Antibody or antigen-binding fragments capable of recognition of ANG-2 and double antibody for

Abstract

Provided are a single-domain antibody or an antigen-binding fragment and a multispecific antibody capable of recognizing ANG-2. The antibody or the antigen binding fragment has a heavy chain variable region as shown in any one of SEQ ID NO: 16-20, and the multispecific antibody comprises a first binding region containing the antibody or the antigen binding fragment and a second binding region with first molecular binding activity.

Description

Antibodies or antigen-binding fragments that recognize ANG-2 and bispecific antibodies that simultaneously recognize VEGF and ANG-2 Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology. Specifically, the present invention relates to an antibody or antigen-binding fragment capable of recognizing ANG-2 and a bispecific antibody that simultaneously recognizes VEGF and ANG-2. More specifically, the present invention relates to an antibody or antigen-binding fragment, a recombinant protein, a multispecific antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a conjugate, a pharmaceutical composition, a kit, and uses thereof, as well as methods for treating or preventing angiogenesis-related diseases, methods for diagnosing and evaluating angiogenesis-related diseases, and methods for evaluating the prognosis of angiogenesis-related diseases. Background Art

[0002] Current studies have found that angiogenesis is involved in the pathogenesis of various diseases, including cancer, intraocular neovascular syndromes (such as proliferative retinopathies and age-related macular degenerative disease (AMD)), and inflammatory diseases (such as rheumatoid arthritis and psoriasis).

[0003] Angiopoietin 2 (ANG-2) is a secreted glycoprotein with a molecular weight of approximately 70 kDa. It plays a complex role in angiogenesis, inflammation, and vascular development. It is expressed in vascular endothelial cells and smooth muscle cells in highly angiogenic tissues, lung epithelial cells, differentiated myotubes, and neural progenitor cells. ANG-2, upon binding to its receptor, TEK tyrosine kinase (TIE2), promotes endothelial cell survival, proliferation, and migration, and promotes sprouting angiogenesis. It induces pericyte loss in the vascular wall and increases vascular permeability. Therefore, inhibition of the ANG-2 signaling pathway has the potential to treat angiogenesis-related diseases, such as tumors and ophthalmology.

[0004] Therefore, developing antibodies with higher binding activity to ANG-2 is of great significance for angiogenesis-related diseases.

[0005] Summary of the Invention

[0006] The present invention aims to at least partially address one of the technical problems existing in the prior art. To this end, the present invention provides an anti-ANG-2 single-domain antibody that has high binding activity to ANG-2 and, by blocking the binding of ANG-2 to TIE2, exerts anti-angiogenic and anti-vascular inflammation effects.

[0007] The present invention is accomplished based on the following findings of the inventors:

[0008] Currently developed drugs that inhibit ANG-2 activity include anti-ANG-2 monoclonal antibodies (WO2013 / 144266) and soluble ANG-2 receptor inhibitors, such as soluble TIE2 (Siemeister, G. et al., Cancer Res, 59:3 (1999) 3185-91). However, there are relatively few single-domain antibody drugs that inhibit ANG-2 activity.

[0009] Compared to traditional antibodies, single-domain antibodies (also known as nanobodies, Nbs) offer numerous advantages, including: 1) small molecular weight and the ability to penetrate the blood-brain barrier; 2) high expression in prokaryotic or eukaryotic systems; 3) strong specificity and high affinity; and 4) low immunogenicity in humans. Consequently, single-domain antibodies are widely used in biopharmaceutical research and development.

[0010] Based on this, the inventors immunized alpacas with ANG-2 recombinant protein and constructed and screened a phage library from the immunized alpacas' peripheral blood. They ultimately obtained a single-domain antibody that binds to ANG-2. This single-domain antibody exhibits high binding activity to ANG-2. Furthermore, a bispecific antibody (BSA) can be prepared by combining this single-domain antibody with the VEGF receptor. This BSA can simultaneously bind to VEGF and ANG-2. By blocking the binding of VEGF to VEGFR and ANG-2 to TIE2, it can exert anti-angiogenic effects and reduce vascular inflammation.

[0011] Therefore, in one aspect, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a heavy chain variable region CDR sequence selected from at least one of the following: SEQ ID NOs: 1-14, or an amino acid sequence having at least 80% identity thereto. The antibody or antigen-binding fragment of the present invention is capable of binding to ANG-2 and exhibits high ANG-2 binding activity. By blocking the binding of ANG-2 to TIE2, it exerts anti-angiogenic effects and reduces vascular inflammation, thereby effectively treating or preventing angiogenesis-related diseases.

[0012] According to an embodiment of the present invention, the antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises:

[0014] the heavy chain variable region CDR1, CDR2, and CDR3 sequences as set forth in the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or amino acid sequences at least 80% identical to SEQ ID NOs: 1, 2, and 3;

[0015] the heavy chain variable region CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively, or an amino acid sequence at least 80% identical to SEQ ID NOs: 4, 5, and 6;

[0016] the heavy chain variable region CDR1, CDR2, and CDR3 sequences as set forth in the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or amino acid sequences at least 80% identical to SEQ ID NOs: 7, 8, and 9;

[0017] The heavy chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NOs: 10, 11, and 12, respectively, or in amino acid sequences that are at least 80% identical to SEQ ID NOs: 10, 11, and 12; or

[0018] The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NOs: 10, 13, and 14, respectively, or in amino acid sequences that are at least 80% identical to SEQ ID NOs: 10, 13, and 14.

[0019] YYSIG (SEQ ID NO: 1);

[0020] CISRTDGSTYYADSVKG(SEQ ID NO:2);

[0021] KGARYSGSYLCGLDEYDY(SEQ ID NO:3);

[0022] SATMS (SEQ ID NO: 4);

[0023] LIAYAGGSTTYADSVKG(SEQ ID NO:5);

[0024] RRYNRDF (SEQ ID NO: 6);

[0025] YYAIG (SEQ ID NO: 7);

[0026] CISSGDGSTYYADSVKG(SEQ ID NO:8);

[0027] VRSGSKCLVTRGTVVAAGQLYEY(SEQ ID NO:9);

[0028] FSTVR (SEQ ID NO: 10);

[0029] TLLDDGSTNYADSVKG(SEQ ID NO:11);

[0030] DGRTGWGPRGDY (SEQ ID NO: 12);

[0031] THDDGSTNYEDSVKG(SEQ ID NO:13);

[0032] DGSTGWGPRGDY (SEQ ID NO: 14).

[0033] According to an embodiment of the present invention, the antibody contains a heavy chain framework region sequence, at least a portion of which is derived from at least one of a murine antibody, a human antibody, a primate antibody, an alpaca antibody or a mutant thereof.

[0034] According to an embodiment of the present invention, the antibody has a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 16 to 20.

[0035]

[0036] According to an embodiment of the present invention, the antibody contains a heavy chain constant region, at least a portion of which is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0037] According to an embodiment of the present invention, the heavy chain constant region is an Fc fragment.

[0038] According to an embodiment of the present invention, the heavy chain constant region of the antibody is derived from a human IgG antibody or a mutant thereof.

[0039] According to an embodiment of the present invention, the heavy chain constant region of the antibody is derived from human IgG1.

[0040] According to an embodiment of the present invention, the antibody comprises a monoclonal antibody or a polyclonal antibody.

[0041] According to an embodiment of the present invention, the monoclonal antibody includes at least one of a Fab antibody, a Fv antibody, a single-chain antibody, a single-domain antibody, and a minimum recognition unit.

[0042] In a preferred embodiment of the present invention, the antibody is a single domain antibody.

[0043] According to an embodiment of the present invention, the antibody has a heavy chain having an amino acid sequence shown in any one of SEQ ID NOs: 21 to 25.

[0044]

[0045] In yet another aspect, the present invention provides a recombinant protein. According to an embodiment of the present invention, the recombinant protein comprises the aforementioned antibody or antigen-binding fragment. The recombinant protein of the embodiment of the present invention is capable of targeted binding to ANG-2 and exhibits high ANG-2 binding activity. It can be used to detect ANG-2 and block the binding of ANG-2 to TIE2, thereby exerting anti-angiogenic and anti-vascular inflammation effects and effectively treating or preventing angiogenesis-related diseases.

[0046] According to an embodiment of the present invention, it further includes at least one of a biologically active protein or a fragment thereof, a biologically active polypeptide or a fragment thereof.

[0047] According to an embodiment of the present invention, the biologically active protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof and Fc fragments.

[0048] In this article, "protein tag" generally refers to a polypeptide or protein that is fused with a target protein (antibody or antigen-binding fragment) and can be used for the expression, detection, disappearance or purification of the target protein. It includes but is not limited to the His tag (also known as His-Tag, sequence HHHHHH), Flag tag (also known as Flag-Tag, sequence DYKDDDDK), GST tag (also known as GST-Tag, glutathione S-transferase tag), MBP tag (also known as MBP-Tag, maltose binding protein tag), SUMO tag and C-Myc tag.

[0049] As used herein, "toxin" generally refers to a substance toxic to the host, including protein toxins and non-protein toxins. Protein toxins include, but are not limited to, abrin, ricin A, Pseudomonas exotoxin, and diphtheria toxin. In the present invention, protein toxins are preferably enzymatically active.

[0050] Herein, "tumor necrosis factor" generally refers to a substance that can cause hemorrhagic necrosis of various tumors, including but not limited to TNF-α and TNF-β.

[0051] In this article, "interferon" generally refers to a glycoprotein that has the ability to directly kill or inhibit viruses, including but not limited to IFN-α, INF-β and IFN-γ.

[0052] In this article, "biological response modifiers" generally refer to a class of protein substances that directly or indirectly enhance the body's anti-tumor response through the immune system, including but not limited to lymphokines, IL-2, IL-6, IL-10, and GM-CSF.

[0053] As used herein, "Fc fragment" generally refers to an Fc region from IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM, including CH2, CH3, and optionally, a hinge region. Preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM is derived from mouse, human, primate, or alpaca.

[0054] In another aspect, the present invention provides a multispecific antibody. According to an embodiment of the present invention, the multispecific antibody comprises: a first binding region comprising the aforementioned antibody or antigen-binding fragment; and a second binding region having binding activity for a first molecule. The multispecific antibody of the present invention can simultaneously bind to ANG-2 and the first molecule and can be used to detect ANG-2 and the first molecule. It can also be used to block the binding of ANG-2 to TIE2, exerting anti-angiogenic effects and reducing vascular inflammation, thereby effectively treating or preventing angiogenesis-related diseases.

[0055] According to an embodiment of the present invention, the multispecific antibody may further include at least one of the following additional technical features:

[0056] According to an embodiment of the present invention, the multispecific antibody comprises at least one selected from a bispecific antibody, a trispecific antibody and a tetraspecific antibody, preferably a bispecific antibody.

[0057] It should be noted that the second binding region may be one or more. When the multispecific antibody is a bispecific antibody, the second binding region is one; when the multispecific antibody is a trispecific antibody, the second binding region is two, and each of the two second binding regions can bind to two different first molecules; when the multispecific antibody is a tetraspecific antibody, the second binding region is three, and each of the three second binding regions can bind to three different first molecules.

[0058] According to an embodiment of the present invention, the first molecule includes at least one selected from immune cell surface antigens, tumor antigens, viruses, bacteria, endotoxins, cytokines and cytokine receptors.

[0059] In this article, "immune cell surface antigens" should be understood in a broad sense and may refer to immunogenic proteins on the surface of immune cells (such as T cells, NK cells, B cells, etc.), including but not limited to BCMA, CTLA-4, LAG-3, TIGIT, CD38, SLAMF7, B7-H3, CD19, CD20, CD30, CD33, CD47, CD52, CD133, RANKL, CD3, and CD16a.

[0060] In this article, "tumor antigen" should be understood broadly and may refer to immunogenic proteins on the surface of tumor cells, including but not limited to PD-L1, PD-1, TGF-β, CEA, GD2, and GD3.

[0061] In this article, "cytokine" should be understood broadly, referring to a class of proteins or small molecule polypeptides that can transmit information between cells and have immune regulatory and effector functions. This includes but is not limited to IL-10, VEGF (including at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF), EpCAM, GM2, and RANKL.

[0062] In this article, "cytokine receptor" should be understood in a broad sense, and may refer to receptors on the cell surface that can bind to cytokines, including but not limited to Her2, EGFR, and IL-10R.

[0063] According to an embodiment of the present invention, the first molecule includes at least one selected from PD-L1, PD-1, IL-10, IL-10R, BCMA, VEGF, TGF-β, CTLA-4, LAG-3, TIGIT, CEA, CD38, SLAMF7, B7-H3, Her2, EpCAM, CD19, CD20, CD30, CD33, CD47, CD52, CD133, EGFR, GD2, GD3, GM2, RANKL, CD3 and CD16a.

[0064] According to an embodiment of the present invention, the second binding region is the binding protein of the first molecule or a fragment thereof.

[0065] It should be noted that the term "binding protein or fragment thereof of a first molecule" generally refers to a protein or protein fragment that can bind to the first molecule. When the first molecule is a protein receptor, such as IL-10R and EGFR, the binding protein or fragment thereof of the first molecule can be a cytokine (IL-10 or EGF) or a fragment thereof, or an antibody or fragment thereof that binds to the protein receptor. When the first molecule is a protein other than a protein receptor, such as IL-10 and VEGF, the binding protein or fragment thereof of the first molecule can be a protein receptor (IL-10R and VEGFR) or a fragment thereof, or an antibody or fragment thereof that binds to the protein.

[0066] According to an embodiment of the present invention, the first molecule is VEGF. The multispecific antibody of the present invention can simultaneously bind to VEGF and ANG-2, and by blocking the binding of VEGF to VEGFR and ANG-2 to TIE2, exerts anti-angiogenic and anti-vascular inflammation effects, thereby effectively treating or preventing angiogenesis-related diseases.

[0067] According to an embodiment of the present invention, the second binding region comprises at least one selected from a VEGF receptor fragment and an anti-VEGF antibody.

[0068] According to an embodiment of the present invention, the VEGF receptor fragment includes at least one selected from a VEGFR1 fragment, a VEGFR2 fragment, and a VEGFR3 fragment.

[0069] It should be noted that the VEGF receptor (VEGFR for short) consists of three parts: the extracellular VEGF binding region, the receptor's transmembrane region (TM) and the intracellular signal transduction domain. Among them, the VEGF binding region is composed of 7 immunoglobulin-like structures, which are specifically divided into immunoglobulin-like region 1 to immunoglobulin-like region 7 (or D1 to D7).

[0070] According to an embodiment of the present invention, the second binding region includes the immunoglobulin-like region 2 in the VEGFR1 fragment and / or the immunoglobulin-like region 3 in the VEGFR2.

[0071] According to an embodiment of the present invention, the C-terminus of the immunoglobulin-like region 2 in the VEGFR1 fragment is connected to the N-terminus of the immunoglobulin-like region 3 in the VEGFR2.

[0072] According to an embodiment of the present invention, the bispecific antibody includes a symmetric bispecific antibody or an asymmetric bispecific antibody.

[0073] According to an embodiment of the present invention, the bispecific antibody is a symmetric bispecific antibody.

[0074] According to an embodiment of the present invention, the second binding region further includes an Fc fragment.

[0075] According to an embodiment of the present invention, the Fc fragment includes CH2, CH3 regions and optionally a hinge region.

[0076] According to an embodiment of the present invention, the C-terminus of the CH2 region and the N-terminus of the CH3 region are connected.

[0077] According to an embodiment of the present invention, the C-terminus of the hinge region and the N-terminus of the CH2 region, and the C-terminus of the CH2 region and the N-terminus of the CH3 region are connected.

[0078] According to an embodiment of the present invention, at least a portion of the Fc fragment is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0079] According to an embodiment of the present invention, at least a portion of the Fc fragment is derived from a human antibody or a mutant thereof.

[0080] According to an embodiment of the present invention, at least a portion of the Fc fragment is derived from human antibody IgG or a mutant thereof.

[0081] According to an embodiment of the present invention, the C-terminus of the immunoglobulin-like region 2 in the VEGFR1 fragment is connected to the N-terminus of the immunoglobulin-like region 3 in the VEGFR2, and the C-terminus of the immunoglobulin-like region 3 in the VEGFR2 is connected to the N-terminus of the Fc fragment; or, the C-terminus of the immunoglobulin-like region 2 in the VEGFR1 fragment is connected to the N-terminus of the immunoglobulin-like region 3 in the VEGFR2, and the N-terminus of the immunoglobulin-like region 2 in the VEGFR1 is connected to the C-terminus of the Fc fragment.

[0082] According to an embodiment of the present invention, the second binding region has an amino acid sequence as shown in SEQ ID NO: 26 or an amino acid sequence having at least 90% identity thereto.

[0083]

[0084] According to an embodiment of the present invention, a connecting peptide is further included.

[0085] According to an embodiment of the present invention, the C-terminus of the immunoglobulin-like region 3 in the VEGFR2 is connected to the N-terminus of the Fc fragment, the C-terminus of the Fc fragment is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the antibody or antigen-binding fragment; or, the C-terminus of the antibody or antigen-binding fragment is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting peptide is connected to the N-terminus of the Fc fragment, and the C-terminus of the Fc fragment is connected to the N-terminus of the immunoglobulin-like region 2 in the VEGFR1.

[0086] According to an embodiment of the present invention, the amino acid sequence of the connecting peptide is (GGGGS) n , wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0087] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in SEQ ID NO: 27.

[0088] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 27).

[0089] According to an embodiment of the present invention, the antibody has an amino acid sequence as shown in any one of SEQ ID NOs: 28 to 32.

[0090]

[0091]

[0092] In another aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, or the aforementioned multispecific antibody. The nucleic acid molecule according to an embodiment of the present invention encodes the aforementioned antibody or antigen-binding fragment, recombinant protein, or multispecific antibody.

[0093] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0094] It should be noted that, for nucleic acid molecules mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one form implies disclosure of the other.

[0095] In another aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. When the aforementioned nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably connected. Herein, "operably connected" means that the exogenous gene is connected to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected function of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, the expression of the aforementioned antibody or antigen-binding fragment, recombinant protein or the aforementioned multi-specific antibody can be effectively achieved under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of the antibody or antigen-binding fragment, recombinant protein or the aforementioned multi-specific antibody.

[0096] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector. Preferably, the expression vector is a plasmid expression vector.

[0097] In another aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises: carrying the aforementioned nucleic acid molecule or the aforementioned expression vector; or expressing the aforementioned antibody or antigen-binding fragment, recombinant protein, or multispecific antibody. Under suitable conditions, the recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment, recombinant protein, or multispecific antibody within the cell.

[0098] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of the antibody or antigen-binding fragment, recombinant protein or the aforementioned multispecific antibody of the present invention. It will be readily understood by those skilled in the art that conditions suitable for the expression of the antibody or antigen-binding fragment, recombinant protein or the aforementioned multispecific antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibody or antigen-binding fragment, recombinant protein or the aforementioned multispecific antibody according to the specific environment of the laboratory.

[0099] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0100] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell.

[0101] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0102] In another aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. The pharmaceutical composition of the present invention can bind to ANG-2, blocking the binding of ANG-2 to its respective receptors, and can effectively treat or prevent diseases associated with angiogenesis.

[0103] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable excipient.

[0104] In another aspect, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises: the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, or the aforementioned multispecific antibody; and a coupling moiety linked to the antibody or antigen-binding fragment, the recombinant protein, or the multispecific antibody. The pharmaceutical composition of the present invention can bind to ANG-2 and can be used to detect ANG-2 or block the binding of ANG-2 to its respective receptors, effectively treating or preventing diseases associated with angiogenesis.

[0105] According to an embodiment of the present invention, the coupling moiety includes but is not limited to at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier and a chemotherapeutic agent.

[0106] Herein, the carrier can be a substance that can be suspended or dispersed in a liquid phase (e.g., solid phase carriers such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., supports such as plates, membranes, test tubes, and containers such as well plates, microfluidics, glass capillaries, nanocolumns, and monolithic columns); it can also be a labeling carrier for labeling antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies, such as an enzyme (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), an affinity substance (e.g., one of streptavidin and biotin, one of mutually complementary sense and antisense nucleic acids), a fluorescent substance (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), a luminescent substance (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridine)ruthenium, luminol), a radioactive isotope (e.g., 3 H. 14 C. 32 P.35 S. 125 I) and gold colloid, etc.

[0107] According to an embodiment of the present invention, the drug is a small molecule drug that can bind to an antibody or antigen-binding fragment, a recombinant protein or a multispecific antibody, including but not limited to EGFR-TKI inhibitors, ALK inhibitors, verteporfin and pegaptanib sodium, etc.

[0108] According to an embodiment of the present invention, the toxins include but are not limited to abrin, ricin A, Pseudomonas exotoxin and diphtheria toxin.

[0109] According to an embodiment of the present invention, the cytokines include but are not limited to IL-10, VEGF, EpCAM, GM2 and RANKL.

[0110] According to an embodiment of the present invention, the protein tag includes but is not limited to a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag.

[0111] According to an embodiment of the present invention, the modifier should be understood in a broad sense and may refer to a substance used to modify a protein, for example, polyethylene glycol or a derivative thereof.

[0112] According to an embodiment of the present invention, the chemotherapy agents include but are not limited to albumin-paclitaxel, cyclophosphamide, ifosfamide, phenylalanine mustard, methotrexate, fluorouracil, actinomycin D and vincristine.

[0113] It should be noted that methods known in the art can be used to bind the conjugated moiety to the antibody, antigen-binding fragment, recombinant protein, or multispecific antibody. Examples include physical adsorption, covalent binding, methods using affinity substances (e.g., biotin, streptavidin), and ion binding.

[0114] In another aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises: the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned conjugate. The antibody or antigen-binding fragment in the kit provided by the present invention is capable of effectively binding to ANG-2. Furthermore, under appropriate conditions, the nucleic acid molecule, expression vector, or recombinant cell is capable of expressing the antibody or antigen-binding fragment. Furthermore, the kit containing the aforementioned substances is capable of effectively binding to ANG-2 and can be used to effectively detect ANG-2. The kit can be used for scientific research, such as for qualitative or quantitative detection of ANG-2 in biological samples, or for determining the status of an individual, such as determining whether the individual's ANG-2 level is excessively high or low after obtaining the individual's ANG-2 level. The biological sample can be cells, tissue, blood, or the like.

[0115] In another aspect, the present invention provides a use of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned conjugate in preparing a kit for detecting ANG-2.

[0116] In another aspect of the present invention, the present invention provides a use of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned pharmaceutical composition or the aforementioned conjugate in the preparation of a drug, wherein the drug is used to treat or prevent related diseases caused by angiogenesis.

[0117] According to an embodiment of the present invention, the angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

[0118] In another aspect of the present invention, the present invention proposes a use of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned pharmaceutical composition or the aforementioned conjugate in treating or preventing related diseases caused by angiogenesis.

[0119] According to an embodiment of the present invention, the angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

[0120] In another aspect of the present invention, the present invention provides a use of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned pharmaceutical composition or the aforementioned conjugate in diagnosing angiogenesis-related diseases, assessing the prognosis of angiogenesis-related diseases and assessing the staging of angiogenesis-related diseases.

[0121] According to an embodiment of the present invention, the angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

[0122] In another aspect, the present invention provides a method for treating or preventing angiogenesis-related diseases. According to an embodiment of the present invention, the method comprises administering a pharmaceutically acceptable amount of the aforementioned antibody or antigen-binding fragment, recombinant protein, multispecific antibody, pharmaceutical composition, or conjugate to a subject. The method of the present invention can effectively treat or prevent angiogenesis-related diseases.

[0123] The effective amount of the antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate or pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0124] The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, conjugates or pharmaceutical compositions of the present invention can be incorporated into drugs suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions. The aforementioned antibodies or antigen-binding fragments, the aforementioned recombinant proteins, the aforementioned multispecific antibodies, the aforementioned pharmaceutical compositions or the aforementioned conjugates can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0125] According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0126] According to an embodiment of the present invention, the angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

[0127] In another aspect, the present invention provides a method for diagnosing angiogenesis-related diseases. According to an embodiment of the present invention, the method comprises: detecting ANG-2 in a test sample using the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned kit, or the aforementioned conjugate; and determining the ANG-2 content in the test sample based on the detection result.

[0128] According to an embodiment of the present invention, VEGF in the test sample is further detected; and based on the detection result, the content of VEGF in the test sample is determined.

[0129] According to an embodiment of the present invention, the angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

[0130] In another aspect, the present invention provides a method for assessing the prognosis of angiogenesis-related diseases. According to an embodiment of the present invention, the method comprises: detecting ANG-2 in a test sample using the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned kit, or the aforementioned conjugate; and determining the ANG-2 content in the test sample based on the detection result.

[0131] According to an embodiment of the present invention, VEGF in the test sample is further detected; and based on the detection result, the content of VEGF in the test sample is determined.

[0132] According to an embodiment of the present invention, the angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

[0133] In another aspect, the present invention provides a method for assessing the staging of angiogenesis-related diseases. According to an embodiment of the present invention, the method comprises: detecting ANG-2 in a test sample using the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned kit, or the aforementioned conjugate; and determining the ANG-2 content in the test sample based on the detection result.

[0134] According to an embodiment of the present invention, VEGF in the test sample is further detected; and based on the detection result, the content of VEGF in the test sample is determined.

[0135] According to an embodiment of the present invention, the content of ANG-2 and / or VEGF in the test sample being not less than the minimum level for stage IV tumor is an indication that the test sample is derived from a patient with stage IV tumor;

[0136] The ANG-2 and / or VEGF content in the test sample being between the levels of patients with stage IV and stage III tumors is an indication that the test sample is derived from a patient with stage III tumors;

[0137] The ANG-2 and / or VEGF content in the test sample being between the standard levels for stage III and stage II tumors indicates that the test sample is derived from a patient with stage II tumors;

[0138] The level of ANG-2 and / or VEGF in the test sample being between the standard levels for stage I and stage II disease is an indication that the test sample is derived from a patient with stage I tumor.

[0139] According to an embodiment of the present invention, the angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

[0140] In another aspect of the present invention, the present invention provides the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned pharmaceutical composition or the aforementioned conjugate for use in treating or preventing diseases related to angiogenesis.

[0141] According to an embodiment of the present invention, the angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

[0142] In another aspect of the present invention, the present invention provides the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, the aforementioned multispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned kit or the aforementioned conjugate for use in diagnosing angiogenesis-related diseases, assessing the prognosis of angiogenesis-related diseases or assessing the staging of angiogenesis-related diseases.

[0143] According to an embodiment of the present invention, the angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases. Beneficial effects:

[0144] 1. The variable regions of the ANG-2 single-domain antibodies screened and obtained in the present invention have high binding activity with ANG-2 and can effectively block the binding of ANG-2 to its receptor TIE2, thereby effectively treating or preventing diseases related to angiogenesis.

[0145] 2. The bispecific inhibitor prepared by using the variable region of the ANG-2 single-domain antibody of the present invention and the VEGF receptor-Fc fusion protein can simultaneously bind to VEGF and ANG-2, and by blocking the binding of VEGF to VEGFR and blocking the binding of ANG-2 to TIE2, exerts anti-angiogenic and anti-vascular inflammation effects, thereby effectively treating or preventing angiogenesis-related diseases. Moreover, the bispecific inhibitor of the present invention has significantly better blocking activity against both VEGF and ANG-2 than the marketed drug Faricimab.

[0146] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0148] FIG1 shows the affinity test results of the ANG-2 single domain antibody in Example 3 of the present invention;

[0149] FIG2 is a schematic structural diagram of the bispecific inhibitor in Example 4 of the present invention;

[0150] FIG3 is the VEGF reporter gene activity detection results of the bispecific inhibitor in Example 6 of the present invention;

[0151] FIG4 is the HUVEC proliferation activity detection result of the bispecific inhibitor in Example 6 of the present invention;

[0152] FIG5 shows the results of the ANG-2 blocking activity assay of the bispecific inhibitor in Example 7 of the present invention. DETAILED DESCRIPTION

[0153] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0154] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0155] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0156] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0157] As used herein, the term "antibody" generally refers to an antibody that can recognize one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, multimeric antibodies, and CDR-grafted antibodies.

[0158] In this article, the term "monoclonal antibody" refers to an antibody that recognizes only one specific antigenic epitope. Common monoclonal antibodies consist of two light chains (lighter in molecular weight) and two heavy chains (heavy in molecular weight). The heavy chain (H chain) and light chain (L chain) are linked by disulfide bonds to form a tetrapeptide chain. The amino acid sequence at the amino terminus (N-terminus) of the heavy or light chain varies greatly and is called the variable region (V region). The carboxyl terminus (C-terminus) of the heavy or light chain is relatively stable and varies little and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively. Monoclonal antibodies can also be small molecule antibodies, which mainly include: Fab antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and minimal recognition units.

[0159] As used herein, the term "Fab antibody" generally refers to an antibody containing only Fab molecules, which are composed of VH and CH1 of the heavy chain and a complete light chain, with the light chain and heavy chain connected by a disulfide bond.

[0160] In this article, the term "Fv antibody" generally refers to an antibody composed only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds. It is the smallest functional fragment of an antibody that retains a complete antigen-binding site.

[0161] In this article, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), which only contain a heavy chain variable region (VH) and conventional CH2 and CH3 regions, and specifically bind to an antigen through the heavy chain variable region.

[0162] Herein, the term "single-chain antibody" generally refers to an antibody composed of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker peptide.

[0163] In this article, the terms "minimum recognition unit" and "MRU" both refer to an antibody consisting of only one CDR, which has a very small molecular weight accounting for only about 1% of the complete antibody.

[0164] In this article, the terms "polyspecific antibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (abbreviated as bispecific antibody), antibodies that recognize three antigenic epitopes, or antibodies that recognize four antigenic epitopes. This is understood in a broad sense and the specific structure is not limited, as long as it can recognize multiple antigenic epitopes. For example, the bispecific antibody shown in Figure 1.

[0165] As used herein, the terms "CDR-grafted antibody" and "modified antibody" both refer to the transplantation of the CDRs of a monoclonal antibody from one species into the variable region of an antibody from another species. For example, the CDRs of a mouse monoclonal antibody can be transplanted into the variable region of a human antibody to replace the human antibody CDRs, allowing the human antibody to acquire the antigen-binding specificity of the mouse monoclonal antibody while reducing its heterologous nature. It should be noted that both polyclonal antibodies and monoclonal antibodies in this application can be CDR-grafted antibodies.

[0166] In this article, the terms "symmetrical bispecific antibody" and "symmetrical bispecific antibody" are synonymous, and generally refer to antibodies having two or more identical peptide chains, with the number of peptide chains not particularly limited; for example, for a bispecific antibody containing an Fc fragment, the two peptide chains of the bispecific antibody Fc fragment are located on two peptide chains respectively, and a bispecific antibody refers to a bispecific antibody in which the structures and sequences of the two peptide chains are exactly the same. Illustratively, in the present application, the bispecific antibody has two identical peptide chains, each peptide chain comprising a first binding region, a connecting peptide, and a second binding region, wherein the first binding region comprises an antibody or antigen-binding fragment capable of recognizing ANG-2, and the second binding region comprises the immunoglobulin-like region 2 in the VEGFR1 fragment, the immunoglobulin-like region 3 in VEGFR2, and the Fc fragment, the C-terminus of the immunoglobulin-like region 2 in the VEGFR1 fragment is connected to the N-terminus of the immunoglobulin-like region 3 in VEGFR2, the C-terminus of the immunoglobulin-like region 3 in VEGFR2 is connected to the N-terminus of the Fc fragment, the C-terminus of the Fc fragment is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the antibody or antigen-binding fragment, specifically the bispecific antibody shown in Figure 1.

[0167] As used herein, the terms "asymmetric bispecific antibody" and "asymmetric bispecific antibody" are synonymous and generally refer to antibodies having two or more different peptide chains, with the number of such chains not being particularly limited. For example, in a bispecific antibody containing an Fc fragment, the two peptide chains of the bispecific antibody Fc fragment are located on two separate peptide chains. An asymmetric bispecific antibody refers to a bispecific antibody in which the two peptide chains have different structures or sequences. For example, the two peptide chains of the bispecific antibody Fc fragment are each connected to two different binding regions (i.e., a first binding region and a second binding region), wherein the first binding region comprises an antibody or antigen-binding fragment capable of recognizing ANG-2, the second binding region comprises immunoglobulin-like region 2 of the VEGFR1 fragment and immunoglobulin-like region 3 of VEGFR2, the C-terminus of immunoglobulin-like region 2 of the VEGFR1 fragment is connected to the N-terminus of immunoglobulin-like region 3 of VEGFR2, the N-terminus of one peptide chain of the Fc fragment is connected to the C-terminus of immunoglobulin-like region 3 of VEGFR2, and the N-terminus of the other peptide chain is connected to the C-terminus of the antibody or antigen-binding fragment.

[0168] Furthermore, the two peptide chains of the Fc fragment of the asymmetric antibody can be connected by a "knob into hole structure", wherein the "knob into hole structure" is a knob (knob) and a hole (hole) mutation formed in the CH3 region of the antibody heavy chain constant region to facilitate the heavy chain bite to form a heterodimer, for example, by mutating the amino acids in the CH3 domain of the human IgG1 heavy chain constant region (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., the "hole"; T366W, S354C mutations in the other chain, i.e., the "knob").

[0169] As used herein, the term "fragment" refers to a target protein or polypeptide, as well as a target protein or polypeptide with an N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and / or internal deletion. For example, a VEGF receptor fragment can be a VEGF receptor (e.g., VEGFR1, VEGFR2, or VEGFR3), or a shortened or truncated VEGF receptor (e.g., VEGFR1, VEGFR2, or VEGFR3). The polypeptide can be. Exemplarily, a VEGF receptor fragment is an immunoglobulin-like region 2 in VEGFR1 and / or an immunoglobulin-like region 3 in VEGFR2.

[0170] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0171] As used herein, the term "at least 80% identity" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to the respective reference sequence.

[0172] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0173] In this article, the term "recombinant cell" generally refers to a cell that has been modified or reorganized using genetic engineering techniques or cell fusion techniques to modify or reorganize the genetic material of a host cell to obtain a cell with a unique trait of stable inheritance. Wherein, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0174] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0175] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0176] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient by a suitable means. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate, or pharmaceutical composition of the present invention can be administered by any common route as long as it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0177] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0178] In this article, the term "angiogenesis-related diseases" generally refers to diseases caused by angiogenesis (or angiogenesis), including but not limited to cancer, ophthalmic diseases and inflammatory diseases. Exemplarily, cancer can be a solid tumor or a blood tumor, including but not limited to hemangioma, vascular tumor, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and glioma; ophthalmic diseases can be intraocular neovascular syndromes (e.g., proliferative retinopathies, age-related macular degeneration (AMD)); inflammatory diseases include but are not limited to rheumatoid arthritis and psoriasis.

[0179] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0180] In the examples of the present invention, the control drug Faricimab was purchased from Shanghai Baiying Biotechnology Co., Ltd. with the product number B936701.

[0181] Example 1: Screening of ANG-2 single-domain antibodies (single-domain antibodies)

[0182] 1. Phage library construction

[0183] Two alpacas were immunized five times with human ANG-2 protein (Sino Biological, Cat. No. 10691-H08H). After the fifth immunization, 50 mL of peripheral blood was collected from each alpaca. Lymphocytes were isolated using a lymphocyte separation buffer. Total RNA from these lymphocytes was extracted using RNAiso Plus, and reverse transcribed into cDNA using the PrimeScript II kit (TAKARA 6210A). Nested PCR was then used to amplify the VHH gene fragment of interest, i.e., the variable region of a single-domain antibody.

[0184] The target gene VHH and the vector pComb3xss were digested with SfiI and ligated with T4 DNA ligase, and then transformed into TG1 electroporated TG1 competent cells to construct a VHH gene library and obtain the transformation solution. At the same time, 0.1 μL, 0.01 μL, 0.001 μL, and 0.0001 μL of the mixed transformation solution were evenly spread on a 90 mm culture dish, and the library capacity was calculated to be 4.75×10 9 100 randomly selected colonies were amplified and sequenced, and the results showed that the insertion efficiency was 100%.

[0185] 10-100 times the library volume of living cells were taken from the above gene library for inoculation and culture. After culture to the logarithmic phase, M13K07 phage was used for rescue. After rescue culture, phages were collected by centrifugation and purified with PEG-NaCl to obtain a phage display library with a titer of 6.8×10 13 cfu / mL. Directly used for subsequent affinity screening of specific phages.

[0186] 2. Phage library screening

[0187] Plates were coated with human ANG-2 protein (Sino Biological, Cat. No. 10691-H08H) at concentrations of 100 μg / mL and 50 μg / mL, incubated at 4°C overnight, and then screened. The next day, the plates were blocked with 1% ovalbumin (OVA) at 37°C for 1 hour. 100 μL of phage (6.8 × 10 13 cfu / mL) and washed five times with PBST (PBS containing 0.05% Tween 20) after incubation to remove unbound phage. Finally, phage specifically bound to the human ANG-2 protein were eluted with 100 μL of 0.1 M Gly-HCl (pH 2.2) and used to infect Escherichia coli TG1 cells in logarithmic phase growth to generate and purify phage for the next round of screening. After two rounds of the same screening process, the resulting phage were infected with E. coli TG1 cells and plated. Single colonies were picked from the plates and sequenced. The protein sequences of each clone were analyzed based on the sequence alignment results, and clones with different CDR1, CDR2, and CDR3 sequences were considered different antibody strains. Finally, a total of 5 different single-domain antibodies were obtained. The heavy chain variable regions (VHH) of the 5 single-domain antibodies were VHH-04 having the amino acid sequence shown in SEQ ID NO: 16, VHH-08 having the amino acid sequence shown in SEQ ID NO: 17, VHH2-03 having the amino acid sequence shown in SEQ ID NO: 18, VHH2-09 having the amino acid sequence shown in SEQ ID NO: 19, and VHH2-14 having the amino acid sequence shown in SEQ ID NO: 20.

[0188] Example 2: Preparation of ANG-2 single domain antibodies

[0189] The nucleotide sequences of the variable regions of the five single-domain antibodies obtained in Example 1 (the nucleotide sequences are shown in SEQ ID NOs: 33 to 37, respectively) were connected to the nucleotide sequence of human IgG1 Fc (the nucleotide sequence is shown in SEQ ID NO: 39). The specific sequences are shown in Table 1 and synthesized into the expression plasmid pcDNA3.1. After transformation into Escherichia coli, the plasmid was extracted. The expression plasmid was filtered through a 0.22 μm filter membrane and transfected into CHO cells using PEI transfection reagent (Polysciences, 08APR22). After culturing for 4 days, the cell supernatant was collected and added to the fully resuspended Protein A gel solution. After thorough mixing, the cells were incubated at 4°C for 1 hour. The mixture of Protein A and cell culture supernatant was transferred to a gravity column. After the liquid in the mixture was drained away, the Protein A gel was washed with 10 column volumes of eluent. The protein eluate was collected and 4 mL of 1 M Tris-HCl neutralizing solution (pH 8.0) was added. The mixture was incubated at room temperature for 5 minutes to obtain the target products, ANG-2 single-domain antibodies, which were named VHH-04, VHH-08, VHH2-03, VHH2-09, and VHH2-14 antibodies, respectively.

[0190] Table 1: Sequences of tetravalent bispecific inhibitors

[0191]

[0192]

[0193]

[0194] Example 3: Affinity detection of ANG-2 single domain antibody

[0195] Human ANG-2 protein (Sino Biologics, 10691-H08H) was diluted to a 1 μg / mL coating solution. 100 μL / well of the ELISA plate was added and coated at 2-8°C for at least 12 hours. The residual coating solution was discarded, and 200 μL of 2% BSA-PBS was added to each well. After blocking at 37°C for 2 hours, the wells were washed three times with 300 μL of PBST. The test sample (VHH-04, VHH-08, VHH2-03, VHH2-09, or VHH2-14 antibodies prepared in Example 2) or the control drug (Faricimab, Bio-Bio, Catalog No. B936701, Lot No. 20220108Z003) was diluted to 20 nM with PBS. Eight three-fold dilutions were then added, and 100 μL / well of the ELISA plate was added. Incubate at 37°C for 1 hour, add 300 μL of PBST to each well, wash 3 times, and then add 10,000-fold diluted Goat anti-Human Fc-HRP (abcam, ab97225), 100 μL / well. After incubation at 37°C for 1 hour, add 300 μL of PBST to each well, wash 3 times, and pat dry. Add TMB colorimetric solution, 100 μL per well. After reacting at room temperature for 5 minutes, add 2M H2SO4 to terminate the reaction, 50 μL / well. Place the terminated microplate on a microplate reader and read the absorbance OD at a wavelength of 450 nm. 450 The data were analyzed using GraphPad Prism 7.0 software, and the binding activity was fitted to obtain the ANG-2 binding activity EC 50 The results are shown in Table 2 and Figure 1. The results showed that VHH-04, VHH-08, VHH2-03, VHH2-09 and VHH2-14 antibodies had significant binding activity to human ANG-2, and their binding activity was better than that of Faricimab.

[0196] Table 2: ANG-2 binding activity of different single domain antibodies

[0197] Sample EC 50 (nM) Relative activity VHH-04 0.04442 50% VHH-08 0.04244 50% VHH2-03 0.09619 50% VHH2-09 0.08821 30% VHH2-14 0.0652880% Faricimab 1.871100%

[0198] Example 4: Preparation of tetravalent bispecific inhibitors (also known as bispecific antibodies or bispecific antibodies)

[0199] The VEGF receptor-Fc fusion protein with the nucleotide sequence set forth in SEQ ID NO:15 and the ANG-2 single-domain antibody variable region with the nucleotide sequence set forth in any one of SEQ ID NOs:33-37 were linked via a linker peptide with the nucleotide sequence set forth in SEQ ID NO:40. The resulting proteins were synthesized into the expression plasmid pcDNA3.1 and transformed into Escherichia coli. The plasmid was then extracted. The expression plasmid was filtered through a 0.22 μm filter and transfected into HEK 293 cells using PEI transfection reagent (Polysciences, 08APR22). Feed (Sino Biological, M293-SUPI) was added 20-24 hours after transfection. Feed was then added every other day for a total of six days, and protein was collected after the sixth day. On the sixth day after transfection, the cell culture supernatant was collected and added to the thoroughly resuspended Protein A gel solution. The cells were mixed thoroughly and incubated at 4°C for 1 hour. The mixture of Protein A and cell culture supernatant was transferred to a gravity column. After the liquid in the mixture was drained away, the Protein A gel was washed with 10 column volumes of eluent. The protein eluate was collected and 4 mL of 1 M Tris-HCl (pH 8.0) neutralizing solution was added. The mixture was incubated at room temperature for 5 minutes to obtain the target tetravalent bispecific inhibitors, which were named GB10-04, GB10-08, GB10-03, GB10-09, and GB10-14, respectively. The specific sequences are shown in Table 3, and the structures are shown in Figure 2.

[0200] Table 3: Sequences of tetravalent bispecific inhibitors

[0201]

[0202]

[0203]

[0204] Example 5: Affinity determination of tetravalent bispecific inhibitors for VEGF and ANG-2

[0205] 1. Affinity determination of tetravalent bispecific antibody to human VEGFa165 and human PLGF

[0206] A ProA sensor (Sartorius, 18-5010) was immersed in 1×KB solution for 10 minutes. The test samples (GB10-04, GB10-08, GB10-03, GB10-09, and GB10-14) prepared in Example 4 and the control Faricimab were diluted to 1 μg / mL using 1×KB solution. The pre-wetted sensor was transferred to 1×KB solution for 60 seconds and then placed in solutions containing the above proteins to allow each protein to couple to the ProA sensor. The capture end trigger point was set to a loading thickness of 1.0 nm and the shaking speed was set to 1000 rpm / min. Human VEGFa165.his (ACRO, VE5-H5248) was diluted to 50 nmol / L using 1×KB and then diluted in a 2-fold gradient to 8 concentrations in a black 96-well plate, with the final well concentration being 0 nmol / L. The protein-coupled sensors were transferred to 1×KB and equilibrated for 120 seconds. The sensors were then placed in solutions of human VEGFa165.his at varying concentrations for binding, with shaking at 1000 rpm / min. The ProA sensors, bound to samples at varying concentrations, were then placed in 1×KB for dissociation, with shaking at 1000 rpm / min. After dissociation, the sensors were placed in 10 mmol / L glycine solution and 1×KB for 5 seconds, respectively, with shaking at 1000 rpm / min. This cycle was repeated three times to elute the complex from the ProA sensor, allowing analysis of the next sample.

[0207] The affinity determination results are shown in Table 4 , wherein the tetravalent bispecific antibody proteins GB10-04, GB10-08, GB10-03, GB10-09 and GB10-14 all have better affinity for VEGF than Faricimab.

[0208] Table 4: Affinity of tetravalent bispecific antibody protein for VEGF

[0209]

[0210] 2. Affinity determination of tetravalent bispecific antibodies for human ANG-2

[0211] An NTA sensor (Sartorius, 18-5101) was immersed in 1×KB solution for 10 minutes. Human ANG-2.his protein (Sino, 10691-H08H) was diluted to 1 μg / mL in 1×KB solution. The pre-wetted sensor was transferred to 1×KB solution for equilibration for 60 seconds and then placed in the aforementioned human ANG-2.his solution to allow coupling of human ANG-2.his to the NTA sensor. The capture end trigger point was set at a loading thickness of 1.0 nm, and the shaking speed was set to 1000 rpm / min. The test samples (GB10-04, GB10-08, GB10-03, GB10-09, and GB10-14) prepared in Example 4 and the control, Faricimab, were diluted to 30 nmol / L in 1×KB. The samples were then serially diluted in a two-fold gradient across eight concentrations in a black 96-well plate, with the final concentration reaching 0 nmol / L. Sensors coupled to human ANG-2.his were transferred to 1×KB and equilibrated for 120 seconds. They were then placed in the aforementioned test sample solutions of varying concentrations for binding, with shaking at 1000 rpm / min. NTA sensors bound to the test samples at varying concentrations were then transferred to 1×KB for dissociation, with shaking at 1000 rpm / min. After dissociation, the sensors were placed in 10 mmol / L glycine solution and 1×KB for 5 seconds, respectively, with shaking at 1000 rpm / min. This cycle was repeated three times to elute the complex from the NTA sensors. The sensors were then activated in 10 mmol / L NiCl₂ solution for 60 seconds before analysis of the next sample. Affinity assay results are shown in Table 5. GB10-04, GB10-08, GB10-03, GB10-09, and GB10-14 all exhibited superior affinity for ANG-2 to faricimab.

[0212] Table 5: Affinity of tetravalent bispecific antibody protein ANG-2

[0213]

[0214] Example 6: Determination of VEGF blocking activity of tetravalent bispecific inhibitors

[0215] 1. VEGF reporter gene activity detection

[0216] The H-VEGF Reporter 293 cell (GM-C09057) cell suspension was added to a 96-well microplate (BeyoGold, FCP968), and 2.5×10 4Cells / 50 μL / well, in a carbon dioxide incubator overnight. Take out the microplate, add 50 μL of a mixed culture medium containing VEGF165 (final concentration 5 ng / mL, Nearshore Bio C083) and different gradient dilutions (highest concentration 25 nM, 3-fold dilution 8 gradients) of the test sample (GB10-04, GB10-08, GB10-03, GB10-09 or GB10-14 prepared in Example 4) or the control drug (Faricimab) to each well, and continue to culture in an incubator at 37°C and 5% CO2. After 6 hours, add 100 μL of luciferase reporter gene detection reagent (Vazyme, DD1203) to each well, incubate for 15 minutes, and read the chemiluminescence value RLU. Use GraphPad Prism 8.0 software to analyze the data, fit the inhibitory activity, and obtain the VEGF blocking activity IC 50 The results are shown in Table 6 and Figure 3. The results showed that GB10-04, GB10-08, GB10-03, GB10-09, GB10-14 and Faricimab had significant blocking activity on VEGF-mediated reporter gene expression, among which the tetravalent bispecific inhibitor of the present invention had better blocking activity on VEGF than Faricimab.

[0217] Table 6: VEGF reporter gene activity IC 50

[0218] Sample IC 50 (nM) Relative activity GB10-04 0.030557% GB10-08 0.048347% GB10-03 0.045366% GB10-09 0.051322% GB10-14 0.045371% Faricimab 0.165100%

[0219] 2. HUVEC proliferation activity detection

[0220] Add the HUVEC cell (Promocell#C12205) suspension to a 96-well microplate (Corning, 3917), 3000 cells / 50μL / well, and incubate in a carbon dioxide incubator overnight. Remove the microplate and add 50μL of a mixed culture medium containing VEGF165 (final concentration 5ng / mL, Nearshore Bio C083) and different gradient dilutions (highest concentration 25nM, 3-fold dilution 8 gradients) of the test sample (GB10-04, GB10-08, GB10-03, GB10-09 or GB10-14 prepared in Example 4) or a control drug (Faricimab) to each well, and continue to culture in an incubator at 37°C and 5% CO2. After 3 days, detect the cell proliferation results. Add 50uL of detection reagent to each well Luminescent Cell Viability Assay (Promega G7570) was used, and the cells were incubated for 15 minutes after shaking. The chemiluminescence value (RLU) was read. The data were analyzed using GraphPad Prism 8.0 software, and the inhibitory activity was fitted to obtain the IC value of HUVEC proliferation inhibitory activity. 50 The results are shown in Table 6 and Figure 4. The results showed that GB10-04, GB10-08, GB10-03, GB10-09, and GB10-14 had significant blocking activity on VEGF-mediated HUVEC proliferation, and GB10-04 had a better blocking activity on VEGF than Faricimab.

[0221] Table 7: HUVEC proliferation activity IC 50

[0222] Sample IC 50 (nM)GB10-040.029Faricimab0.040

[0223] Example 7: Determination of ANG-2 blocking activity by tetravalent bispecific inhibitors

[0224] TIE2 protein (Acro, TI2-H5255) was diluted to a 1 μg / mL coating solution. 100 μL / well of the microtiter plate was added and coated at 2-8°C for at least 12 hours. The residual coating solution was discarded, and 200 μL of 2% BSA-PBS was added to each well. After blocking at 37°C for 2 hours, the wells were washed three times with 300 μL of PBST per well. A sample diluent containing 100 ng / mL of ANG-2 protein (Sino Biological, 10691-H08H) was prepared. The test sample (GB10-04, GB10-08, GB10-03, GB10-09, or GB10-14 prepared in Example 4) or the control drug (Faricimab) was diluted to 20 nM. Eight 3-fold dilutions were then added, and 100 μL / well of the microtiter plate was added. Incubate at 37°C for 1 hour, add 300 μL of PBST to each well, wash 3 times, and then add 10,000-fold diluted Goat anti-His-HRP (abcam, ab1187), 100 μL / well. After incubation at 37°C for 1 hour, add 300 μL of PBST to each well, wash 3 times, and pat dry. Add TMB colorimetric solution, 100 μL per well. After reacting at room temperature for 5 minutes, add 2M H2SO4 to terminate the reaction, 50 μL / well. Place the terminated ELISA plate on an ELISA reader and read the absorbance OD at a wavelength of 450 nm. 450 The data were analyzed using GraphPad Prism 7.0 software, and the inhibitory activity was fitted to obtain the IC value of ANG-2 inhibitory activity. 50The results are shown in Table 8 and Figure 5. The results showed that GB10-04, GB10-08, GB10-03, GB10-09, and GB10-14 had significant blocking activity against the binding of ANG-2 to its receptor TIE2, and the five tetravalent bispecific inhibitors of the present invention had better blocking activity against ANG-2 than Faricimab.

[0225] Table 8. ANG-2 blocking activity IC50

[0226] Sample IC50 (nM) Relative activity GB10-04 0.1127069% GB10-08 0.0819775% GB10-03 0.5181528% GB10-09 0.5331485% GB10-14 0.3961999% Faricimab 7.918100%

[0227] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0228] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment, characterized in that The invention also comprises a heavy chain variable region CDR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity thereto: SEQ ID NO: 1~14.

2. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment comprises: the heavy chain variable region CDR1, CDR2, and CDR3 sequences as set forth in the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, or amino acid sequences at least 80% identical to SEQ ID NOs: 1, 2, and 3; the heavy chain variable region CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively, or an amino acid sequence at least 80% identical to SEQ ID NOs: 4, 5, and 6; the heavy chain variable region CDR1, CDR2, and CDR3 sequences as set forth in the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, or amino acid sequences at least 80% identical to SEQ ID NOs: 7, 8, and 9; The heavy chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NOs: 10, 11, and 12, respectively, or in amino acid sequences that are at least 80% identical to SEQ ID NOs: 10, 11, and 12; or The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NOs: 10, 13, and 14, respectively, or in amino acid sequences that are at least 80% identical to SEQ ID NOs: 10, 13, and 14.

3. The antibody or antigen-binding fragment according to claim 1, wherein The antibody contains a heavy chain framework region sequence, at least a portion of which is derived from at least one of a murine antibody, a human antibody, a primate antibody, an alpaca antibody, or a mutant thereof.

4. The antibody or antigen-binding fragment according to claim 1, wherein The antibody has a heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NOs: 16 to 20.

5. The antibody or antigen-binding fragment according to claim 1, wherein The antibody contains a heavy chain constant region, at least a portion of which is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

6. The antibody or antigen-binding fragment according to claim 5, characterized in that The heavy chain constant region of the antibody is derived from a human IgG antibody or a mutant thereof.

7. The antibody or antigen-binding fragment according to claim 6, characterized in that The heavy chain constant region of the antibody is derived from human IgG1.

8. The antibody or antigen-binding fragment according to claim 1, wherein The antibodies include monoclonal antibodies or polyclonal antibodies.

9. The antibody or antigen-binding fragment according to claim 8, wherein The monoclonal antibody includes at least one of a Fab antibody, a Fv antibody, a single-chain antibody, a single-domain antibody, and a minimum recognition unit.

10. The antibody or antigen-binding fragment according to claim 1, wherein The antibody has a heavy chain having an amino acid sequence shown in any one of SEQ ID NOs: 21 to 25.

11. A recombinant protein, characterized in that include: The antibody or antigen-binding fragment according to any one of claims 1 to 10.

12. The recombinant protein according to claim 11, characterized in that It further comprises at least one selected from a biologically active protein or a fragment thereof, a biologically active polypeptide or a fragment thereof.

13. The recombinant protein according to claim 12, characterized in that The biologically active protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof and Fc fragments.

14. A multispecific antibody, characterized in that include: a first binding region, the first binding region comprising the antibody or antigen-binding fragment of any one of claims 1 to 10; A second binding region having a first molecule binding activity.

15. The multispecific antibody according to claim 14, characterized in that The multispecific antibody comprises at least one selected from a bispecific antibody, a trispecific antibody and a tetraspecific antibody, preferably a bispecific antibody.

16. The multispecific antibody according to claim 15, characterized in that The first molecule comprises at least one selected from immune cell surface antigens, tumor antigens, viruses, bacteria, endotoxins, cytokines, and cytokine receptors.

17. The multispecific antibody according to claim 16, characterized in that The first molecule includes at least one selected from PD-L1, PD-1, IL-10, IL-10R, BCMA, VEGF, TGF-β, CTLA-4, LAG-3, TIGIT, CEA, CD38, SLAMF7, B7-H3, Her2, EpCAM, CD19, CD20, CD30, CD33, CD47, CD52, CD133, EGFR, GD2, GD3, GM2, RANKL, CD3 and CD16a.

18. The multispecific antibody according to claim 14, characterized in that The second binding region is the binding protein of the first molecule or a fragment thereof.

19. The multispecific antibody according to claim 14, characterized in that The first molecule is VEGF.

20. The multispecific antibody according to claim 19, wherein The second binding region comprises at least one selected from the group consisting of a VEGF receptor fragment and an anti-VEGF antibody.

21. The multispecific antibody according to claim 20, characterized in that The VEGF receptor fragment includes at least one selected from the group consisting of a VEGFR1 fragment, a VEGFR2 fragment, and a VEGFR3 fragment.

22. The multispecific antibody according to claim 21, wherein The second binding region includes the immunoglobulin-like region 2 in the VEGFR1 fragment and / or the immunoglobulin-like region 3 in the VEGFR2.

23. The multispecific antibody according to claim 22, characterized in that The C-terminus of the immunoglobulin-like region 2 in the VEGFR1 fragment is connected to the N-terminus of the immunoglobulin-like region 3 in the VEGFR2.

24. The multispecific antibody according to claim 23, characterized in that The bispecific antibody includes a symmetric bispecific antibody or an asymmetric bispecific antibody.

25. The multispecific antibody according to claim 23, characterized in that The bispecific antibody is a symmetrical bispecific antibody.

26. The multispecific antibody according to claim 25, characterized in that The second binding region further comprises an Fc fragment.

27. The multispecific antibody according to claim 26, characterized in that The C-terminus of the immunoglobulin-like region 2 in the VEGFR1 fragment is connected to the N-terminus of the immunoglobulin-like region 3 in the VEGFR2, and the C-terminus of the immunoglobulin-like region 3 in the VEGFR2 is connected to the N-terminus of the Fc fragment; or The C-terminus of the immunoglobulin-like region 2 in the VEGFR1 fragment is connected to the N-terminus of the immunoglobulin-like region 3 in the VEGFR2, and the N-terminus of the immunoglobulin-like region 2 in the VEGFR1 is connected to the C-terminus of the Fc fragment.

28. The multispecific antibody according to claim 14, wherein The second binding region has the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 90% identity thereto.

29. The multispecific antibody according to claim 27, wherein Further included is a connecting peptide.

30. The multispecific antibody according to claim 29, wherein The C-terminus of the immunoglobulin-like region 3 in the VEGFR2 is connected to the N-terminus of the Fc fragment, the C-terminus of the Fc fragment is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the antibody or antigen-binding fragment; or The C-terminus of the antibody or antigen-binding fragment is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting peptide is connected to the N-terminus of the Fc fragment, and the C-terminus of the Fc fragment is connected to the N-terminus of the immunoglobulin-like region 2 in the VEGFR1.

31. The multispecific antibody according to claim 29, wherein The amino acid sequence of the connecting peptide is (GGGGS) n , wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

32. The multispecific antibody according to claim 29, wherein The connecting peptide has the amino acid sequence shown in SEQ ID NO:

27.

33. The multispecific antibody according to claim 14, characterized in that The antibody has an amino acid sequence as shown in any one of SEQ ID NOs: 28 to 32.

34. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, or the multispecific antibody of any one of claims 14 to 33.

35. The nucleic acid molecule according to claim 34, characterized in that The nucleic acid molecule is DNA.

36. An expression vector, characterized in that Carrying the nucleic acid molecule according to claim 34 or 35.

37. The expression vector according to claim 36, characterized in that The expression vector is a eukaryotic expression vector, preferably, the expression vector is a plasmid expression vector.

38. A recombinant cell, characterized in that include: Carrying the nucleic acid molecule of claim 34 or 35 or the expression vector of claim 36 or 37; or, Expressing the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, or the multispecific antibody of any one of claims 14 to 33.

39. The recombinant cell according to claim 38, characterized in that The recombinant cell is obtained by introducing the expression vector according to claim 36 or 37 into a host cell.

40. The recombinant cell according to claim 38, wherein The recombinant cell is a eukaryotic cell.

41. The recombinant cell according to claim 38, wherein The recombinant cell is a mammalian cell.

42. A pharmaceutical composition, characterized in that include: Expressing the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, or the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, or the recombinant cell of any one of claims 38 to 41.

43. The pharmaceutical composition according to claim 42, characterized in that Further includes pharmaceutically acceptable excipients.

44. A conjugate, characterized in that Include: The antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, or the multispecific antibody of any one of claims 14 to 33; and A coupling portion is connected to the antibody or antigen-binding fragment, recombinant protein or multispecific antibody.

45. The conjugate according to claim 44, characterized in that The coupling portion comprises at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier and a chemotherapeutic agent.

46. ​​A kit, characterized in that include: The antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, or the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, or the conjugate of claim 44 or 45.

47. Use of the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, or the conjugate of claim 44 or 45 in the preparation of a kit for detecting ANG-2.

48. The use according to claim 47, characterized in that The kit is used for detecting VEGF.

49. Use of the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, the pharmaceutical composition of claim 42 or 43, or the conjugate of claim 44 or 45 in the preparation of a medicament for treating or preventing angiogenesis-related diseases.

50. Use of the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, the pharmaceutical composition of claim 42 or 43, or the conjugate of claim 44 or 45 in treating or preventing diseases related to angiogenesis.

51. Use of the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, the conjugate of claim 44 or 45, or the kit of claim 46 in diagnosing angiogenesis-related diseases, assessing the prognosis of angiogenesis-related diseases, and assessing the staging of angiogenesis-related diseases.

52. The use according to any one of claims 49 to 51, characterized in that The angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

53. A method for treating or preventing angiogenesis-related diseases, characterized in that: include: Administering a pharmaceutically acceptable amount of the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, the multispecific antibody of any one of claims 14 to 33, the pharmaceutical composition of claim 42 or 43, or the conjugate of claim 44 or 45 to a subject.

54. The method according to claim 53, wherein The administration route of the method is subcutaneous injection or intravenous injection.

55. A method for diagnosing angiogenesis-related diseases, characterized in that: include: Detecting ANG-2 in a sample using the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, the conjugate of claim 44 or 45, or the kit of claim 46; Based on the test results, the content of ANG-2 in the test sample is determined.

56. The method according to claim 55, characterized in that Further detecting VEGF in the sample to be tested; Based on the detection results, the content of VEGF in the test sample is determined.

57. A method for evaluating the prognosis of angiogenesis-related diseases, characterized in that: include: Detecting ANG-2 in a sample using the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, the conjugate of claim 44 or 45, or the kit of claim 46; Based on the test results, the content of ANG-2 in the test sample is determined.

58. The method according to claim 57, wherein Further detecting VEGF in the sample to be tested; Based on the detection results, the content of VEGF in the test sample is determined.

59. A method for evaluating the staging of angiogenesis-related diseases, characterized in that: include: Detecting ANG-2 in a sample using the antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, the conjugate of claim 44 or 45, or the kit of claim 46; Based on the test results, the content of ANG-2 in the test sample is determined.

60. The method according to claim 59, wherein Further detecting VEGF in the sample to be tested; Based on the detection results, the content of VEGF in the test sample is determined.

61. The method according to claim 60, characterized in that The content of ANG-2 and / or VEGF in the test sample being not less than the minimum level for stage IV tumor is an indication that the test sample is derived from a patient with stage IV tumor; The ANG-2 and / or VEGF content in the test sample being between the levels of patients with stage IV and stage III tumors is an indication that the test sample is derived from a patient with stage III tumors; The ANG-2 and / or VEGF content in the test sample being between the standard levels for stage III and stage II tumors indicates that the test sample is derived from a patient with stage II tumors; The level of ANG-2 and / or VEGF in the test sample being between the standard levels for stage I and stage II disease is an indication that the test sample is derived from a patient with stage I tumor.

62. The method according to any one of claims 53 to 61, characterized in that The angiogenesis-induced related diseases include at least one selected from cancer, ophthalmic diseases and inflammatory diseases.

63. The antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, or the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, the pharmaceutical composition of claim 42 or 43, or the conjugate of claim 44 or 45, for treating or preventing diseases related to angiogenesis.

64. The antibody or antigen-binding fragment of any one of claims 1 to 10, the recombinant protein of any one of claims 11 to 13, the multispecific antibody of any one of claims 14 to 33, the nucleic acid molecule of claim 34 or 35, the expression vector of claim 36 or 37, the recombinant cell of any one of claims 38 to 41, the conjugate of claim 44 or 45, or the kit of claim 46, for use in diagnosing angiogenesis-related diseases, assessing the prognosis of angiogenesis-related diseases, or assessing the staging of angiogenesis-related diseases.

Images