.

[0250] The candidate antibodies and control antibody nesvacumab were serially diluted using FACS buffer (1×PBS + 2% FBS). 100 μL of the antibody dilution was added to each well of a 96-well round-bottom plate. hANG2-Fc-biotin protein was also diluted to 2 μg / mL using FACS buffer. 100 μL was added to the corresponding 96-well plate, mixed, and incubated at 4°C for 1 hour. The density of hTIE2-HEK293 cells was adjusted to 1×10 6 Cells were plated at 100 μL per well of a new 96-well round-bottom plate at 4°C / mL. The cells were centrifuged at 300 g at 4°C, and the supernatant removed. 180 μL of the pre-incubated test antibody and hANG2-Fc-biotin mixture was added to the corresponding wells, mixed thoroughly, and incubated at 4°C for 30 min. The incubated cell mixture was washed three times, followed by the addition of 200 μL of a 1:200 dilution of PE-labeled Streptavidin (Invitrogen, 12-4317-87). The cells were incubated at 4°C in the dark for 30 min, washed three times, and the amount of hANG2-Fc-biotin bound to the cells (expressed as mean fluorescence intensity (MFI)) was determined using a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0251] The experimental results show that Figure 6 and Table 4: The blocking activity of antibodies 78A46, 78A74, 78A51, and 78A14 is superior to that of the control antibody nesvacumab.

[0252] Table 4 Blocking activity of candidate antibodies based on FACS method

[0253] Antibody name <![CDATA[Blocking IC 50 Value (μg / mL)]]> nesvacumab 0.362 78A46 0.234 78A74 0.298 78A51 0.227 7 0.377 78A14 0.265 78A5 0.480 17 0.323 35 0.424

[0254] Example 7 Detection of TIE2 phosphorylation inhibition activity by candidate antibodies

[0255] hTIE2-HEK293 cells were plated into polylysine-coated 96-well plates (WHB, WHB-96-LC) at 1×10 cells per well. 4Cells were plated and incubated at 37°C for 24 hours. After incubation, the cell culture supernatant was discarded and the plate was rinsed once with serum-free DMEM. Then, 50 μL of a mixture of hANG2-Fc and serially diluted test antibodies was added to each well and incubated at 37°C for 30 minutes. The supernatant was then removed, and the plate was rinsed once with 4°C pre-chilled PBS. 100 μL of 1× cell lysis buffer (CST, 9803) and 1× protease inhibitors (Thermo, 78430) were added to each well and lysed on ice for 30 minutes. The lysate was collected and the phosphorylated TIE2 in the protein lysate was quantified using the Human Phospho-Tie-2 DuoSet ICELISA kit (R&D, DYC2720-2). Detailed assay methods and procedures are provided in the kit instructions.

[0256] Test results are shown in Figure 7 Table 5 shows that the activity of most candidate antibodies in inhibiting ANG2-mediated TIE2 phosphorylation is better than or equivalent to that of the positive control antibody nesvacumab, among which antibodies 78A46, 78A74 and 17 are significantly better than nesvacumab.

[0257] Table 5 Inhibitory activity of candidate antibodies against TIE2 phosphorylation

[0258] Antibody name <![CDATA[IC 50 (μg / mL)]]> Nesvacumab 8.308 78A74 5.467 7 9.761 78A51 8.052 78A14 7.311 78A46 3.568 78A5 7.089 17 6.357 35 11.93

[0259] Example 8 In vivo efficacy evaluation of candidate antibodies

[0260] In this example, the tumor inhibition effects of six candidate antibodies and the positive control antibody nesvacumab in animals were verified. The tumor cells used were colon cancer cells Colo205 ( CCL-222 TM Male BALB / c nude mice aged 6-8 weeks and weighing about 20 g (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were used and 5×10 6 Colo205 cells, wait until the tumor volume reaches 150mm 3 Around 30 minutes later, the mice were divided into groups and cages and given the drug. Each group consisted of 8 tumor-bearing nude mice, for a total of 8 groups: 6 candidate antibody groups, 1 negative control group, and 1 positive control antibody nesvacumab group. The drug was administered by intraperitoneal injection at a dose of 5 mg / kg, once every 3-4 days, twice a week, and the tumor volume was measured twice, for a total of 6 doses / 3 weeks. Tumor volume (V) was calculated as follows: V = L × W 2= / 2 (where L is the longest tumor diameter and W is the shortest tumor diameter). One week after dosing, mice were euthanized, and tumors were removed and weighed. Tumor volume, tumor weight, and mouse body weight changes were analyzed to calculate the tumor inhibition rate (TGI) = (1 - average tumor volume of experimental group / average tumor volume of PBS control group) × 100%.

[0261] The results are shown in Figures 8A-8C and Table 6. Figure 8C It can be seen that there is no significant difference in the body weight of mice in each group, and there is no significant change in the body weight of mice in each group during the treatment period, indicating that the mice have good tolerance to the antibody. Figure 8A 、 8B As can be seen from Table 6, the tumors in the mice in the PBS negative control group grew the fastest. Compared with the PBS group, all antibody groups had significant tumor inhibition effects. Among them, the tumor volumes and weights of the mice in the antibody 17, 35, 78A14, 78A46, and 78A74 groups were lower than those in the positive control antibody nesvacumab group, indicating a better tumor inhibition effect.

[0262] Table 6 Tumor inhibition rate TGI (%) of candidate antibodies

[0263]

[0264] Example 9: Detection of drug metabolism rate of candidate antibodies in Balb / C mice

[0265] In this example, the drug metabolism rates of 6 candidate antibodies in Balb / C mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were determined, and the positive control antibody nesvacumab was used as a control. The experimental animals were divided into 7 groups, with 6 Balb / C mice in each group. Each mouse was intraperitoneally injected with the drug at a dose of 40 mg / kg; the administration volume was 10 mL / kg. Blood samples collected at time points 2h, 4h, 8h, 1d, 2d, 3d, 4d, 5d, 9d, and 14d in each group were placed at room temperature for 2 hours. Three mice were selected from each group at each time point to collect blood samples. The mice in the group were alternately bled and then centrifuged at 10,000g for 5 minutes at 4°C. The supernatant was collected and the experiment was performed immediately or the samples were aliquoted and stored at -80°C.

[0266] Drug concentrations in mouse serum were quantitatively determined using an ELISA method. Half-well 96-well microtiter plates were coated with hANG2-Fc (2 μg / mL) overnight at 4°C. After washing three times with PBST, the plates were blocked with 160 μL / well of 2% BSA for 1 hour at room temperature. After washing three times with PBST, the mouse serum samples were diluted to various concentrations with 2% BSA, 30 μL per well, and incubated at room temperature for 1 hour. After washing three times, a mixture of anti-human IgG kappa HRP secondary antibodies (Millipore, AP502P) and anti-human Lambda HRP secondary antibodies (Millipore, AP506P) at a dilution of 1:5000 was added and incubated at room temperature for 1 hour. Following incubation, the plates were washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). According to the color development results, 2M HCl was added to terminate the reaction, and the OD450 value was read using a microplate reader (Molecular Devices, SpecterMax 190).

[0267] The results are shown in Figure 9 and Table 7: The drug metabolism rates of antibodies 78A74, 78A14, 7 and 17 are lower than that of the control antibody nesvacumab. Therefore, the bioavailability of antibodies 78A74, 78A14, 7 and 17 is higher than that of the control antibody nesvacumab.

[0268] Table 7 Blood concentrations of candidate antibodies at different time points (μg / mL)

[0269] Antibody name 2h 4h 8h 24h 48h 72h 96h 120h 216h 336h Nesvacumab 693 819 877 419 225 159 150 164 45 17 78A46 232 264 216 173 203 158 138 105 63 29 78A14 473 492 628 560 516 488 500 301 273 38 78A74 1135 1811 1272 1351 1461 1257 1194 906 507 252 7 531 726 569 477 428 431 371 366 88 25 17 441 586 537 413 432 387 396 353 274 127 35 303 316 323 244 248 168 156 158 61 8

[0270] Example 10 Engineering of candidate antibodies

[0271] In the data from the previous examples, antibody 78A74 demonstrated good drug metabolism in mice and demonstrated good ANG2-TIE2 phosphorylation blocking activity. Affinity maturation was performed using M13 phage display technology, using codon-based primers (a single codon consisting of NNK during primer synthesis) to introduce mutations in the CDR regions. Four phage display libraries were constructed for each parent molecule. Library 1 and Library 2 contained single-point combination mutations: Library 1 contained CDRL1+CDRL3+CDRH3 combination mutations, while Library 2 contained CDRL2+CDRH1+CDRH2 combination mutations. Library 3 and Library 4 contained double-point saturation mutations: Library 3 contained double-point saturation mutations in CDRL3, while Library 4 contained double-point saturation mutations in CDRH3.

[0272] Using antibody 78A74 as a template, a single CDR region mutation fragment was obtained by PCR, and then the Fab fragment (VL-CL-linker-VH-CH1) was obtained by Overlapping PCR. The point mutation antibody was connected to the phage display vector by double enzyme digestion (HindⅢ and NotⅠ) and double sticky end ligation. Finally, the antibody sequence with the mutation site was transferred into Escherichia coli SS320 by electroporation.

[0273] After the four constructed libraries were packaged into phage, library screening, primary screening, affinity sorting, and sequence analysis were performed (see Example 2 for methods). Fab supernatants expressed from 28 positive clones were selected for affinity sorting and sequence analysis. Finally, five preferred Fab fragments were selected for sample preparation and affinity evaluation. Fully human antibodies were prepared based on these five Fab fragments (see Example 4 for methods), and their variable region amino acid sequences are shown in Table 8.

[0274] Table 8 Amino acid sequences of the variable regions of fully human anti-ANG2 antibodies obtained by affinity maturation (SEQ ID NO:

[0275] Antibody name HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 VH VL 78A74-7 62 5 6 7 8 80 69 74 78A74-21 62 5 81 82 8 83 75 76 78A74-25 62 5 6 7 8 84 69 77 78A74-35 62 5 6 7 8 85 69 78 78A74-36 62 5 6 7 8 86 69 79

[0276] Example 11 Affinity kinetics evaluation of anti-ANG2 antibodies

[0277] In this example, the binding affinity of candidate antibodies to the antigen ANG2 was detected based on the Gator device, and nesvacumab was used as a positive control.

[0278] The antibody to be tested was diluted to 20 nM with 10×KB (10×PBS buffer containing 1% BSA and 0.5% Tween 20). hANG2-His, the antigen, was diluted 2-fold with 10×KB buffer to concentrations of 100 nM, 50 nM, 25 nM, and 0 nM, respectively. Under light-proof conditions, the sensor (Gator, 20-5006) was pre-wetted with 10×KB buffer. After at least 10 minutes, the sample plate (Gator, 06-0153) was tested. After the test was correct, the procedure was followed. First, the antibody and sensor were combined for 120 seconds. After the combination was complete, the sensor was equilibrated in 10×KB buffer for 30 seconds. The sensor with the antibody was then transferred to different concentrations of antigen dilutions for 120 seconds. After the signal stabilized, it was transferred to 10×KB buffer for a dissociation time of 120 seconds. Finally, the K value was obtained by fitting the binding and dissociation data of the anti-antigen at different concentrations. D (affinity kinetic constant), Kon (binding constant) and Koff (dissociation constant), Kon can be written as Ka, Koff can be written as Kd.

[0279] The test results are shown in Table 9. The results show that antibodies 78A74-7, 78A74-21, and 78A74-25 have higher affinities than antibody 78A74 and are approximately twice that of the control antibody nesvacumab. Antibodies 7, 35, 78A46, 78A14, 78A74, 78A74-35, and 78A74-36 have similar affinities for human ANG2 as the control antibody.

[0280] Table 9 Affinity kinetics evaluation of candidate antibodies

[0281] Antibody name <![CDATA[K D (M)]]> ka(1 / Ms) kd(1 / s) nesvacumab 1.17E-09 1.80E+05 2.11E-04 7 1.56E-09 1.97E+05 3.07E-04 35 2.36E-09 2.04E+05 4.82E-04 78A46 9.08E-10 1.99E+05 1.81E-04 78A14 1.62E-09 1.79E+05 2.89E-04 78A74 1.22E-09 1.95E+05 2.38E-04 78A74-7 5.44E-10 1.89E+05 1.03E-04 78A74-21 6.87E-10 1.92E+05 1.32E-04 78A74-25 8.28E-10 1.94E+05 1.61E-04 78A74-35 1.52E-09 1.87E+05 2.84E-04 78A74-36 1.32E-09 2.00E+05 2.63E-04

[0282] Those skilled in the art will further appreciate 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. Instead, reference should be made to the appended claims for an indication of the scope and content of the present invention. Sequence Listing <110> Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. <120> Anti-ANG2 antibodies and their uses <130> I2021TC6149CS <160> 111 <170> PatentIn version 3.5 <210> 1 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 Fc <400> 1 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> 2 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> nesvacumab HC <400> 2 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 Thr Phe Ser Ser Tyr 20 25 30 Asp Ile His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Pro Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Leu Ile Thr Phe Gly Gly Leu Ile Ala Pro Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 3 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> nesvacumab LC <400> 3 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Thr 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asp Asn Ser Gln 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 16 HCDR1 <400> 4 Gly Phe Thr Phe Ser Ser Tyr Gly Met His 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 16 HCDR2 <400> 5 Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr 1 5 10 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 16 HCDR3 <400> 6 Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 16 LCDR1 <400> 7 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 16 LCDR2 <400> 8 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 16 LCDR3 <400> 9 Gln His Arg Thr Asn Trp Pro Thr 1 5 <210> 10 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 16 VH <400> 10 Gln Val Gln Leu Val 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 Ser Ser 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> 11 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 16 VL <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val 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 Ser Ala Val Tyr Tyr Cys Gln His Arg Thr Asn Trp Pro Thr 85 90 95 Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 78A14 HCDR1 <400> 12 Gly Phe Thr Phe Asn Val Tyr Gly Met His 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A14 LCDR2 <400> 13 Gly Pro Ser Asn Arg Ala Thr 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 78A14 LCDR3 <400> 14 Gln His Tyr Gly Val Ser Gln His Thr 1 5 <210> 15 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 78A14 VH <400> 15 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> 16 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 78A14 VL <400> 16 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> 17 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 1JIU LCDR1 <400> 17 Arg Ala Ser Gln Ser Val Ala Ser Ser Tyr Leu Ala 1 5 10 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 1JIU LCDR2 <400> 18 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 1JIU LCDR3 <400> 19 Gln Gln Tyr Gly Ser Ser Pro Pro Trp Thr 1 5 10 <210> 20 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 1JIU VL <400> 20 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Gln Ser Leu Ser Pro Gly 1 5 10 15 Asp Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ala Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Lys Lys Pro Asp Gln Ala Pro Arg Leu Ile 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 21 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> 78A51 LCDR1 <400> 21 Thr Gly Thr Thr Ser Asp Val Gly Gly Phe Asn Tyr Val Ser 1 5 10 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A51 LCDR2 <400> 22 Asp Val Thr Asn Arg Pro Ser 1 5 <210> 23 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A51 LCDR3 <400> 23 Thr Ser Tyr Thr Ser Arg Asn Thr Leu Tyr Val 1 5 10 <210> 24 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 78A51 VL <400> 24 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Thr Ser Asp Val Gly Gly Phe 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Ile Ile Phe Asp Val Thr Asn Arg Pro Ser Gly Val Ser Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Thr Ser Tyr Thr Ser Arg 85 90 95 Asn Thr Leu Tyr Val Phe Gly Thr Ala Thr Lys Val Thr Val Leu 100 105 110 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 7 LCDR1 <400> 25 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 7 LCDR3 <400> 26 Gln Gln Arg Ser Asn Ser Leu Thr 1 5 <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 7 VL <400> 27 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A42 LCDR2 <400> 28 Gly Ala Ser Asn Arg Ala Thr 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 78A42 LCDR3 <400> 29 Gln His Tyr Gly Ser Ser Leu Phe Thr 1 5 <210> 30 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 78A42 VH <400> 30 Glu Val Gln Leu Val 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 Ser Ser 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 Arg 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 Ala 115 120 125 <210> 31 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> 78A42 VL <400> 31 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln His Tyr Gly Ser Ser Leu 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 9 LCDR2 <400> 32 Gly Ala Thr Ser Arg Ala Ile 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 9 LCDR3 <400> 33 Glu Gln Tyr Asp Asn Ser Pro Tyr Thr 1 5 <210> 34 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 9 VH <400> 34 Glu Val Gln Leu Val 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 Ser Ser 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 Ala 115 120 125 <210> 35 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> 9 VL <400> 35 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Asp Thr Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Thr Ser Arg Ala Ile Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser Gly Leu Glu 65 70 75 80 Pro Glu Asp Ile Ala Val Tyr Tyr Cys Glu Gln Tyr Asp Asn Ser Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 2 LCDR3 <400> 36 Gln Gln Tyr Gly Ser Ser Pro Tyr Thr 1 5 <210> 37 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 2 VH <400> 37 Glu Val Gln Leu Val 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 Ser Ser 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 Met Val Thr Val Ser Ala 115 120 125 <210> 38 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> 2 VL <400> 38 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 21 LCDR3 <400> 39 Gln Gln Arg Ser Asn Trp Pro Pro Ser Tyr Thr 1 5 10 <210> 40 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 21 VH <400> 40 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 Ser Ser 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 Ala 115 120 125 <210> 41 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 21 VL <400> 41 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 Gln Arg Ser Asn Trp Pro Pro 85 90 95 Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A27 LCDR1 <400> 42 Arg Ala Ser Gln Gly Ile Ser Ser Gly Leu Val 1 5 10 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A27 LCDR2 <400> 43 Asp Ala Ser Ser Val Gln Ser 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 78A27 LCDR3 <400> 44 Gln Gln Gly Asn Ser Phe Pro Pro Thr 1 5 <210> 45 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 78A27 VL <400> 45 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Gly 20 25 30 Leu Val Trp Tyr Gln Val Lys Pro Gly Lys Ala Pro Gln Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Val Gln Ser Gly Val Pro Pro Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Phe Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 46 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A40 LCDR1 <400> 46 Arg Ala Ser Gln Asp Ile Arg Asn Glu Leu Ser 1 5 10 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A40 LCDR2 <400> 47 Ala Ala Ser Asn Leu Glu Ser 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 78A40 LCDR3 <400> 48 Leu Gln Asp Asn Asn Tyr Pro Arg Thr 1 5 <210> 49 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 78A40 VL <400> 49 Asn Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Val Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Glu 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln Asp Asn Asn Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Asp Ile Arg 100 105 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A45 LCDR1 <400> 50 Arg Ala Ser Gln Val Ile Asn Ser Asp Leu Asn 1 5 10 <210> 51 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A45 LCDR2 <400> 51 Asp Ala Ser Thr Leu Lys Ser 1 5 <210> 52 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 78A45 LCDR3 <400> 52 Gln Gln Asn Tyr Arg Thr Leu Ser Tyr Thr 1 5 10 <210> 53 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> 78A45 VL <400> 53 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Ser Asp 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Tyr Arg Thr Leu Ser 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 54 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A46 LCDR1 <400> 54 Gly Gly Asn Ile Leu Gly Ser Asn Thr Val His 1 5 10 <210> 55 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A46 LCDR2 <400> 55 Tyr Asp Asn Asp Arg Pro Ser 1 5 <210> 56 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A46 LCDR3 <400> 56 His Val Trp Asp Arg Val Ala Thr Gln Tyr Val 1 5 10 <210> 57 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 78A46 VH <400> 57 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 Ser Ser 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> 58 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> 78A46 VL <400> 58 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Ile Leu Gly Ser Asn Thr Val 20 25 30 His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Thr Val Ile Tyr 35 40 45 Tyr Asp Asn Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asp Thr Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys His Val Trp Asp Arg Val Ala Thr Gln 85 90 95 Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu 100 105 <210> 59 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 8 LCDR3 <400> 59 Gln Gln Tyr Gly Ser Ser Pro Ser Tyr Thr 1 5 10 <210> 60 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 8 VH <400> 60 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 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 Leu Val Thr Val Ser Ala 115 120 125 <210> 61 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 8 VL <400> 61 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Ser Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 62 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 6 HCDR1 <400> 62 Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn 1 5 10 <210> 63 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 6 LCDR1 <400> 63 Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 <210> 64 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 6 LCDR2 <400> 64 Asp Val Ser Asn Arg Ala Thr 1 5 <210> 65 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 6 LCDR3 <400> 65 Gln Gln Arg Ala Ser Trp Pro Leu Thr 1 5 <210> 66 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 6 VH <400> 66 Glu Val Gln Leu Leu 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 Ala 115 120 125 <210> 67 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 6 VL <400> 67 Glu Ile Val Met 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 Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Val 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 Phe Tyr Tyr Cys Gln Gln Arg Ala Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 68 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A74 LCDR3 <400> 68 Gln Gln Arg Ser Asn Trp Pro Pro Glu Trp Thr 1 5 10 <210> 69 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 78A74 VH <400> 69 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> 70 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 78A74 VL <400> 70 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 Gln Arg Ser Asn Trp Pro Pro 85 90 95 Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 71 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> hIgG1 heavy chain constant region <400> 71 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> 72 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> hIgG1 CL (Kappa) <400> 72 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> 73 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> hIgG1 CL (Lambda) <400> 73 Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 74 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 VL <400> 74 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> 75 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 78A74-21 VH <400> 75 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 Glu 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> 76 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 78A74-21 VL <400> 76 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 Gly 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 Gln Arg Ser Asn Gly Pro Pro 85 90 95 Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 77 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 78A74-25 VL <400> 77 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 Gln Arg Ser Asn Trp Pro Pro 85 90 95 Glu Leu Tyr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 78 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 78A74-35 VL <400> 78 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 Gln Arg Ser Asn Trp Pro Pro 85 90 95 Ala Met Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 79 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 78A74-36 VL <400> 79 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 Gln Arg Thr Met Trp Pro Pro 85 90 95 Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 80 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A74-7 LCDR3 <400> 80 Gln Leu Met Ser Asn Trp Pro Pro Glu Trp Thr 1 5 10 <210> 81 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 78A74-21 HCDR3 <400> 81 Glu Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 82 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A74-21 LCDR1 <400> 82 Arg Ala Ser Gln Ser Val Gly Ser Tyr Leu Ala 1 5 10 <210> 83 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A74-21 LCDR3 <400> 83 Gln Gln Arg Ser Asn Gly Pro Pro Glu Trp Thr 1 5 10 <210> 84 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A74-25 LCDR3 <400> 84 Gln Gln Arg Ser Asn Trp Pro Pro Glu Leu Tyr 1 5 10 <210> 85 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A74-35 LCDR3 <400> 85 Gln Gln Arg Ser Asn Trp Pro Pro Ala Met Thr 1 5 10 <210> 86 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A74-36 LCDR3 <400> 86 Gln Gln Arg Thr Met Trp Pro Pro Glu Trp Thr 1 5 10 <210> 87 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 78A5 HCDR1 <400> 87 Gly Tyr Thr Phe Thr Asn Tyr Asp Phe Ser 1 5 10 <210> 88 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 78A5 HCDR2 <400> 88 Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn 1 5 10 <210> 89 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 78A5 HCDR3 <400> 89 Asp Val Ile Pro Tyr Gly Tyr Pro Ala Phe Asp Ile 1 5 10 <210> 90 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 78A5 LCDR1 <400> 90 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 91 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 78A5 LCDR2 <400> 91 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 78A5 LCDR3 <400> 92 Gln Gln Ala Asn Ser Phe Pro Ile Thr 1 5 <210> 93 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 78A5 VH <400> 93 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Asp Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Asp Val Ile Pro Tyr Gly Tyr Pro Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 94 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 78A5 VL <400> 94 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 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 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 95 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 17 HCDR1 <400> 95 Gly Phe Thr Phe Asn Lys Tyr Thr Ile Asn 1 5 10 <210> 96 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 17 HCDR2 <400> 96 Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn 1 5 10 <210> 97 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 17 HCDR3 <400> 97 Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser 1 5 10 <210> 98 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 17 LCDR1 <400> 98 Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala 1 5 10 <210> 99 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 17 LCDR2 <400> 99 Ala Ala Ser Thr Leu Gln Ser 1 5 <210> 100 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 17 LCDR3 <400> 100 Gln Gln Phe Glu Ser Tyr Pro Leu Thr 1 5 <210> 101 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 17 VH <400> 101 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> 102 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 17 VL <400> 102 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> 103 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 35 HCDR1 <400> 103 Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 10 <210> 104 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 35 HCDR2 <400> 104 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr 1 5 10 <210> 105 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> 35 HCDR3 <400> 105 Asp Leu Leu Asp Phe Trp Ser Gly Pro Gly Ala Phe Asp Ile 1 5 10 <210> 106 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 35 LCDR1 <400> 106 Arg Ala Ser Gln His Ile Ser Ser Trp Leu Ala 1 5 10 <210> 107 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 35 LCDR3 <400> 107 Gln Gln Phe Asn Ser Tyr Pro Leu Thr 1 5 <210> 108 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 35 VH <400> 108 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Leu Leu Asp Phe Trp Ser Gly Pro Gly Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 109 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 35 VL <400> 109 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 110 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is S or N <220> <221> MISC_FEATURE <222> (6) <223> Xaa is S or V <220> <221> MISC_FEATURE <222> (8) <223> Xaa is S or G <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa is N or H <400> 110 Gly Phe Thr Phe Xaa Xaa Tyr Xaa Met Xaa 1 5 10 <210> 111 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is A or E <400> 111 Xaa Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15

Claims

1. An anti-ANG2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region polypeptide and a light chain variable region polypeptide, wherein the heavy chain variable region polypeptide comprises the HCDR1 sequence of SEQ ID NO: 12, the HCDR2 sequence of SEQ ID NO: 5, and the HCDR3 sequence of SEQ ID NO: 6, and the light chain variable region polypeptide comprises the LCDR1 sequence of SEQ ID NO: 7, the LCDR2 sequence of SEQ ID NO: 13, and the LCDR3 sequence of SEQ ID NO:

14.

2. The anti-ANG2 antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region polypeptide consists of the amino acid sequence of SEQ ID NO:

15.

3. The anti-ANG2 antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region polypeptide consists of the amino acid sequence of SEQ ID NO:

16.

4. The anti-ANG2 antibody or antigen-binding fragment thereof of claim 1 , wherein the heavy chain variable region polypeptide consists of the amino acid sequence of SEQ ID NO: 15, and the light chain variable region polypeptide consists of the amino acid sequence of SEQ ID NO:

16.

5. The anti-ANG2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a fully human antibody, scFv, Fab, Fab', F(ab')2, Fv fragment or dsFv.

6. The anti-ANG2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, further comprising a heavy chain constant region and / or a light chain constant region.

7. The anti-ANG2 antibody or antigen-binding fragment thereof according to claim 6, wherein The heavy chain constant region is a heavy chain constant region of human IgG1; and / or The light chain constant region is a human kappa or lambda light chain constant region.

8. The anti-ANG2 antibody or antigen-binding fragment thereof of claim 6, wherein The heavy chain constant region consists of the amino acid sequence of SEQ ID NO: 71; and / or The light chain constant region consists of the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO:

73.

9. The anti-ANG2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which specifically binds to ANG2 but does not bind to ANG1.

10. A polynucleotide encoding the anti-ANG2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9. An expression vector comprising the polynucleotide of claim 10 .

12. A host cell comprising the polynucleotide of claim 10 or the expression vector of claim 11.

13. A pharmaceutical composition comprising the anti-ANG2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

14. Use of the anti-ANG2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 13, 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, neovascular glaucoma, macular edema, retinal edema, or choroidal neovascular disease.

15. The use according to claim 14, wherein the angiogenesis-related eye disease is retinopathy of prematurity, diabetic retinopathy or diabetic macular edema.

16. Use of the anti-ANG2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 14, 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, esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue sarcoma, bladder cancer, or a central nervous system tumor.

17. The use according to claim 16, wherein the cancer is glioma.

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