An-ANG2 antibodies and uses thereof

Abstract

The invention relates to the field of biological medicine. Specifically, the invention provides an anti-ANG2 heavy chain variable region polypeptide, an antibody or an antigen binding fragment thereof, especially a fully human anti-ANG2 heavy chain variable region polypeptide, an antibody or an antigen binding fragment thereof. The invention also provides polynucleotide for coding the anti-ANG2 heavy chain variable region polypeptide, the antibody or the antigen binding fragment thereof, and an expression vector and a host cell for expressing the anti-ANG2 heavy chain variable region polypeptide, the antibody or the antigen binding fragment thereof. The invention further provides methods of producing the anti-ANG2 heavy chain variable region polypeptides, antibodies or antigen binding fragments thereof, and uses thereof.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202111422308.1, titled "Anti-ANG2 Antibody and Its Use", filed on November 26, 2021. Technical Field

[0002] The present invention relates to the field of biomedicine. Specifically, the present invention provides an anti-ANG2 heavy chain variable region polypeptide, an antibody or an antigen-binding fragment thereof, particularly a fully human anti-ANG2 heavy chain variable region polypeptide, an antibody or an antigen-binding fragment thereof. Background Art

[0003] Angiopoietin is part of the vascular growth factor family. As a member of the angiopoietin family, angiopoietin-2 (Angiopoietin-2, also known as ANG2 or ANGPT2) is a secreted glycoprotein, and its overexpression promotes the formation of new blood vessels. The direct receptor of ANG2 is TIE2, which can bind angiopoietin ANG1, ANG2, and ANG4. TIE1, an orphan receptor, cannot be directly activated by binding to ANG. After the binding of ANG-TIE2, TIE1 and TIE2 form a complex and are activated. The signal pathway after the binding of the ANG-TIE ligand can regulate vascular permeability, inflammation, and pathological remodeling of blood vessels in tumors.

[0004] ANG proteins can stimulate the trafficking and localization of TIE receptors expressed on endothelial cells to cell-cell and cell-matrix junctions. ANG1 and ANG2 have opposite functions in regulating angiogenesis. ANG1 is expressed by mesenchymal cells and activates TIE2 function as an agonist, promoting the survival of vascular endothelial cells, tight junctions, and stability of blood vessels; while ANG2 is expressed by endothelial cells and is usually stored in endothelial cell Weibel–Palade bodies. When encountering situations that promote the formation of new blood vessels such as hypoxia and inflammation, ANG2 is released from the Weibel–Palade bodies and competes with ANG1. As an antagonist of TIE2, it inhibits the stability of blood vessels and promotes the formation of new blood vessels together with VEGF and others. At the same time, many studies have shown that the overexpression of ANG2 plays a key role in promoting the lymph node metastasis of tumors. Therefore, ANG2 is an important target for the treatment of diseases related to pathological angiogenesis and tumors.

[0005] In 2004, it was first confirmed that blocking the activity of ANG2 by antibodies that neutralize the interaction between ANG2 and its receptor TIE2 could effectively inhibit tumor angiogenesis and tumor growth (Oliner, J., et al. (2004) Cancer Cell 6(5):507-516). Therefore, the development of monoclonal antibodies targeting the ANG2 target has great application value in the treatment of solid tumors and the treatment of eye diseases related to pathological angiogenesis.

[0006] There are anti-ANG2 antibodies in the prior art (see, for example, WO2015179166A1 and WO2011014469A1), including the clinical in-development fully human antibody nesvacumab that targets ANG2. The art still hopes to develop new and improved anti-ANG2 fully human antibodies. SUMMARY OF THE INVENTION

[0007] In one aspect, the present invention provides a heavy chain variable region polypeptide that specifically binds to ANG2, which comprises HCDR1, HCDR2, and HCDR3 sequences, wherein

[0008] (a) The HCDR1 sequence is as shown in SEQ ID NO:110

[0009] GFTFX1X2YX3MX4 (SEQ ID NO:110)

[0010] wherein X1 is S or N; X2 is S or V; X3 is S or G; X4 is N or H;

[0011] (b) The HCDR2 sequence is as shown in VISYDGSNKY (SEQ ID NO:5);

[0012] (c) The HCDR3 sequence is as shown in SEQ ID NO:111

[0013] X5TLDGYTAGYYYGMDV (SEQ ID NO:111)

[0014] wherein X5 is A or E.

[0015] In another aspect, the present invention provides an anti-ANG2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region polypeptide and a light chain variable region polypeptide. 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 described above, and the LCDR1, LCDR2, and LCDR3 sequences are selected from any one of (1)-(21):

[0016] (1) 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; (2) 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; (3) 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; (4) 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; (5) 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; (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; (7) 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; (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:26; (14) 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; (15) 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; (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:54; the LCDR2 sequence shown in SEQ ID NO:55; and the LCDR3 sequence shown in SEQ ID NO:56;.

[0017] In yet another aspect, the present invention provides an anti-ANG2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region polypeptide and a light chain variable region polypeptide, the heavy chain variable region polypeptide comprising HCDR1, HCDR2 and HCDR3 sequences, and the light chain variable region polypeptide comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences are selected from any one of (1)-(3): (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; (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; (3) the HCDR1 sequence shown in SEQ ID NO:103; the HCDR2 sequence shown in SEQ ID NO:104; the HCDR3 sequence shown in SEQ ID NO:105; the LCDR1 sequence shown in SEQ ID NO:106; the LCDR2 sequence shown in SEQ ID NO:91; and the LCDR3 sequence shown in SEQ ID NO:107.

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

[0019] The present invention also provides a polynucleotide that encodes the heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof or multispecific antibody of the present invention.

[0020] The present invention also provides an expression vector that comprises the polynucleotide of the present invention.

[0021] The present invention also provides a host cell that comprises the polynucleotide or expression vector of the present invention.

[0022] The present invention also provides a pharmaceutical composition, which comprises the heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof or multispecific antibody of the present invention, and a pharmaceutically acceptable carrier.

[0023] 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) eye diseases related to angiogenesis; or (2) cancer. In one embodiment, the eye diseases related to angiogenesis are macular degeneration, retinal vein occlusion, retinopathy, retinopathy of prematurity, diabetic retinopathy, neovascular glaucoma, pathologic 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 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

[0024] Figure 1A-1C Shows the binding activity of the candidate monoclonal supernatant binding to the antigen ANG2 measured by ELISA method.

[0025] Figure 2A-2C Shows the blocking activity of the candidate monoclonal supernatant blocking the binding of ANG2 and the receptor TIE2 measured by ELISA.

[0026] Figure 3A-3B Shows the binding activity of the candidate antibody binding to the antigen ANG2 measured by ELISA method.

[0027] Figure 4 Shows the binding activity of the candidate antibody binding to the antigen ANG1 measured by ELISA method.

[0028] Figure 5A-5B Shows the blocking activity of the candidate antibody blocking the binding of ANG2 and the receptor TIE2 measured by ELISA.

[0029] Figure 6 Shows the blocking activity of the candidate antibody blocking the binding of ANG2 and hTIE2-HEK293 cells measured by FACS.

[0030] Figure 7 Shows the inhibitory activity of the candidate antibody inhibiting ANG2-mediated TIE2 phosphorylation.

[0031] Figure 8A-8C Shows the inhibitory effect of the candidate antibody on tumor growth in a mouse subcutaneous xenograft tumor model; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0032] Figure 9 Shows the in vivo plasma concentration-time curve of the candidate antibody in Balb / C mice. Detailed implementation

[0033] Definition

[0034] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation procedures used herein are all widely used terms and conventional procedures in the corresponding fields. At the same time, for a better understanding of the present invention, the definitions and explanations of relevant terms are provided below.

[0035] As used herein, the expressions "comprising", "including", "containing", and "having" are open-ended, indicating the inclusion of the recited elements, steps, or components but not excluding other unrecited elements, steps, or components. The expression "consisting of" does not include any unspecified elements, steps, or components. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, plus optionally present 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 "including".

[0036] As used herein, "antibody" refers to an immunoglobulin or a fragment thereof that specifically binds to an epitope through at least one antigen-binding site. The term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single-domain antibodies, and antigen-binding fragments. Antibodies can be synthetic (e.g., produced by chemical conjugation or bioconjugation), enzymatically treated, or recombinantly produced. The 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).

[0037] The molecular, antibody, or product numbers used herein are only for differentiating or identifying molecules or products and are not intended to indicate that such identification is a characteristic of the molecules or products of the present invention. Those skilled in the art should understand that, for example, for the purpose of differentiation or identification, other molecules, antibodies, or products may also use such identification, but it does not mean the same or equivalent molecules, antibodies, or products. Similarly, the similar numbers or identifications used in the examples are only for the convenience of illustration, and the molecular antibodies or products of the present invention are defined by the features described in the appended claims.

[0038] As used herein, "antigen-binding fragment" refers to a portion of a full-length antibody that is less than full-length but that includes at least a portion of the variable region of the full-length antibody (e.g., includes one or more CDRs and / or one or more antigen-binding sites) and thus retains at least a portion of the ability of the full-length antibody to specifically bind an antigen. Examples of antigen-binding fragments include, but are not limited to, sdAbs (e.g., variable domains of heavy-chain antibodies), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab')2, diabodies, Fd, and Fd' fragments, and other fragments (e.g., fragments that include modifications).

[0039] As used herein, "full-length antibody" typically includes four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain includes a light-chain variable region (VL) and a light-chain constant region (CL). Each heavy chain includes a heavy-chain variable region (VH) and a heavy-chain constant region (CH).

[0040] The light-chain variable region and the heavy-chain variable region each can include three highly variable "complementary determining regions (CDRs)" and four relatively conserved "framework regions (FRs)", and are linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. In this document, the CDRs of the light-chain variable region (CDRL or LCDR) can be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy-chain variable region (CDRH or HCDR) can be referred to as HCDR1, HCDR2, and HCDR3.

[0041] In the present invention, the amino acid sequences of the CDRs are 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, as is well known to those skilled in the art, there are various methods in the art for defining the CDRs of antibodies, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (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 the sequence variability of antibodies (see, for example, Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), AbM (Martin, A. C. R. 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, R. M. et al., (1996) J. Mol. Biol. 262:732-745), IMGT (Lefranc, M.-P., 2011(6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art should understand that, unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or its region (such as the variable region) should be understood to cover the complementary determining regions defined by any of the above-known schemes described in the present invention.Although the scope claimed in the claims of the present invention is based on the sequences shown by the AbM definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the protection scope of the present invention.

[0042] Therefore, when referring to antibodies defined by the specific CDR sequences of the present invention, the scope of the antibodies also encompasses antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined by the present invention due to the application of different schemes (such as different assignment system rules or combinations).

[0043] As used herein, the terms "framework region" and "framework segment" can be used interchangeably. As used herein, the terms "framework region", "framework segment" or "FR" residues refer to those amino acid residues in the antibody variable region other than the CDR sequences defined as above.

[0044] It is generally considered that the "Fv" fragment composed of one VH and one VL through non-covalent interaction is the smallest antigen-binding fragment containing the antigen-binding site. However, single variable domains (single-domain antibodies) also have antigen-binding ability. The "single-chain Fv (scFv)" can be obtained by linking VH and VL through a peptide linker. By introducing disulfide bonds into Fv or scFv, "disulfide-stabilized Fv (dsFv)" or "single-chain disulfide-stabilized Fv (scdsFv or dsscFv)" can be obtained respectively.

[0045] As used herein, "Fab" contains a complete antibody light chain (VL-CL) and the variable region of the antibody heavy chain and one heavy chain constant region (VH-CH1, also known as Fd). Linking CL and CH1 in "Fab" with a peptide linker can obtain single-chain "Fab (scFab)". "F(ab')2" basically contains two Fab fragments linked by disulfide bonds in the hinge region. "Fab'" is half of F(ab')2, which can be obtained by reducing the disulfide bonds in the hinge region of F(ab')2.

[0046] As used herein, the terms "fully human antibody", "completely human antibody" and "human antibody" can be used interchangeably, which refer to antibodies produced by humans or antibodies having amino acid sequences corresponding to those produced by humans prepared using any technique known in the art. The definition of fully human antibodies encompasses intact or full-length antibodies, their fragments and / or antibodies containing at least one human heavy chain and / or light chain polypeptide. Fully human antibodies have low immunogenicity in the human body. In some embodiments, the anti-ANG2 antibody or its antigen-binding fragment of the present invention is a fully human antibody.

[0047] As used herein, an "affinity matured" antibody contains one or more modifications (e.g., substitutions of amino acid residues) in one or more CDRs such that the affinity matured antibody has an improved affinity for an antigen as compared to a parental antibody that does not contain such modifications. Methods for affinity maturing 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); Schier et al., Gene 169:147-155 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0048] As used herein, "percent (%) sequence identity" and "sequence identity" of an amino acid sequence have their recognized definitions in the art, which refer to the percentage of identity between two polypeptide sequences determined by sequence alignment (e.g., by manual inspection or well-known algorithms). Methods known to those skilled in the art can be used to determine, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0049] A polypeptide (e.g., the CDR region, framework region, and constant region of an antibody) can be modified, for example, by one or more amino acid substitutions, additions, and / or deletions without altering the function of the polypeptide. The substitutions are preferably conservative substitutions of amino acids. Suitable conservative substitutions are well known to those skilled in the art. In addition, methods known in the art can be used to modify antibodies to change their properties, such as changing the type of antibody glycosylation modification, changing the ability to form interchain disulfide bonds, or providing reactive groups for the preparation of antibody conjugates. Such modified antibodies are also encompassed within the scope of the antibodies of the present invention.

[0050] "Affinity" or "binding affinity" is used to measure the strength of the non-covalent interaction between an antibody and an antigen. The magnitude of "affinity" can typically be reported as the equilibrium dissociation constant K D or EC 50 . K D can be calculated by measuring the equilibrium association constant (ka) and the equilibrium dissociation constant (kd): K D = kd / ka. Affinity can be determined using conventional techniques known in the art, such as biolayer interferometry (e.g., using a ForteBio Octet or Gator detection system), surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS), etc.

[0051] As used herein, "specific binding" between an antibody and an antigen means that the antibody binds to the antigen with relatively high affinity. Generally, the K D value between specifically binding antibody and antigen can be at least about 10 -7 M to at least about 10 -10 M or lower, 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 M or lower, such as determined by biolayer interferometry. In some embodiments, the anti-ANG2 antibody or antigen-binding fragment thereof of the present invention binds to ANG2 with a K D value of 5 nM, 2 nM, 1 nM or lower.

[0052] As used herein, the term "isolated" means that a substance (such as a polynucleotide or polypeptide) is separated from its source or environment in which it exists, i.e., it substantially does not contain any other components. The anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof or polynucleotide encoding the same of the present invention can be isolated.

[0053] As used herein, the terms "polynucleotide" and "nucleic acid" can be used interchangeably to denote an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives. Polynucleotides can include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0054] As used herein, a "vector" is a vehicle for introducing an exogenous polynucleotide into a host cell, and when the vector is transformed into a suitable host cell, the exogenous polynucleotide can be amplified or expressed. As used herein, the definition of a vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (such as 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, herpesviral vectors, poxviral vectors, baculoviral vectors, etc. As used herein, an "expression vector" refers to a vector capable of expressing a polypeptide of interest. An expression vector generally can contain a polynucleotide sequence encoding the polypeptide of interest and regulatory sequences (such as a promoter and ribosome binding site) operably linked thereto.

[0055] As used herein, "host cell" refers to 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. A host cell can be a eukaryotic cell or a prokaryotic cell. Prokaryotic cells such as Escherichia coli (E. coli) or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus species, insect cells (such as Drosophila S2 cells or Sf9), and animal cells (such as fibroblasts, CHO cells, COS cells, HeLa cells, NSO cells, or HEK293 cells).

[0056] As used herein, the term "treatment" refers to the amelioration of a disease / symptom, such as reducing or eliminating the disease / symptom, preventing or slowing the onset, progression, and / or worsening of the disease / symptom.

[0057] As used herein, "effective amount" means the amount of an active substance (such as an antibody or pharmaceutical composition of the present invention) that elicits a biological or medical response or a desired therapeutic effect in a tissue, system, animal, mammal, or human. Thus, an "effective amount" can be the amount required to prevent, cure, ameliorate, arrest, or partially arrest a disease or symptom (such as cancer). Those skilled in the art can determine the effective amount based on factors such as the age, physical condition, gender, severity of the symptom, specific composition, or route of administration of the subject, etc.

[0058] 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, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0059] Examples of mammals as used herein 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.

[0060] Anti-ANG2 polypeptide, antibody or antigen-binding fragment thereof

[0061] In one aspect, the present invention provides a heavy chain variable region polypeptide that specifically binds to ANG2, which comprises HCDR1, HCDR2, and HCDR3 sequences, wherein

[0062] (a) the HCDR1 sequence is as shown in SEQ ID NO: 110

[0063] GFTFX1X2YX3MX4 (SEQ ID NO:110)

[0064] wherein X1 is S or N; X2 is S or V; X3 is S or G; X4 is N or H;

[0065] (b) The HCDR2 sequence is as shown in VISYDGSNKY (SEQ ID NO:5);

[0066] (c) The HCDR3 sequence is as shown in SEQ ID NO:111

[0067] X5TLDGYTAGYYYGMDV (SEQ ID NO:111)

[0068] wherein X5 is A or E.

[0069] In a specific embodiment, the HCDR1, HCDR2 and HCDR3 sequences are selected from any one of (1)-(4):

[0070] (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;

[0071] (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;

[0072] (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;

[0073] (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.

[0074] In another embodiment, the anti-ANG2 antibody or its antigen-binding fragment further comprises a light chain variable region polypeptide, and the light chain variable region polypeptide comprises LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences are selected from any one of (1)-(21):

[0075] (1) 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;

[0076] (2) 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;

[0077] (3) 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;

[0078] (4) 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;

[0079] (5) 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;

[0080] (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;

[0081] (7) 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;

[0082] (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;

[0083] (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;

[0084] (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;

[0085] (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;

[0086] (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;

[0087] (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:26;

[0088] (14) 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;

[0089] (15) 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;

[0090] (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;

[0091] (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;

[0092] (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;

[0093] (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;

[0094] (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;

[0095] (21) 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.

[0096] 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, the heavy chain variable region polypeptide comprising HCDR1, HCDR2 and HCDR3 sequences, and the light chain variable region polypeptide comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences are selected from any one of (1)-(21):

[0097] (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;

[0098] (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;

[0099] (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;

[0100] (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;

[0101] (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;

[0102] (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;

[0103] (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;

[0104] (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;

[0105] (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;

[0106] (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;

[0107] (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;

[0108] (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;

[0109] (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;

[0110] (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;

[0111] (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;

[0112] (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;

[0113] (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;

[0114] (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;

[0115] (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;

[0116] (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;

[0117] (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.

[0118] 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 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: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 as 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.

[0119] 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 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity 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.

[0120] 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; 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.

[0121] In a specific embodiment, the heavy chain variable region polypeptide and the light chain variable region polypeptide are selected from any one of (1)-(21):

[0122] (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;

[0123] (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;

[0124] (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;

[0125] (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;

[0126] (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;

[0127] (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;

[0128] (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;

[0129] (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;

[0130] (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;

[0131] (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;

[0132] (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;

[0133] (12) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 40; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 49;

[0134] (13) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 40; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 53;

[0135] (14) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 75; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 76;

[0136] (15) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 15; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 16;

[0137] (16) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 30; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 31;

[0138] (17) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 34; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 35;

[0139] (18) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 37; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 38;

[0140] (19) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 57; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 58;

[0141] (20) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 60; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 61;

[0142] (21) Heavy chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 66; light chain variable region polypeptide, which comprises the amino acid sequence of SEQ ID NO: 67.

[0143] In another aspect, the present invention also provides an anti-ANG2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region polypeptide and a light chain variable region polypeptide, the heavy chain variable region polypeptide comprising HCDR1, HCDR2 and HCDR3 sequences, and the light chain variable region polypeptide comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences are selected from any one of (1)-(3):

[0144] (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;

[0145] (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;

[0146] (3) The HCDR1 sequence shown in SEQ ID NO:103; the HCDR2 sequence shown in SEQ ID NO:104; the HCDR3 sequence shown in SEQ ID NO:105; the LCDR1 sequence shown in SEQ ID NO:106; the LCDR2 sequence shown in SEQ ID NO:91; and the LCDR3 sequence shown in SEQID NO:107.

[0147] 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.

[0148] 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 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:94, SEQ ID NO:102, or SEQ ID NO:109. 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: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.

[0149] 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.

[0150] In a specific embodiment, the heavy chain variable region polypeptide and the light chain variable region polypeptide are selected from any one of (1)-(3):

[0151] (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;

[0152] (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;

[0153] (3) A heavy chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 108; a light chain variable region polypeptide comprising the amino acid sequence of SEQ ID NO: 109.

[0154] In some embodiments, the anti-ANG2 antibody or antigen-binding fragment thereof of the present invention is an scFv, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv (dsFv) or diabody.

[0155] 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.

[0156] 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 immunoglobulins of any species. The heavy chain constant region can be derived from the heavy chain constant region of immunoglobulins of any subtype (such as IgA, IgD, IgE, IgG and IgM), class (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass (such as, IgG2a and IgG2b) or combinations thereof. The light chain constant region can be derived from the lambda (Lambda) light chain or kappa (Kappa) light chain constant region.

[0157] Suitable immunoglobulin constant regions (such as CH1 and light chain constant region, hinge region-CH2-CH3, CH1-hinge region-CH2-CH3 and light chain constant region or Fc region), as well as types (such as IgG, such as IgG1, IgG2, IgG3 and IgG4) can be selected and optionally modified to obtain antibodies with desired properties.

[0158] In some embodiments, the heavy chain constant region is the heavy chain constant region (such as Fc region or CH1-hinge region-CH2-CH3) of human IgG (such as IgG1, IgG2a, IgG2b, IgG3 or IgG4). In one embodiment, the heavy chain constant region is the heavy chain constant region of human IgG1 (the 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.

[0159] In a preferred embodiment, the light chain constant region is the human kappa light chain constant region (exemplary amino acid sequence shown in SEQ ID NO: 72) or the human lambda light chain constant region (exemplary amino acid sequence 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.

[0160] In some embodiments, the anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof of the present invention specifically binds to ANG2, but does not bind or substantially does not bind to ANG1. As used herein, the expression "does not bind" or "substantially does not bind" means that the binding ability of the polypeptide, antibody or antigen-binding fragment thereof of the present invention to ANG1 is significantly lower than its binding ability to ANG2. For example, as Figure 4 shown, the molecules of the present invention exhibit weak binding or no binding to ANG1.

[0161] Antibodies or antigen-binding fragments thereof can be prepared and produced using methods known in the art. Such methods can include, for example, preparing and isolating the coding nucleic acids of antibodies or antigen-binding fragments from phage display libraries, yeast display libraries, immortalized B cells (e.g., mouse B cell hybridoma cells or EBV-immortalized B cells). Antibodies can also be generated by immunizing animals, such as immunizing an animal (e.g., humanized mouse) with an antigen or DNA encoding the antigen, and then isolating the antibody-expressing B cells from the immunized animal. Polynucleotides encoding antibodies or antigen-binding fragments thereof can also be isolated from immunized animals or humans or prepared by chemical synthesis, and then expression vectors for expressing antibodies or antigen-binding fragments are constructed using the polynucleotides.

[0162] Bispecific antibody

[0163] In another aspect, the present invention 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 the anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof of the present invention.

[0164] As used herein, the term "multispecific antibody" refers to an antibody capable of specifically binding two or more (e.g., 2, 3, 4, 5 or 6) different antigenic epitopes. Multispecific antibodies can be, for example, bispecific, trispecific or tetra-specific antibodies, which are capable of specifically binding 2, 3 or 4 antigenic epitopes, respectively. As used herein, the term "antigenic epitope" or "epitope" refers to a region of an antigen that specifically binds to the antigen-binding site of an antibody. Antigenic epitopes usually consist of chemically active surface groups of the antigen (such as amino acids or sugar side chains) and usually have specific three-dimensional structural properties as well as specific charge properties. Multispecific antibodies can be multivalent (e.g., 2, 3, 4-valent) antibodies, i.e., they have multiple antigen-binding sites. Multispecific antibodies can be, for example, chimeric antibodies, humanized antibodies, fully human antibodies, scFabs, F(ab')2 or diabodies.

[0165] Methods for constructing multispecific antibodies using antibodies or antigen-binding fragments of interest are well known to those skilled in the art (see, for example, WO 93 / 08829; Suresh et al., (1986) Methods in Enzymology, 121:210; and Traunecker et al., (1991) EMBO, 10:3655-3659).

[0166] As used herein, "first antigen-binding portion" and "second antigen-binding portion" refer to amino acid sequences that contain antigen-binding sites and are capable of binding to antigenic epitopes, and their definitions fall within the meaning of antibodies or antigen-binding fragments. The first antigen-binding portion and the second antigen-binding portion 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.

[0167] The first antigen-binding portion and the second antigen-binding portion can optionally be linked by a linker. In some embodiments, the first antigen-binding portion and the second antigen-binding portion are not linked by a linker. In other embodiments, the first antigen-binding portion and the second antigen-binding portion are linked 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 linked by a peptide linker. Exemplary peptide linkers can include but are not limited to polyglycine (G), polyalanine (A), polyserine (S) or combinations thereof, such as GGAS, GGGS, GGGSG or (G4S) n , where n is an integer from 1 to 20.

[0168] Polynucleotide, vector and host cell

[0169] 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.

[0170] The polynucleotides of the present invention can be obtained by methods known in the art. For example, the polynucleotides of the present invention can be isolated from humans, phage display libraries, yeast display libraries, immunized animals, immortalized cells (e.g., murine B cell hybridoma cells, EBV-mediated immortalized B cells) or chemically synthesized. The polynucleotides can be codon-optimized for the host cell used for expression.

[0171] In yet another aspect, the present invention further provides an expression vector comprising the polynucleotide of the present invention. The expression vector can further comprise additional polynucleotide sequences, such as transcriptional regulatory sequences and antibiotic resistance genes.

[0172] The present invention also provides a host cell comprising the 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 by various methods known in the art. Such methods include but are not limited to viral transduction, liposome transfection, electroporation, and calcium phosphate transfection, etc. In a preferred embodiment, the host cell is used for expressing 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, 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 expressing antibodies.

[0173] 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, which comprises:

[0174] (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

[0175] (II) isolating the heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof or multispecific antibody from the host cell or its culture.

[0176] Pharmaceutical composition

[0177] 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.

[0178] Pharmaceutically acceptable carriers can 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 bisulfite, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.), solubilizers, gelling agents, softeners, solvents (e.g., water, alcohol, acetic acid and syrup), buffers (e.g., phosphate buffer, histidine buffer and acetate buffer), surfactants (e.g., nonionic surfactants such as polysorbate 80, polysorbate 20, poloxamer or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delaying agents, chelating agents and emulsifying agents. For compositions comprising an antibody or an antibody conjugate, suitable carriers can be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer) or combinations thereof.

[0179] The pharmaceutical compositions provided herein can be in a variety of dosage forms, including but not limited to solid, semi-solid, 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).

[0180] 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), local (e.g., intratumoral), epidural or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual or topical). The method of administration can be, for example, injection or infusion.

[0181] As a general guide, the dosage range for administration of the anti-ANG2 antibody or its antigen-binding fragment of the present invention can be 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 can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight or 20 mg / kg body weight, or within the range of 1 - 20 mg / kg. Exemplary treatment regimens require administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3 - 6 months, or an initial dosing interval that is slightly shorter and a later dosing interval that is longer.

[0182] Treatment

[0183] The anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, or multispecific antibody of the present invention blocks the binding of human ANG2 to the human TIE2 receptor and inhibits the phosphorylation of TIE2 by binding to human ANG2, thereby inhibiting angiogenesis related to ANG2 activity. As used herein, "angiogenesis" refers to the formation of new blood vessels. Studies have shown that angiogenesis is associated with a variety of diseases, such as cancer and angiogenesis-related eye diseases.

[0184] The anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, multispecific antibody or pharmaceutical composition of the present invention can be used to treat cancer or angiogenesis-related eye diseases. The present invention also provides the use of the anti-ANG2 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 drug 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, which comprises administering to the subject an effective amount of the anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, multispecific antibody or pharmaceutical composition of the present invention.

[0185] Angiogenesis-related eye diseases can be eye diseases related to choroidal and retinal vascular diseases, including but not limited to choroidal neovascular diseases, retinal neovascular diseases and diseases related to vascular leakage. In one embodiment, the angiogenesis-related eye disease is macular degeneration (such as dry or wet age-related macular degeneration (AMD)), retinal vein occlusion, retinopathy, retinopathy of prematurity (ROP), diabetic retinopathy, neovascular glaucoma, pathologic myopia, macular edema, retinal edema, diabetic macular edema (DME) or choroidal neovascular disease.

[0186] 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. Cancers can include primary cancers and metastatic cancers. Non-limiting examples of cancers 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 lymphoma, esophageal cancer, anal cancer, cancers of the endocrine system, thyroid cancer, parathyroid cancer, kidney cancer (e.g., renal cell carcinoma, renal pelvic carcinoma, 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.

[0187] For the treatment of cancer, the anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, multispecific antibody, or pharmaceutical composition of the present invention can be used in combination with one or more therapeutic agents selected from the following: chemotherapeutic agents, immune checkpoint inhibitors, and angiogenesis inhibitors. Examples of chemotherapeutic agents include, but are not limited to: cyclophosphamide, ifosfamide, melphalan, busulfan, nitrogen mustard, chlorambucil, lomustine (CCNU), carmustine (BCNU), 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, liposomal paclitaxel, actinomycin D, idarubicin, doxorubicin, epirubicin, mitomycin, bleomycin, and adriamycin. 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 (e.g., Bevacizumab), anti-VEGFR antibodies (e.g., Ramucirumab), small molecule inhibitors of VEGFR, and VEGF inhibitory fusion proteins (e.g., aflibercept).

[0188] Kit

[0189] The present invention also provides a kit, which comprises the anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof, multispecific antibody or pharmaceutical composition of the present invention, as well as instructions for use. The kit may also comprise a suitable container, such as an ampoule. In some embodiments, the kit further includes a device for administration. The kit may also comprise a label for indicating the intended use and / or method of use of the contents of the kit. The term "label" includes any written or recorded material provided on or with the kit or otherwise provided with the kit.

[0190] Beneficial effect

[0191] The anti-ANG2 heavy chain variable region polypeptide, antibody or antigen-binding fragment thereof or multispecific antibody of the present invention can at least achieve the following beneficial effects:

[0192] (1) Specifically bind to ANG2, but not bind or substantially not bind to ANG1;

[0193] (2) Block the binding of ANG2 to TIE2 and inhibit TIE2 phosphorylation;

[0194] (3) Inhibit angiogenesis related to ANG2 activity; and / or

[0195] (4) Inhibit tumor growth.

[0196] The anti-ANG2 antibody or antigen-binding fragment thereof of the present invention can be a fully human antibody, and thus has low immunogenicity.

[0197] Example

[0198] The present 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 present invention. The experimental methods without specific conditions noted in the following examples were carried out according to conventional methods and conditions, or according to the product specifications.

[0199] Example 1 Preparation of Raw Materials

[0200] 1.1 Preparation of Antigen Proteins ANG2, ANG1 and Receptor Protein TIE2

[0201] The coding sequences of the extracellular domains 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 Bioscience Co., Ltd. The C-terminals of their gene sequences were respectively ligated to the gene sequence of human IgG1 Fc segment (SEQ ID NO: 1) and His tag by PCR amplification, and then constructed into the eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen) by homologous recombination method. The constructed recombinant protein expression vectors were respectively transformed into Escherichia coli DH5α, cultured overnight at 37 °C, and then the plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). They were expressed through the Expi293 transient expression system (ThermoFisher, A14635). The transient transfection method refers to the Expi293 TM ExpressionSystem USER GUIDE.

[0202] Seven days after transfection, the cell expression supernatant was centrifuged at 15,000 g for 10 min at high speed. The obtained Fc-tagged protein expression supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100 mM sodium acetate (pH 3.0), followed by neutralization with 1 M Tris-HCl; the obtained His-tagged protein expression supernatant was affinity purified using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with gradient concentrations of imidazole. The eluted proteins were respectively exchanged into PBS buffer using an ultrafiltration concentrator tube (Millipore, UFC901096), and stored at -80 °C for use after being qualified by SDS-PAGE identification and activity identification. Each protein is hereinafter simply referred to as hANG2-Fc, hANG2-His, cANG2-His, mANG2-Fc, hANG1-Fc and hTIE2 ECD-Fc.

[0203] 1.2 Preparation of positive control antibodies

[0204] The positive control antibody used in this application is the anti-ANG2 antibody nesvacumab, which was synthesized according to the sequence disclosed in US2011027286A1. Plasmids containing the nesvacumab heavy chain (SEQ ID NO:2) gene and the nesvacumab light chain (SEQ ID NO:3) gene were constructed respectively by molecular cloning methods. The remaining steps refer to Example 1.1.

[0205] 1.3 Preparation of hTIE2-HEK293 cell line

[0206] The DNA sequence of full-length human TIE2 (NCBI Gene ID:7010) was constructed onto the pLVX-puro plasmid (Clontech, Cat#632164). Then, the resulting plasmid was electrotransformed into HEK293 cells . Through selection under 2 μg / mL puromycin pressure, the single cell clones that grew out were identified using the antibody nesvacumab (conventional FACS method), and the HEK293 cell line overexpressing human TIE2 was successfully obtained, which is also referred to as the "hTIE2-HEK293 cell line" in this article.

[0207] Example 2 Construction and screening of a natural human antibody phage display library

[0208] In this example, an antibody gene phage display library was constructed, and the library was screened using the antigen proteins ANG2 (including hANG2-Fc and hANG2-His) prepared in Example 1.1 as screening antigens, and multiple antibody molecules with specific binding to ANG2 were obtained.

[0209] 2.1 Construction of a gene library of human antibodies

[0210] The Ficoll-Paque density gradient separation solution (purchased from GE, catalog number: 17144003S) was used to separate peripheral blood mononuclear cells (PBMC) from normal human blood. Total RNA was extracted from the isolated PBMC cells by conventional methods, and the extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa, catalog number: 6210A). Degenerate primers were designed at the front end of the V region and the rear end of the first constant region of the heavy chain and light chain respectively based on the sequence similarity of the heavy chain and light chain germline genes (Xiaolin Li, Construction and preliminary screening of a large-capacity non-immune human-derived Fab phage antibody library, Master's thesis of Peking Union Medical College, June 2007). After PCR, the heavy chain variable region gene fragment and light chain variable region gene fragment of the antibody were obtained. The fragment containing the variable regions of the light chain and heavy chain of the antibody was amplified by fusion PCR. The PCR product and the vector for phage display were digested, recovered and ligated, and the ligation product was recovered using a recovery kit (Omega, catalog number: D6492-02). Finally, it was transformed into competent Escherichia coli SS320 (Lucigen, MC1061 F) by an electroporator (Bio-Rad, MicroPulser), and the transformed Escherichia coli SS320 bacterial solution was spread on a 2-YT solid plate with ampicillin resistance. By gradient dilution plating, the library capacity of this library was determined to be 3×10 11 cfu, that is, an antibody gene library of 3×10 11 antibody genes (the calculation method of the library capacity refers to Example 2.2 in CN112250763B). It was packaged using the VSCM13 helper phage (purchased from Stratagene) to obtain an antibody gene phage display library (the preparation of the antibody gene phage display library refers to Example 2.3 in CN112250763B).

[0211] 2.2 Screening of the antibody gene phage display library

[0212] 2.2.1 Screening of the antibody gene phage display library by magnetic bead method

[0213] The screening by magnetic bead method is based on the process of biotin-labeling hANG2-Fc and then binding it to magnetic beads conjugated with streptavidin, and performing a panning process of incubating, washing and eluting the magnetic beads bound with the antigen and the antibody gene phage display library. Usually, 3 - 4 rounds of panning are experienced, and specific monoclonal antibodies against the antigen can be enriched in large quantities. In this example, biotin-labeled hANG2-Fc was used for screening the phage display library, and after 3 rounds of panning, the primary screening of monoclonal antibodies against ANG2 was carried out. The specific method refers to Example 2.4.1 in CN112250763B.

[0214] 2.2.2 Screening of Antibody Gene Phage Display Library by Immunotube Method

[0215] The purposes of both the immunotube method and the magnetic bead method are to enrich specific antibodies against antigens, and they are two mutually complementary and verifying experimental methods. The principle of screening by the immunotube method is to coat hANG2-Fc on the surface of an immunotube with high adsorption capacity. Through the panning process of adding the phage display antibody library into the immunotube and incubating, washing, and eluting with the antigen protein adsorbed on the surface of the immunotube, after 2 - 4 rounds of panning, finally, specific monoclonal antibodies against the antigen are enriched. In this example, after 3 rounds of panning, the primary screening of monoclonal antibodies against ANG2 was carried out, and the specific method refers to Example 2.4.2 in CN112250763B.

[0216] 2.3 Selection of Monoclonal

[0217] ELISA detection was performed on the phage pool eluted in each round to evaluate the enrichment effect, and 10 clones were randomly selected from the phage pool screened in each round for sequence analysis. Combining the enrichment effect and the repeatability ratio of the measured sequences for comprehensive analysis, a suitable round was selected for monoclonal selection.

[0218] For the primary screening of ELISA monoclonal, hANG2-His was used. The positive antibodies that bound to hANG2-His obtained from the primary screening were rechecked using cANG2-His and mANG2-Fc. The ELISA detection data (OD450 values for binding to human, monkey, and mouse ANG2 proteins) of exemplary monoclonal are shown in Table 1. The candidate fully human antibodies were named according to the clone number, and the variable region amino acid sequences of the candidate fully human antibodies are shown in Table 2. The complementarity-determining region sequences were determined by the AbM-defined CDR method.

[0219] Table 1 OD450 (ELISA) of the Screened Exemplary Monoclonal Antibodies Binding to Antigen

[0220]

[0221] Table 2 Variable Region Amino Acid Sequences of Candidate Antibodies

[0222]

[0223] Example 3 Preliminary Identification of Antigen-Binding Activity and Blocking Activity of Candidate Monoclonal Supernatants

[0224] 3.1 Specific Binding of Candidate Monoclonal Supernatants to hANG2-Fc

[0225] Coat hANG2-Fc (2 μg / mL, 30 μL / well) on a 96-well ELISA plate and incubate overnight at 4°C. The next day, wash the plate 3 times with PBST and then block it with 5% skim milk for 2 h. After washing the plate 3 times with PBST, add the serially diluted candidate monoclonal supernatant (obtained by overnight expression of Escherichia coli SS320 containing the plasmid of the monoclonal Fab fragment) and incubate for 1 h. Then, after washing 3 times with PBST, add the anti-human Kappa HRP and anti-human Lambda HRP secondary antibody mixture (Millipore, AP502P, AP506P) and incubate for 1 h. After incubation, wash the plate 6 times with PBST, add TMB for color development, terminate the reaction by adding 2 M HCl according to the color development result, and read the OD450 value using an ELISA reader.

[0226] The detection results are shown in Figure 1A-1C : All candidate monoclonal supernatants specifically bind to hANG2-Fc and have comparable binding abilities.

[0227] 3.2 Blocking activity of candidate monoclonal supernatants against the binding of ANG2 and receptor TIE2.

[0228] Coat hTIE2 ECD-Fc (4 μg / mL, 30 μL / well) on the plate and incubate overnight at 4°C. The next day, wash the plate 3 times with PBST and then block it with 5% skim milk for 2 h. Then, serially dilute the candidate monoclonal supernatants and premix them with biotinylated hANG2-Fc (4 μg / mL) for 0.5 h in advance. After blocking and washing are completed, add them to the 96-well ELISA plate and incubate for 1 h. Then, after washing 3 times with PBST, add NeutrAvidin-HRP (Therofisher, 31001) and incubate for 1 h. After incubation, wash the plate 6 times with PBST, add TMB (SurModics, TMBS-1000-01) for color development, terminate the reaction by adding 2 M HCl according to the color development result, and read the OD450 value using an ELISA reader (Molecular Devices, SpecterMax 190).

[0229] The results show in Figure 2A-2C : All candidate monoclonal supernatants have good ability to block the binding of ANG2 and receptor TIE2, and the abilities are comparable.

[0230] Example 4 Construction, expression and purification of candidate antibodies

[0231] 4.1 Plasmid construction

[0232] The VH in the Fab sequences of the monoclonal 7, 78A46, 78A74, 78A14, 78A51, 78A5, 17, and 35 obtained by screening was ligated 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 ligated 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 the 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 overnight at 37°C. The plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody plasmids for eukaryotic expression.

[0233] 4.2 Expression and Purification of Antibodies

[0234] The candidate antibody was expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133). The specific method is as follows: On the day of transfection, confirm that the cell density is about 7×10 6 to 1×10 7 viable cells / mL, and the cell viability > 98%. At this time, use fresh ExpiCHO expression medium pre-warmed at 37°C to adjust the cells to a final concentration of 6×10 6 cells / mL. Dilute the target plasmid with OptiPRO TM SFM (add 1 μg plasmid to 1 mL of the medium), and at the same time dilute ExpiFectamine TM CHO with OptiPRO TM SFM. Then mix the two in equal volumes and gently pipette to prepare the ExpiFectamine TM CHO / plasmid DNA mixture, incubate at room temperature for 1 - 5 min, slowly add it to the prepared cell suspension and gently shake at the same time. Finally, place it in a cell culture shaker and culture at 37°C and 8% CO2.

[0235] At 18 - 22 h after transfection, add ExpiCHO TM Enhancer and ExpiCHO TM Feed to the culture medium, and place the shake flask in a shaker at 32°C and 5% CO2 for continued culture. On the 5th day after transfection, add the same volume of ExpiCHO TMFeed, slowly add while gently mixing the cell suspension. Seven days after transfection, centrifuge the cell culture supernatant expressing the target protein at 15,000 g for 10 min at high speed. The obtained supernatant is affinity purified using MabSelect SuRe LX (GE, 17547403), and then the target protein is eluted with 100 mM sodium acetate (pH 3.0), followed by neutralization with 1 M Tris-HCl. Finally, the obtained protein is exchanged into PBS buffer using an ultrafiltration concentrator tube (Millipore, UFC901096).

[0236] Example 5 Determination of the Antigen-Binding Activity and Blocking Activity of Candidate Antibodies Based on the ELISA Method

[0237] In this example, the binding activities of 8 candidate antibodies 7, 78A46, 78A74, 78A14, 78A51, 78A5, 17, and 35 to hANG2-His and hANG1-Fc were detected based on the ELISA method. The activity of the candidate antibodies to block the binding of ANG2 and the receptor TIE2 was also detected based on the ELISA method.

[0238] 5.1 Detection of the Binding Activity of Candidate Antibodies to hANG2-His Based on ELISA

[0239] Coat hANG2-His (2 μg / mL, 30 μL / well) on a 96-well ELISA plate and incubate overnight at 4°C. The next day, wash the well plate 3 times with PBST and then block it with 5% skim milk for 2 h. After washing the plate 3 times with PBST, add serially diluted candidate antibodies and the positive control antibody nesvacumab and incubate for 1 h. Then, after washing 3 times with PBST, add the anti-human Fc HRP secondary antibody (Jackson Immuno Research, 109-035-008) and incubate for 1 h. After incubation is completed, wash the plate 6 times with PBST, add TMB (SurModics, TMBS-1000-01) for color development. According to the color development result, add 2 M HCl to terminate the reaction, and read the OD450 value using an ELISA reader (Molecular Devices, SpecterMax 190).

[0240] The results showed that Figure 3A-3B and Table 3: All candidate antibodies had high antigen-binding activities to hANG2-His and were all significantly superior to the positive control antibody nesvacumab.

[0241] 5.2 Detection of the Binding Activity of Candidate Antibodies to hANG1-Fc Based on ELISA

[0242] Coat the 96-well ELISA plate with hANG1-Fc (2 μg / mL, 30 μL / well) overnight at 4°C. The next day, wash the wells 3 times with PBST and then block with 5% non-fat milk for 2 h. After washing the plate 3 times with PBST, add serially diluted candidate antibodies and the positive control antibody nesvacumab and incubate for 1 h. Then, after washing 3 times with PBST, add the secondary antibody mixture of anti-human Kappa HRP and anti-human Lambda HRP (Millipore, AP502P, AP506P) and incubate for 1 h. After incubation, wash the plate 6 times with PBST, add TMB for color development, add 2 M HCl to terminate the reaction according to the color development result, and read the OD450 value using an ELISA reader.

[0243] The results are shown in Figure 4 : Except for a weak binding between antibody 78A5 and hANG1-Fc, the remaining 7 antibodies and the positive control antibody did not bind to hANG1.

[0244] 5.3 Blocking activity of candidate antibodies against the binding of ANG2 and receptor TIE2 detected by ELISA

[0245] Coat the plate with hTIE2 ECD-Fc (4 μg / mL, 30 μL / well) overnight at 4°C. The next day, wash the wells 3 times with PBST and then block with 5% non-fat milk for 2 h. Then, serially dilute the candidate antibodies or the positive control antibody nesvacumab and premix with biotinylated hANG2-Fc (4 μg / mL) for 0.5 h in advance. After blocking and washing are completed, add them to the 96-well ELISA plate and incubate for 1 h. Then, after washing 3 times with PBST, add the secondary antibody NeutrAvidin-HRP (Therofisher, 31001) and incubate for 1 h. After incubation, wash the plate 6 times with PBST, add TMB (SurModics, TMBS-1000-01) for color development, add 2 M HCl to terminate the reaction according to the color development result, and read the OD450 value using an ELISA reader (Molecular Devices, SpecterMax 190).

[0246] The results are shown in Figure 5A-5B and Table 3: All candidate antibodies have good ability to block the binding of ANG2 and receptor TIE2, and all candidate antibodies show better blocking activity than the control antibody nesvacumab.

[0247] Table 3 Binding activity and blocking activity of candidate antibodies based on ELISA method

[0248] 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

[0249] Example 6 Detection of the blocking activity of candidate antibodies based on the FACS method

[0250] In this example, the activity of a candidate antibody to block the binding of ANG2 and the cell surface-expressed receptor TIE2 was evaluated based on the FACS method.

[0251] The candidate antibody and the control antibody nesvacumab were serially diluted using FACS buffer (1×PBS + 2% FBS), and 100 μL of the antibody diluent was added to each well of a 96-well round-bottom plate. The hANG2-Fc-biotin protein was also diluted to 2 μg / mL using FACS buffer, and 100 μL was added to the corresponding 96-well plate. After mixing, the 96-well plate was incubated at 4°C for 1 h. The density of hTIE2-HEK293 cells was adjusted to 1×10 6 cells / mL, and 100 μL was added to each well of a new 96-well round-bottom plate. After centrifugation at 4°C and 300 g, the supernatant was removed. 180 μL of the pre-incubated antibody to be tested and hANG2-Fc-biotin mixture was added to the corresponding wells, and after mixing, the cells were incubated at 4°C for 30 min. After washing the incubated cell mixture three times, 200 μL of 1:200 diluted PE-labeled Streptavidin (Invitrogen, 12-4317-87) was added, and the cells were incubated at 4°C in the dark for 30 min. After washing three times, the amount of hANG2-Fc-biotin bound to the cells (expressed as mean fluorescence intensity, MFI) was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0252] The experimental results are shown in Figure 6 and Table 4: The blocking activities of antibodies 78A46, 78A74, 78A51, and 78A14 were superior to those of the control antibody nesvacumab.

[0253] Table 4 Blocking activities of candidate antibodies based on the FACS method

[0254] Antibody name <![CDATA[Blocking IC 50 Value (μg / mL) <!-- 22 -->]]> 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

[0255] Example 7 Detection of the activity of a candidate antibody to inhibit TIE2 phosphorylation

[0256] hTIE2-HEK293 cells were plated onto polylysine-coated 96-well plates (WHB, WHB-96-LC), 1×10 4Cells were cultured at 37°C for 24 h. After the culture, the cell culture supernatant was discarded, and the cells were rinsed once with serum-free DMEM medium. Then, 50 μL of a mixed DMEM medium containing hANG2-Fc and serially diluted test antibodies was added to each well, and the cells were incubated at 37°C for 30 min. Then, the supernatant was removed, and the cell plate was rinsed once with ice-cold PBS at 4°C. 100 μL of 1× cell lysis buffer (CST, 9803) and 1× protease inhibitor (Thermo, 78430) were added to each well, and the cells were lysed on ice for 30 min. The lysate was collected, and the phosphorylation of TIE2 in the protein lysate was quantitatively detected using the Human Phospho-Tie-2 DuoSet IC ELISA kit (R&D, DYC2720-2). The detailed detection method and operation steps are described in the kit instructions.

[0257] The detection results are shown in Figure 7 and Table 5: It 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, and among them, antibodies 78A46, 78A74, and 17 are significantly better than nesvacumab.

[0258] Table 5 Inhibitory activity of candidate antibodies on TIE2 phosphorylation

[0259] 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

[0260] Example 8 In vivo efficacy evaluation of candidate antibodies

[0261] In this example, the antitumor effects of 6 candidate antibodies and the positive control antibody nesvacumab in animals were verified. The tumor cells used were colon cancer cells Colo205. Male BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) at 6-8 weeks of age and weighing about 20 g were used. Each nude mouse was subcutaneously injected with 5×10 6 Colo205 cells. When the tumor volume reached about 150 mm 3 , grouping, caging, and drug administration operations were carried out. There were 8 tumor-bearing nude mice in each group, and a total of 8 groups: including 6 candidate antibody groups, 1 negative control group, and 1 positive control antibody nesvacumab group. The drug administration method was intraperitoneal injection, and the dose was 5 mg / kg. The drug was administered once every 3-4 days, 2 times a week, and the tumor volume was measured 2 times. A total of 6 doses were administered in 3 weeks. The calculation method of tumor volume (V): V = L × W. 2 / 2 (where L is the longest of the tumor diameters and W is the shortest of the tumor diameters). The mice were euthanized 1 week after the end of drug administration, and the tumors were taken and weighed. The data of tumor volume, tumor weight and mouse body weight changes were analyzed, and the tumor inhibition rate was calculated. Tumor growth inhibition rate TGI (%) = (1 - average tumor volume of the experimental group / average tumor volume of the PBS control group) × 100%.

[0262] The results are shown respectively in Figure 8A-8C and Table 6. It can be seen from Figure 8C that there were no significant differences in the body weights of the mice in each group, and there were no significant changes in the body weights of the mice in each group during the treatment period, indicating that the mice had good tolerance to the antibody. It can be seen from Figure 8A , 8B and Table 6 that the tumors of the mice in the PBS negative control group grew the fastest, and 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 of the positive control antibody nesvacumab group, showing good tumor inhibition effects.

[0263] Table 6 Tumor growth inhibition rate TGI (%) of candidate antibodies

[0264]

[0265] Example 9 Detection of the drug metabolism rate of candidate antibodies in Balb / C mice

[0266] In this example, the drug metabolism rates of 6 candidate antibodies in Balb / C mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were measured, and the positive control antibody nesvacumab was used as a control at the same time. The experimental animals were divided into 7 groups, with 6 Balb / C mice in each group. Each mouse was administered intraperitoneally, and the administration dose was 40 mg / kg; the administration volume was 10 mL / kg. The blood samples collected from each group at the time points of 2 h, 4 h, 8 h, 1 d, 2 d, 3 d, 4 d, 5 d, 9 d, and 14 d were placed at room temperature for 2 hours. Three mice were selected from each group at each time point to collect blood samples, and the mice in the group were alternately bled. Then, they were centrifuged at 10000 g for 5 minutes at 4 °C. The supernatant was collected and immediately used for the experiment or the sample was aliquoted and stored at -80 °C.

[0267] The ELISA method was used to quantitatively determine the drug concentration in mouse serum. hANG2-Fc (2 μg / mL) was coated on a 96-well half-well ELISA plate and incubated overnight at 4 °C; after washing the plate 3 times with PBST, it was blocked with 2% BSA, 160 μL / well at room temperature for 1 h; after washing the plate 3 times with PBST, mouse serum samples to be tested were diluted to different concentrations with 2% BSA, and 30 μL was added to each well and incubated at room temperature for 1 h; after washing the plate 3 times, a mixture of anti-human IgG kappa HRP secondary antibody (Millipore, AP502P) and anti-human Lambda HRP secondary antibody (Millipore, AP506P) diluted 1:5000 was added and incubated at room temperature for 1 h; after incubation, the plate was washed 6 times with PBST and developed with TMB (SurModics, TMBS-1000-01). According to the color development results, 2 M HCl was added to terminate the reaction, and the OD450 value was read using an ELISA reader (Molecular Devices, SpecterMax 190).

[0268] The results are shown in Figure 9 and Table 7: The drug metabolism rates of antibodies 78A74, 78A14, 7, and 17 were lower than that of the control antibody nesvacumab. Therefore, the bioavailability of antibodies 78A74, 78A14, 7, and 17 was higher than that of the control antibody nesvacumab.

[0269] Table 7 Plasma drug concentration values (μg / mL) of candidate antibodies at different time points

[0270] 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

[0271] Engineering modification of candidate antibodies in Example 10

[0272] In the data of the previous examples, antibody 78A74 showed good drug metabolism rate in mice, ability to block ANG2-TIE2 phosphorylation activity, etc. Affinity maturation modification was based on the M13 phage display technology, and codon-based primers (during primer synthesis, a single codon was composed of NNK) were used to introduce mutations in the CDR regions. Four phage display libraries were constructed for each parental molecule. Library 1 and Library 2 were single-point combinatorial mutations. Library 1 was a combination mutation of CDRL1 + CDRL3 + CDRH3, and Library 2 was a combination mutation of CDRL2 + CDRH1 + CDRH2; Library 3 and Library 4 were double-point saturation mutations. Library 3 was a double-point saturation mutation of CDRL3, and Library 4 was a double-point saturation mutation of CDRH3.

[0273] Using antibody 78A74 as a template, a single CDR region mutant fragment was obtained by PCR. Then, a Fab fragment (VL-CL-linker-VH-CH1) was obtained by Overlapping PCR. The point mutant antibody was ligated into the phage display vector by double digestion (HindⅢ and NotⅠ) and blunt-end ligation. Finally, the antibody sequence with the mutation site was transferred into Escherichia coli SS320 by electroporation.

[0274] After packaging the constructed 4 libraries into phages, panning, primary screening, affinity ranking and sequence analysis of the libraries were carried out (the method is shown in Example 2). 28 positive clone bacteria expressing Fab supernatants were selected for affinity ranking and sequence analysis. Finally, 5 preferred Fab fragments were selected for sample preparation and affinity evaluation. Based on these 5 Fab fragments, fully human antibodies were prepared (the method is shown in Example 4), and the amino acid sequences of their variable regions are shown in Table 8.

[0275] Table 8 Amino acid sequences of the variable regions of fully human antibodies against ANG2 obtained by affinity maturation (SEQ ID NO:)

[0276] 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

[0277] Example 11 Affinity kinetics evaluation of anti-ANG2 antibody

[0278] In this example, the binding affinity of the candidate antibody against the antigen ANG2 was detected based on the Gator device, and nesvacumab was used as a positive control.

[0279] The antibody to be tested was diluted to 20 nM with 10×KB (10×PBS buffer containing 1% BSA and 0.5% Tween 20). The hANG2-His used as the antigen was serially diluted 2-fold with 10×KB buffer, and the concentrations were 100 nM, 50 nM, 25 nM, and 0 nM. Under light protection conditions, the sensor (Gator, 20-5006) was pre-wetted with 10×KB buffer. At least 10 min later, the sample plate (Gator, 06-0153) was tested. After the test was correct, it was carried out according to the preset program. First, the antibody and the sensor were combined for 120 s. After the combination was completed, it was continued to equilibrate in 10×KB buffer for 30 s. Then, the sensor bound with the antibody was transferred to different concentrations of antigen diluents for binding for 120 s. After the signal was stable, it was transferred to 10×KB buffer, and the dissociation time was 120 s. Finally, the K D (affinity kinetic constant), Kon (association constant), and Koff (dissociation constant) were obtained by fitting the binding and dissociation data of different concentrations of antigen-antibody. Kon can be written as Ka, and Koff can be written as Kd.

[0280] The detection results are shown in Table 9. The results show that the affinities of antibodies 78A74-7, 78A74-21, and 78A74-25 are higher than that of antibody 78A74 and are about twice that of the control antibody nesvacumab. The affinities of antibodies 7, 35, 78A46, 78A14, 78A74, 78A74-35, and 78A74-36 for human ANG2 are similar to those of the control antibody.

[0281] Table 9 Affinity Kinetics Evaluation of Candidate Antibodies

[0282] 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

[0283] Those skilled in the art will further recognize that the present invention may be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the present disclosure has only disclosed its exemplary embodiments, 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, the scope and content of the present invention should be indicated by reference to the appended claims.

Claims

1. An anti-ANG2 antibody or an antigen-binding fragment thereof, which comprises a heavy-chain variable region polypeptide and a light-chain variable region polypeptide, wherein the heavy-chain variable region polypeptide comprises an HCDR1 sequence shown in SEQ ID NO:95, an HCDR2 sequence shown in SEQ ID NO:96, and an HCDR3 sequence shown in SEQ ID NO:97, and the light-chain variable region polypeptide comprises an LCDR1 sequence shown in SEQ ID NO:98, an LCDR2 sequence shown in SEQ ID NO:99, and an LCDR3 sequence shown in SEQ ID NO:

100.

2. The anti-ANG2 antibody or an antigen-binding fragment thereof according to claim 1, wherein the heavy-chain variable region polypeptide comprises: (1) the amino acid sequence of SEQ ID NO:101; or (2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:

101.

3. The anti-ANG2 antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein the light-chain variable region polypeptide comprises: (1) the amino acid sequence of SEQ ID NO:102; or (2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:

102.

4. The anti-ANG2 antibody or an antigen-binding fragment thereof according to claim 1, wherein the heavy-chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO:101, and the light-chain variable region polypeptide comprises the amino acid sequence of SEQ ID NO:

102.

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

6. The anti-ANG2 antibody or an antigen-binding fragment thereof according to any one of claims 1-5, which further comprises a heavy-chain constant region and / or a light-chain constant region; Preferably, the heavy-chain constant region is the heavy-chain constant region of human IgG1 and / or the light-chain constant region is the human κ or λ light-chain constant region; More preferably, the heavy-chain constant region comprises the amino acid sequence of SEQ ID NO:71; and / or the light-chain constant region comprises the amino acid sequence of SEQ ID NO:72 or SEQ ID NO:

73.

7. The anti-ANG2 antibody or an antigen-binding fragment thereof according to any one of claims 1-6, which specifically binds to ANG2, but does not bind or substantially does not bind to ANG1.

8. 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 an anti-ANG2 antibody or an antigen-binding fragment thereof according to any one of claims 1-7.

9. A polynucleotide encoding an anti-ANG2 antibody or an antigen-binding fragment thereof according to any one of claims 1-7 or the multispecific antibody of claim 8.

10. An expression vector comprising the polynucleotide of claim 9.

11. A host cell comprising the polynucleotide of claim 9 or the expression vector of claim 10.

12. A pharmaceutical composition comprising an anti-ANG2 antibody or an antigen-binding fragment thereof according to any one of claims 1-7 or the multispecific antibody of claim 8, and a pharmaceutically acceptable carrier.

13. Use of an anti-ANG2 antibody or an antigen-binding fragment thereof according to any one of claims 1-7, the multispecific antibody of claim 8, or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating the following diseases: (1) Eye diseases related to angiogenesis; or (2) Cancer; Preferably, the eye diseases related to angiogenesis are age-related macular degeneration, retinal vein occlusion, retinopathy, retinopathy of prematurity, diabetic retinopathy, neovascular glaucoma, pathologic myopia, macular edema, retinal edema, diabetic macular edema or choroidal neovascularization disease; the cancer is lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, prostate cancer, uterine cancer, Hodgkin lymphoma, esophageal cancer, anal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue sarcoma, bladder cancer, central nervous system (CNS) tumor, mesothelioma, glioma, meningioma or pituitary adenoma.

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