RU2809746C2 - Humanized anti-vegf monoclonal antibody - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to isolated anti-VEGF antibodies or antigen-binding fragments thereof. The antibody described in the invention can specifically bind to VEGF with high affinity and block the binding of VEGF to VEGFR2 receptor. These antibodies also neutralize the proliferation effect of VEGF165 protein and multiple VEGF subtypes in HUVEC cells and can be used in the clinical treatment of diseases associated with increased expression of VEGF. The claimed invention also relates to a nucleic acid encoding an anti-VEGF antibody or an antigen-binding fragment thereof, an expression vector containing the nucleic acid, a host cell, a method of producing an anti-VEGF antibody or an antigen-binding fragment thereof, a pharmaceutical composition for the treatment of a disease associated with overexpression of VEGF, a method of treating colorectal cancer, the use of an anti-VEGF antibody or an antigen-binding fragment thereof for the preparation of a medicament for the treatment of colorectal cancer, a pharmaceutical combination for the treatment of a disease associated with overexpression of VEGF, and a kit for treatment a disease associated with increased expression of VEGF.

EFFECT: obtaining a humanized anti-VEGF monoclonal antibody.

24 cl, 5 ex, 12 dwg, 8 tbl

Description

TECHNICAL FIELD

The present invention relates to the field of tumor immunotherapy, in particular relates to a humanized monoclonal antibody that binds to VEGF.

BACKGROUND OF THE ART

The development of the vascular system underlies many physiological and pathological processes. Vascular endothelial growth factor (VEGF) is a group of growth factors with important proangiogenic activity that promote endothelial cell mitosis and antiapoptosis, increase vascular permeability, and promote cell migration. The human VEGF gene is located on chromosome 6p21.3 and belongs to the VEGF/PDGF supergene family, which encodes VEGF linked by disulfide bonds in the form of a dimer. In humans, the VEGF family includes several members with different functions: VEGFA (VEGF, with several different splice variants), VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and placental growth factor (PIGF). Recently, endocrine vascular endothelial growth factor (EG-VEGF) has also been included in this family. (Samson M et al., J Clin Endocrinol Metab. 2004; 89(8):4078-4088). VEGF is widely distributed in human tissues and organs, including retinal pigment epithelial cells, vascular endothelial cells, nerve cells, etc. (Goel N L et al. Nat Rev Cancer. 2013; 13(12): 871). There are three types of VEGF receptors: VEGFR1, VEGFR2 and VEGFR3. Binding of VEGF to the extracellular domain of the receptor triggers dimerization of the receptor and promotes autophosphorylation of tyrosine residues in the intracellular domain, thereby activating downstream signals that promote cell proliferation, migration, antiapoptosis, and increased vascular permeability. VEGFR1 and VEGFR2 are expressed mainly in vascular endothelial cells, and VEGFR3 is mainly expressed in lymphatic endothelial cells.

VEGF has been confirmed to play an important role in the regulation of normal and pathological angiogenesis (Melincovici S et al., Rom J Morphol Embryol. 2018; 59(2): 455-467). VEGF is overexpressed in various tumors that can cause malignant ascites, and VEGF expression in tumors correlates with the migratory ability of tumor cells. VEGF concentrations in patients with solid tumors with lower survival rates, such as gastrointestinal, ovarian, breast and lung cancers, are positively correlated with disease stage (Sebastian, K et al., Oncologist. 2009; 14(12): 1242 -1251). Hypoxic conditions in the tumor microenvironment induce the penetration of the tumor cell transcription factor HIF-1a into the nucleus, then HIF-1a binds to the HRE element of VEGFA, thereby increasing the level of VEGFA transcription and stimulating the secretion of large amounts of VEGF into the tumor microenvironment by tumor cells. In turn, high concentrations of VEGF act on the VEGFR of vascular endothelial cells, inducing large amounts of neovascularization, increasing blood supply, and providing sufficient nutrients for tumor cell growth. More VEGF is secreted from rapidly growing tumor cells, which further promotes the proliferation and migration of vascular endothelial cells and causes tumor metastasis. Additionally, VEGF also stimulates the transformation of monocytes in tumor tissues into M2 suppressor macrophages, which produce more negative immune factors and simultaneously activate Treg cells, which synergistically reduces the damaging ability of T cells.

By inhibiting the interaction of VEGF with endothelial cell surface receptors VEGFR2 and VEGFR1, anti-VEGF monoclonal antibody blocks downstream signaling pathways, inhibits endothelial cell proliferation and neovascularization, deprives tumor tissues of blood supply and controls the internal supply of tumor nutrients, thereby limiting tumor growth and ultimately achieving antitumor effectiveness. Avastin (bevacizumab, approved 2009) is the first antibody drug approved to inhibit tumor angiogenesis and is primarily used to treat breast cancer, cervical cancer, colorectal cancer, glioblastoma, glioma, non-small cell lung cancer, ovarian cancer and renal cell carcinoma. Although Avastin has been used to treat various types of cancer, there is still a need in this field for more potent antibodies with stronger VEGF inhibition and higher efficacy.

The present invention provides a new human anti-VEGF antibody for the treatment of colorectal cancer.

Short description

In one aspect, the present invention provides an isolated anti-VEGF antibody or antigen-binding fragment thereof comprising a heavy chain variable region, comprising a heavy chain CDR1 region comprising the amino acid sequence set forth in SEQ ID NO: 30, a heavy chain CDR2 region comprising the amino acid sequence, specified in SEQ ID NO: 31, and the CDR3 region of the heavy chain containing the amino acid sequence specified in SEQ ID NO: 32; and a light chain variable region comprising a light chain CDR1 region containing the amino acid sequence specified in SEQ ID NO: 27, a light chain CDR2 region containing the amino acid sequence specified in SEQ ID NO: 28, and a light chain CDR3 region containing the amino acid sequence , specified in SEQ ID NO: 29.

In one embodiment, said anti-VEGF antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 39, or an amino acid sequence that is at least 90%, 92%, 95% identical, 98% or 99% of the sequence SEQ ID NO: 39; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40, or an amino acid sequence that is at least 90%, 92%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO: 40.

In one embodiment, said antibody further includes a light chain constant region and a heavy chain constant region, preferably said light chain constant region is said light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 42, or an amino acid sequence identical in at least 90%, 92%, 95%, 98% or 99% of the sequence of SEQ ID NO: 42; and/or said heavy chain constant region is an IgG1 heavy chain constant region containing the amino acid sequence set forth in SEQ ID NO: 41, or an amino acid sequence that is at least 90%, 92%, 95%, 98%, or 99 identical % sequence SEQ ID NO:41.

In one embodiment, said anti-VEGF antibody or antigen-binding fragment thereof is an IgG antibody, preferably an IgG1 antibody.

In one embodiment, said anti-VEGFR antibody or antigen-binding fragment thereof is a monoclonal antibody.

In one embodiment, the binding affinity K _D of said anti-VEGF antibody or antigen-binding fragment thereof to recombinant human VEGF165 protein is 1-100 pM, preferably 5-50 pM, and more preferably 19.5 pM.

In one embodiment, said antigen-binding fragment is Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single chain antibody molecule, or single domain antibody; wherein said single chain antibody molecule is preferably a scFv, di-scFv, tri-scFv, diabody or scFab.

In another aspect, the present invention provides an antibody-drug conjugate comprising said anti-VEGF antibody or antigen-binding fragment thereof as described herein and an additional therapeutic agent, preferably said anti-VEGF antibody or antigen-binding fragment thereof linked to said additional therapeutic agent through a link.

In one aspect, the present invention provides an isolated anti-VEGF antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a heavy chain CDR1 region comprising the amino acid sequence set forth in SEQ ID NO: 16 and a heavy chain CDR2 region comprising the amino acid sequence , specified in SEQ ID NO: 17, and the CDR3 region of the heavy chain containing the amino acid sequence specified in SEQ ID NO: 18; and a light chain variable region comprising a light chain CDR1 region containing the amino acid sequence specified in SEQ ID NO: 13, a light chain CDR2 region containing the amino acid sequence specified in SEQ ID NO: 14, and a light chain CDR3 region containing the amino acid sequence , specified in SEQ ID NO: 15.

In one embodiment, said anti-VEGF antibody or antigen-binding fragment thereof comprises a heavy chain constant region containing the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence that is at least 90%, 92%, 95% identical, 98%, or 99% of the sequence SEQ ID NO: 25; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 26, or an amino acid sequence that is at least 90%, 92%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO: 26.

In one embodiment, said anti-VEGF antibody or antigen-binding fragment thereof is a humanized antibody or chimeric antibody.

In another aspect, the present invention provides a nucleic acid encoding the specified anti-VEGF antibody or antigen-binding fragment thereof described herein.

In one embodiment, said nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 7 and/or the nucleotide sequence set forth in SEQ ID NO: 8; or includes the nucleotide sequence specified in SEQ ID NO: 23 and/or the nucleotide sequence specified in SEQ ID NO: 24; or includes the nucleotide sequence set forth in SEQ ID NO: 47 and/or the nucleotide sequence set forth in SEQ ID NO: 48.

In another aspect, the present invention provides an expression vector containing the specified nucleic acid described herein.

In another aspect, the present invention provides a host cell containing said nucleic acid described herein or said expression vector described herein.

In another aspect, the present invention provides a method of producing said anti-VEGF antibody or antigen-binding fragment thereof as described herein, comprising growing said host cell as described herein under conditions suitable for expressing the antibody and isolating the expressed antibody from the culture cell. liquids.

In another aspect, the present invention provides a pharmaceutical composition comprising said anti-VEGF antibody or antigen-binding fragment thereof described herein, or said antibody-drug conjugate described herein, or a nucleic acid described herein, or specified the expression vector described herein and a pharmaceutically acceptable carrier.

In one embodiment, the present invention provides said anti-VEGF antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, for use in the treatment of colon cancer. and rectum.

In another aspect, the present invention provides a method of treating colorectal cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a specified anti-VEGF antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described in herein, or a pharmaceutical composition described herein.

In another aspect, the present invention provides the use of said anti-VEGF antibody or antigen-binding fragment thereof described herein or an antibody-drug conjugate described herein or a pharmaceutical composition described herein in the preparation of a medicament for the treatment of colon cancer. rectum.

In another aspect, the present invention provides a pharmaceutical combination comprising an anti-VEGFR2 antibody or an antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein, or a pharmaceutical composition described herein, and one or more other therapeutic agents.

In another aspect, the present invention provides a kit comprising an anti-VEGF antibody or antigen-binding fragment as described herein, or an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, preferably further comprising a device for introduction.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is illustrated in combination with the accompanying drawings, in which:

In FIG. 1 demonstrates that a rabbit anti-VEGF165 antibody blocks the binding of VEGF165 to the VEGFR2 protein.

In FIG. 2 demonstrates that a rabbit anti-VEGF165 antibody neutralizes VEGF165-induced HUVEC proliferation.

In FIG. 3 shows the binding of the humanized antibody VEGF165-H988 to VEGF165 detected by ELISA.

In FIG. 4 demonstrates the binding of the humanized antibody VEGF165-H988 to mVEGF165 by ELISA.

In FIG. 5 demonstrates that the VEGF-H988 antibody blocks the binding of VEGF165 to the VEGFR2 protein detected by ELISA.

In FIG. 6 demonstrates the effect of the VEGF-H988 antibody on neutralizing VEGF165 at various concentrations.

In FIG. 7 demonstrates the effect of VEGF-H988 in neutralizing the proliferation of HUVEC cells caused by VEGF165, VEGFC and VEGFD.

In FIG. Figure 8 shows a curve of mean blood concentration versus time for a group of G2 mice injected with VEGF-H988 (50 mg/kg) intraperitoneally.

In FIG. Figure 9 shows a curve of mean blood concentration versus time for a group of G4 mice injected with VEGF-H988 (50 mg/kg) intraperitoneally.

In FIG. 10 shows a curve of mean blood concentration versus time for groups of G2 and G4 mice injected with VEGF-H988 (50 mg/kg) intraperitoneally.

In FIG. 11 demonstrates a comparison of the efficacy of VEGF-H988 and a control drug in a xenograft model of human colorectal cancer with the HCT-116 cell line.

In FIG. 12 shows a comparison of the efficacies of VEGF-H988 and a control drug in a xenograft model of human colorectal cancer with the HCT-116 cell line.

DETAILED DESCRIPTION

Various aspects of the present invention relate to an isolated anti-VEGF antibody or an antigen-binding fragment thereof, an antibody-drug conjugate containing said antibody or an antigen-binding fragment thereof, a nucleic acid and expression vector encoding said antibody or an antigen-binding fragment thereof, and a host cell containing said nucleic acid or expression vector, a method of producing said anti-VEGF antibody or an antigen-binding fragment thereof, a pharmaceutical composition containing said anti-VEGF antibody or an antigen-binding fragment thereof, and a method of using said anti-VEGF antibody or an antigen thereof -binding fragment for the treatment of colon and rectal cancer.

Definitions

Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention relates. For purposes of the present invention, the following terms are defined in accordance with the meanings commonly understood in the art.

As used in this specification and in the accompanying claims, the singular forms “one,” “other,” and “said” include the plural designation of the subject matter unless the context clearly indicates otherwise.

The term "antibody" refers to an immunoglobulin molecule and refers to any form of antibody that exhibits the desired biological activity. These include, but are not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (eg, bispecific antibodies), and even antibody fragments. In general, full-length antibody structures preferably comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, typically linked by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this conventional full-length antibody structure, this structure also includes other derivative forms.

Said heavy chain variable region and light chain variable region can be further subdivided into more conserved regions (called framework regions (FR)) and hypervariable regions (called complementarity determining regions (CDR)) separated by spaces.

The term “complementarity determining region” (CDR, e.g. CDR1, CDR2 and CDR3) refers to those amino acid residues in a specified variable region of an antibody whose presence is required for antigen binding. Each variable region typically contains three CDR regions, designated CDR1, CDR2 and CDR3. Each complementarity determining region may contain amino acid residues from a “complementarity determining region” according to Kabat numbering (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD , 1991) and/or residues from the “highly variable loop” (Chothia and Lesk; J MolBiol 196: 901-917(1987)).

The term “framework” or “FR” residues means residues in a specified variable region other than CDR residues as defined herein.

Each heavy chain variable region and light chain variable region typically contains 3 CDRs and up to 4 FRs, with said CDRs and FRs arranged from amino terminus to carboxyl terminus in the following order, for example: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 .

The complementarity determining region (CDR) and framework region (FR) of the antibody can be determined using the Kabat system (Kabat et al.: Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, PHS, NIH , NIH Publication No. 91-3242, 1991).

The term “constant region” refers to that sequence of amino acids in the light and heavy chains of an antibody that is not directly involved in the binding of the antibody to an antigen, but influences various effector functions such as antibody-induced cytotoxicity.

According to the antigenic differences in the constant region amino acid sequence, the antibody heavy chain can be classified into one of five classes: α, δ, ε, γ, and μ. When it forms a full-length light chain antibody, it can be classified into one of five classes: IgA, IgD, IgE, IgG and IgM, which can further be classified into five subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4 , IgA and IgA2. Based on the amino acid sequence of its constant region, the light chain of an antibody can be classified as κ and λ.

“Antigen-binding antibody fragment” means a portion of an intact antibody molecule that retains at least some of the binding specificity of the parent antibody and typically includes at least a portion of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody. Examples of antigen binding fragments include Fv fragment, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fd', single chain antibody molecules (e.g. scFv, di-scFv or tri-scFv, diabody or scFab ), single domain antibodies, but are not limited to them.

The term “antibody fragment” refers to a partial-length antibody molecule that retains at least some of the biological properties of the original antibody, including an Fc fragment, in addition to, but not limited to, the fragments described above as “antigen-binding fragments.”

The term "antibody-drug conjugate" or "ADC" refers to a binding protein, such as an antibody or an antigen-binding fragment thereof, that is chemically linked to one or more chemical agents (also referred to herein as "agents"), which optionally may be a therapeutic agent or a cytotoxic agent. In one preferred embodiment, the ADC includes an antibody, cytotoxic or therapeutic drug, and a linker by which said drug can be bound or conjugate with said antibody. ADCs can typically contain from 1 to 8 conjugate drugs with the specified antibody, including 2, 4, 6 or 8 drugs. Non-limiting examples of drugs that may be included in ADCs include mitotic inhibitors, antitumor antibiotics, immunomodulators, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron agents, chemotherapeutic protective agents, hormones, antihormonal agents, corticosteroids, photoactive therapeutics agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors and radiosensitizers.

The term "chimeric antibody" refers to an antibody in which a portion of said heavy chain and/or light chain is derived from one particular source or species and the remaining portion is derived from another source or species. Said “chimeric antibody” may also be a functional fragment described above. "Humanized antibodies" are a subset of "chimeric antibodies".

The term “humanized antibody” or “humanized antigen-binding fragment” as used herein means an antibody or antibody fragment that: (i) is derived from a non-human source (eg, a transgenic mouse having a heterologous immune system) and is based on the sequence human germline; or (ii) is a chimeric antibody in which the variable region is of non-human origin and the constant region is of human origin; or (iii) a CDR graft in which said variable region CDR is of non-human origin, one or more variable region framework regions are of human origin, and the constant region, if any, is of human origin. The goal of “humanization” is to eliminate the immunogenicity of antibodies of non-human origin in the human body while maintaining the highest possible affinity. As a template for humanization, it is advisable to select the human backbone sequence that is most similar to the backbone sequence of the antibody originating from a non-human source. In some cases, it may be necessary to replace one or more amino acids in the human framework sequence with corresponding residues in the non-human construct to avoid loss of affinity.

The term "monoclonal antibody" refers to an antibody obtained from an essentially homogeneous population of antibodies, i.e. each individual antibody within a given population is identical, except for possible mutations (eg natural mutations) which may be present in very small quantities. Thus, the term "monoclonal" indicates the nature of the specified antibody in question, i.e., and not a mixture of unrelated antibodies. Unlike polyclonal antibody preparations, which typically include different antibodies against different epitopes, all monoclonal antibodies in monoclonal antibody preparations are directed against a single epitope of the antigen. In addition to its specificity, monoclonal antibody preparations have the advantage that they are usually not contaminated with other antibodies. The term "monoclonal" should not be understood as requiring the production of the specified antibody by any particular method.

Said antibody "specifically binds" to a target antigen, such as a tumor-associated peptide antigen target (in this case, VEGF), i.e. binds said antigen with sufficient affinity to enable the use of said antibody as a therapeutic agent by targeting a cell or tissue expressing said antigen and does not significantly cross-react with other proteins or does not significantly cross-react with proteins other than homologs and variants of the target proteins mentioned above (for example, mutant forms, splice variants, or truncated forms of the protein resulting from hydrolysis).

The term "binding affinity" refers to the strength of the sum of non-covalent interactions between the individual binding sites of a molecule and its binding partners. Unless otherwise specified, “binding affinity” as used herein refers to intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). As used herein, the term "K _D " refers to the equilibrium dissociation constant of the antibody-antigen interaction. As used herein, the term " _kon " refers to the rate constant at which an antibody binds to an antigen. As used herein, the term " _koff " refers to the rate constant at which an antibody dissociates from the antibody/antigen complex. " _KD ", "binding rate constant _kon " and "dissociation rate constant koi" are commonly used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen). Affinity, i.e., the degree of strength at which a ligand binds to a specific protein. Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonds, electrostatic interactions, hydrophobic and van der Waals forces between two molecules. In addition, the binding affinity between a ligand and its target molecule can be influenced by the presence of other molecules. Affinity can be analyzed by traditional methods known in the art, including the ELISA described herein.

The term "epitope" includes any cluster of protein determinants that specifically binds to an antibody or T-cell receptor. Epitope determinant clusters typically consist of reactive surface groups of the molecule (eg, amino acid or sugar side chains or combinations thereof) and often have specific three-dimensional structural features as well as specific charge characteristics.

The term “isolated” antibody is an antibody that has been identified and isolated from components of the cell where the antibody is expressed. However, typically, the isolated antibody is obtained through at least one purification step.

"Sequence identity" between two polypeptide or nucleic acid sequences indicates the number of residues that are identical between the specified sequences as a percentage of the total number of residues, and is calculated based on the size of the smaller of the molecules being compared. When calculating percent identity, the compared sequences are compared to obtain the maximum match between the specified sequences, with gaps in the comparison (if any) resolved using a specific algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program suites, including GAP, BLASTP, BLASTN, and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410). The above procedures are publicly available from the International Center for Biotechnology Information (NCBI) and other sources. The well-known Smith-Waterman algorithm can also be used to determine identity.

The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Preferred are natural human FcR sequences and preferably receptors that bind to IgG antibodies (gamma receptors), which include FcγRI, FcγRII and FcγRIII isoforms, as well as variants of these receptors. All other FcRs are included under the term "FcR". The term also includes the neonatal receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus (Guyer et al., Journal of Immunology 117: 587 (1976) and Kim et al., Journal of Immunology 24: 249 (1994)).

The term "neonatal Fc receptor", abbreviated as "FcRn", binds to the Fc region of an IgG antibody. The neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG-like antibodies in vivo. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its serum clearance and prolonging its half-life. Thus, the FcRn binding/IgG characteristics in vitro indicate its pharmacokinetic properties in the bloodstream in vivo.

The term "effector function" refers to that biological activity attributable to the Fc region of an antibody, which varies from isotype to isotype. Examples of antibody effector function include C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, suppression cell surface receptors (eg, B cell receptors) and B cell activation.

The term "effector cell" refers to a cell that expresses one or more FcRs and performs effector functions. In one aspect, said effector cells express less FcγRIII and perform ADCC effector functions. Examples of human cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood. Effector cells are typically lymphocytes associated with the effector phase and function to produce cytokines (helper T cells), kill cells infected by pathogens (cytotoxic T cells), or secrete antibody (differentiated B cells).

"Immune cells" include cells that are of hematopoietic origin and play a role in the immune response. Immune cells include: lymphocytes such as B cells and T cells; natural killer cells; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fey receptors present on certain cytotoxic cells (eg, NK cells, neutrophils and macrophages), allowing these cytotoxic effector cells to specifically bind to target cells bearing antigens, and subsequently destroy said target cells using, for example, a cytotoxin. In vitro ADCC assays, such as the in vitro ADCC assays documented in US Patent No. 5,500,362 or 5,821,337 or US Patent No. 6,737,056 (Presta), can be performed to evaluate the ADCC activity of a target antibody. Useful effector cells for use in such cases include PBMCs and NK cells.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. The classical pathway of complement activation is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass), which binds to the corresponding antigen. To assess complement activation, a CDC assay is performed, such as the CDC assay described in Gazzano-Santoro et al., J. Immunol Methods 202: 163 (1996). For example, US Patent No. 6,194,551 B1 and WO 1999/51642 describe polypeptide variants containing an altered Fc region amino acid sequence (Fc region variant polypeptides) and polypeptide variants having increased or decreased C1q binding.

Amino acid sequence and nucleotide sequence of said antibody according to the present invention

In the present invention, recombinant human VEGF165 protein was used to immunize a rabbit, and then antibody clones VEGF-R859, VEGF-R988, VEGF-R613 and VEGF-R812, which specifically bind to human recombinant VEGF165 protein, were prepared by screening a phage display library. The nucleotide sequence encoding the heavy and light chain variable regions of the scFv VEGFR2-MK19 antibody was then introduced by PCR into the pSTEP2 vector containing the nucleotide sequence encoding the rabbit IgG1 constant region or the rabbit kappa constant region and grown for expression. Highly purified antibodies were obtained through protein A column purification. ELISA showed that the rabbit antibody was able to block the binding of VEGF165 protein to VEGFR2 protein, and VEGF-R988 and VEGF-R613 could effectively reduce the ability of VEGF165 to stimulate the proliferation of HUVEC, and VEGF -R988 showed the highest maximum degree of inhibition.

Then, using the classical humanized CDR transplantation method, human antibody light chain or heavy chain variable regions whose sequence is closer to the specified rabbit light or heavy chain variable region sequence, a humanized light chain variable region (VL) and a heavy chain variable region (VL) were selected as a template. chain (VH) in turn by inserting each of the three CDRs (Table 1) of the light chain or heavy chain of a rabbit antibody into the variable regions of said human antibody. Because the key sites of the rabbit antibody framework are required to maintain the stability of CDR activity, the key sites were mutated back to the corresponding rabbit antibody sequence. The light/heavy chain expression vector VEGF-H988-10 was prepared by whole gene synthesis, transfected into HEK-293 cells and cultured for expression, the culture supernatant was purified using a protein A column, resulting in high purity antibodies. To improve the affinity of VEGF-H988-10, SDM libraries of the heavy and light chain variable region CDR regions (including LCDR1, LCDR3, HCDR2 and HCDR3) were constructed, and libraries of the four mutants were constructed in scFv form and cloned into phage vectors as an svFv-gIII fusion protein ; For each CDR, CDR clones with optimal binding ability to soluble VEGF antigen were screened to produce the final antibody VEGF-H988, which had optimized CDR affinity and stability. Nucleic acids of the present invention

The present invention also relates to nucleic acid molecules encoding antibodies or fragments thereof according to the present invention. The sequences of these nucleic acid molecules include, but are not limited to, SEQ ID NOs: 11, 19-20, 23-24, 43-51 and 53-54.

The nucleic acid molecules of the present invention are not limited to the sequences described herein, but also include variants thereof. The variants in the present invention may be described with reference to their physical hybridization properties. One skilled in the art will appreciate that using nucleic acid hybridization techniques, nucleic acids can be used to identify their complements, as well as their equivalents and homologues. It is also understood that hybridization can occur at a complementarity of less than 100%. However, with appropriate selection of conditions, hybridization techniques can be used to distinguish between specified DNA sequences based on structural relevance to the DNA sequence of a particular probe. For guidance on such conditions, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989 and Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons.

Recombinant vectors and expression

The present invention also provides recombinant constructs containing one or more nucleotide sequences of the present invention. Said recombinant construct of the present invention is produced by inserting said nucleic acid molecule encoding said antibody of the present invention into a vector such as a plasmid, phagemid, phage or viral vector.

The antibodies provided herein can be produced by recombinant expression of the nucleotide sequence encoding the light and heavy chains or portions thereof in a host cell. To recombinantly express said antibody, said host cell may be transfected with one or more recombinant expression vectors carrying a nucleotide sequence encoding the light and/or heavy chains or portions thereof, such that said light and heavy chains are expressed in said host cell. Standard recombinant DNA methodology is used to produce nucleic acids encoding heavy and light chains, to incorporate said nucleic acids into recombinant expression vectors, and to introduce said vectors into host cells, for example, Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and described in US Patent No. 4,816,397 Boss et al. .

Suitable host cells include prokaryotic and eukaryotic cells. Examples of prokaryotic host cells are bacteria, and examples of eukaryotic host cells are yeast, insect or mammalian cells. It should be understood that the design of an expression vector, including the choice of regulatory sequence, is determined by a number of factors, such as the choice of host cell, the level of expression of the desired protein, and whether expression is constitutive or inducible.

Bacterial expression

An expression vector for use in bacteria is constructed by inserting a structural DNA sequence encoding the desired antibody along with appropriate translation initiation and termination signals and functional promoters into the actual reading frame. The vector contains one or more phenotypic selection markers and an origin of replication to ensure vector persistence and allow amplification in the host as needed. Suitable prokaryotic hosts for transformation include several species of E. coli, Bacillus subtilis, Salmonella typhimurium, as well as Pseudomonas, Streptomyces and Staphylococcus.

Said bacterial vector may be based on, for example, a phage, plasmid or phagemid. These vectors may contain selection markers and bacterial origins of replication that are derived from commercially available plasmids, which typically contain elements of the well-known cloning vector pBR322 (ATCC 37017). After transforming the appropriate host strain and growing the host strain to the appropriate cell density, the selected promoter is derepressed/induced by an appropriate method (eg, temperature change or chemical induction), and the cells are cultured for some more time. The cells are usually collected by centrifugation, disrupted by physical or chemical methods, and the resulting crude extract is stored for further purification.

In a bacterial system, a variety of expression vectors can be advantageously selected according to the intended use of the expressed protein. For example, when large quantities of such proteins need to be produced for antibody production or for screening a peptide library, for example, a vector may be required that allows high-level expression of the product fusion protein for easy purification.

Expression in mammalian cells and purification

Preferred regulatory sequences for expression in mammalian host cells include viral elements that allow high-level expression of the protein in mammalian cells, such as cytomegalovirus (CMV)-derived promoters and/or enhancers (eg, CMV promoter/enhancer), promoters and/or or simian virus 40 (SV40) enhancers (eg, SV40 promoter/enhancer), adenovirus promoters and/or enhancers (eg, adenovirus major late promoter (AdMLP)) and polyoma virus promoters and/or enhancers. For further description of viral regulatory elements and their sequences, see, for example, US Pat. No. 5,168,062, Stinski, US Pat. No. 4,510,245, Bell et al., and US Pat. No. 4,968,615, Schaffher et al. These recombinant expression vectors may also include a point origin of replication and selection marker (see, for example, US Patents No. 4,399,216, No. 4,634,665 and No. 5,179,017, Axel et al.), suitable selection markers include genes that confer resistance to drugs, such as G418, hygromycin or methotrexate, to cells -hosts into which the specified vector is introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, while the neo gene confers resistance to G418.

Transfection of the expression vector into host cells can be carried out using standard methods such as electroporation, calcium phosphate precipitation and DEAE-dextran transfection.

Suitable mammalian host cells for expression of the antibody provided herein include Chinese hamster ovary cells (CHO cells) [including dhfr-CHO cells described by Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, using the DHFR selection marker as described, for example, in R.J. Kaufinan and R.A. Sharp (1982) Mol. Biol. 159:601-621], NSO myeloma cells, COS cells and SP2 cells.

Antibodies of the present invention can be isolated and purified from recombinant cell cultures by known methods, including ammonium sulfate or ethanol precipitation, acid extraction, Protein A affinity chromatography, Protein G affinity chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interactions, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, but not limited to. High performance liquid chromatography (HPLC) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), for example, chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety.

Characteristics and Functions of the Antibody of the Present Invention The characteristics and functional analysis of the humanized antibody VEGF-H988 according to the present invention were carried out. The analysis showed that the antibody of the present invention has the following advantages: (1) the ability of VEGF-H988 to bind VEGF165 protein is slightly better than Avastin; (2) the binding affinity of VEGF165-H988 to recombinant human VEGF165 protein is slightly higher than that of Avastin, namely approximately 1.5 times that of Avastin; (3) VEGF165-H988 specifically binds to recombinant human VEGF165 protein and cross-links to recombinant mouse mVEGF164 protein; (4) VEGF-H988 antibody can effectively inhibit the binding of VEGFR2 protein to VEGF165 protein, and its inhibitory ability is weaker than EYLEA but better than Avastin; (5) VEGF-H988 antibody can effectively reduce the ability of VEGF165 to initiate HUVEC proliferation; (6) the VEGF-H988 antibody has a stronger neutralizing effect than Avastin in the case of different VEGF isoforms (VEGF165, VEGFC, VEGFD), acting simultaneously on HUVEC cells; (7) a multiple-drug toxicity study in mice showed that no significant drug-related toxicity reactions were detected with the VEGF-H988 antibody; and (8) a tumor suppression test in the HCT-116 xenograft tumor model showed that VEGF-H988 had a better tumor suppression effect than Avastin.

Applications

The antibodies of the present invention can be used to treat colorectal cancer. The antibody of the present invention can also be used for the production of drugs for the treatment of these diseases.

Pharmaceutical compositions

Antibodies of the present invention can be prepared with at least one other agent (eg, a stable compound) to form a pharmaceutical composition comprising an antibody of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients. Said pharmaceutical composition may optionally contain additional therapeutic agents.

Sets

The present invention also relates to a pharmaceutical kit and a kit containing one or more containers, wherein said containers contain the above pharmaceutical compositions according to the present invention. Such containers may be accompanied by specifications in a form prescribed by the government agency regulating the manufacture, use, or distribution of the drug or biological product, which reflect the approval of its administration to humans by the specified agency of the country in which the product is manufactured, used, or distributed.

Receipt and storage

Said pharmaceutical composition according to the present invention can be prepared by a method known in the art, for example, by conventional methods of mixing, dissolving, granulating, lozenges, grinding, emulsifying, encapsulating, pouring or lyophilizing.

Once prepared, pharmaceutical compositions containing the compounds of the present invention, formulated in a dosage form in a suitable carrier, can be placed in appropriate containers and labeled for the treatment of the specified condition. Such labeling should include the amount, frequency, and route of administration of the drug.

Combinations

Said pharmaceutical composition containing the antibodies of the present invention described above may also be combined with one or more other therapeutic agents, such as antineoplastic agents, as long as the resulting combination does not cause unacceptable side effects.

The following examples provide a better understanding of the present invention, but are not intended to limit the scope of the present invention. All experimental methods in the following examples, unless otherwise noted, are conventional methods. Experimental materials used in the following examples, unless otherwise noted, were purchased from conventional biochemical reagent distributors.

EXAMPLES

Example 1: Screening for rabbit antibodies that block VEGF165 binding to VEGFR1/VEGFR2 using a phage display antibody library

1.1 Immunization of rabbits

Recombinant human VEGF165 protein (from Sino Biological, Inc, cat. no. 11066-HNAH) was used to immunize rabbits. The amino acid sequence of the extracellular Met1-Arg191 region of the human VEGF165 protein (UniProt P15692-4) is given in SEQ ID NO: 1.

A detailed description of the method is as follows: recombinant human VEGF165 protein was mixed with Freund's adjuvant, rabbits were immunized subcutaneously with the mixture 4 times at intervals of 3 weeks, 2 weeks and 2 weeks, respectively, at a dosage of 500 μg each time. After the fourth immunization, blood was collected 4 days after immunization through the medial canthal plexus of the eye. Anti-VEGF165 antibody titer in rabbit serum was measured by ELISA using recombinant human VEGF165 protein coating. The serum titer after the fourth immunization reached 1:250,000, and rabbits were boosted intravenously with 25 μg of recombinant human VEGF165 protein 9 weeks after the fourth immunization. After 7 days, the rabbits were sacrificed and the spleen tissue was removed and frozen in liquid nitrogen.

1.2 Antibody phage display library screening

RNA was extracted from rabbit spleen tissue using TriPure Isolation Reagent (from Roche, cat. no. 11 667 165 001), and cDNA was obtained by reverse transcription of RNA using a reverse transcription kit (from Invitrogen cat. no. 18080-051). 10 pairs of primers were designed to amplify the sequence of the rabbit antibody light chain variable region and 4 pairs of primers to amplify the sequence of the heavy chain variable region (Barbas C F et al., CSHL Press. 2004). These sequences encoding the variable regions of the light and heavy chains of the rabbit antibody were combined with the nucleotide sequence encoding the scFv using overlapping extension PCR, the variable regions of the light and heavy chains were joined (Jones S T et al., Bio/technology. 1991; 9 (1): 88) using the following linker:

TCTAGTGGTGGCGGTGGTTCGGGCGGTGGTGGAGGTGGTAGTTCTAGATCTTCC (SSGGGGSGGGGGGSSRSS) (SEQ ID NO: 2);

then ligated into the phage vector pColb3x (from Sino Biological, Inc.) using Sfi I restriction endonuclease (from Fermentas), and electrotransformed into competent X-Blue cells to produce a rabbit scFv antibody phage display library. Recombinant human VEGF165 protein was coated on an ELISA plate, and a phage library enriched with positive anti-VEGF165 antibodies was screened according to the specified phage antibody sorting method (O'Brien, PM, & Aitken, R. (Eds.), Springer Science & Business Media. 2002; ISBN: 9780896037113). Single phage colonies were selected for expression from the enriched library, and their binding to recombinant human VEGF165 protein was determined using ELISA. ScFv antibody clones that specifically bind to recombinant human VEGF165 were selected and submitted to a sequencing company, resulting in antibody nucleotide sequences, with several scFv antibody clones yielding derivatives VEGF-R859, VEGF-R988, VEGF- R613, VEGF-R812 by the method described in Example 1.3. The nucleotide sequences of the scFV clone antibodies are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

1.3 Preparation of rabbit antibodies that bind to VEGF165

10 Using VEGF-R988 as an example, the indicated nucleotide sequence encoding the heavy chain variable region of the scFv antibody VEGF-R988 was amplified by PCR and inserted into the Sea I + Kpn I cut vector pSTEP2 (Fermentas), carrying the nucleotide sequence encoding the signal heavy chain peptide (SEQ ID NO: 45) and constant

15 region of rabbit IgGl (SEQ ID NO: 9), by fusion to obtain a heavy chain expression vector (SEQ ID NO: 53). The indicated nucleotide sequence encoding the variable region of the scFv light chain of the VEGF-R988 antibody was amplified by PCR and inserted into the pSTEP2 vector, cut by Sea I + BathH I (Fermentas), carrying the nucleotide sequence

20 encoding a light chain signal peptide (SEQ ID NO: 46) and a rabbit kappa constant region (SEQ ID NO: 10) by fusion to produce a light chain expression vector (SEQ ID NO: 54). These recombinant plasmids were extracted, transfected into HEK-293 cells and grown for expression for 7 days; the culture supernatant was purified on a protein A 25 column, resulting in highly purified antibodies.

Primers for amplification of the heavy chain variable region:

Primers for amplification of the light chain variable region:

1.4 Functional analysis of rabbit antibodies binding to VEGF165

1.4.1 Rabbit antibody blocks VEGF165 binding to VEGFR2-his

VEGF165 protein (from SinoBiological, Inc.) at a concentration of 1 μg/ml was coated on a 96-well plate at 100 μl per well overnight at 4°C. The plate was washed the next day and blocked at room temperature for 1 hour. 100 μl of a solution of 5 μg/ml VEGFR2 protein (from SinoBiological, Inc.) and the indicated VEGF165 binding antibody at various concentrations were added and co-incubated. The plate was washed to remove unbound antibodies, incubated with streptavidin/HRP (from Beijing ZSGB-Bio Co., Ltd.), and then washed several times, and the chromogenic substrate solution was added to develop color. OD ₄₅₀ was measured after the color appeared. Taking the concentration of rabbit antibody binding to VEGF165 as the horizontal coordinate and the degree of inhibition P1% as the vertical coordinate, graphPad Prism 6.0 software was used to analyze the data and a curve plot was plotted. Inhibition rate (%) = (OD _control - OD _sample )/OD _control × 100%, where OD _control denotes the OD value of the wells spiked with VEGFR2-biotin only but without the rabbit antibody, and OD _sample denotes the OD value of the wells spiked with VEGFR2 -biotin and rabbit antibody.

As can be seen in FIG. 1, VEGFR2 protein can effectively bind to the VEGF165 protein coat, and rabbit antibodies VEGF-R859, VEGF-R988, VEGF-R613, VEGF-R812 can effectively inhibit the binding of VEGFR165 protein to VEGFR2 protein.

Antibody inhibits the proliferation effect of VEGF165 on umbilical vein endothelial cells

1.4.2 Rabbit antibody inhibits HUVEC proliferation

The effect of an antibody neutralizing the proliferation of umbilical vein endothelial cells induced by VEGF165 was determined using the WST-8 method. Human umbilical vein endothelial cells HUVEC were inoculated into a 96-well plate at 4 x 10 ³ cells per well, grown in M199 medium containing 10% FBS and 5% L-Gln for 4 hours, and then various concentrations of rabbit antibodies were added to amount of 50 μl per well, then VEGF-165 at a final concentration of 10 ng/ml was added in an amount of 10 μl per well, the 96-well plate was incubated at 37°C, 5% CO ₂ in a cell incubator for 3 days, and an empty well B (no cells), negative control M (cells inoculated, no antibody sample, VEGF-165 added) and M' (cells inoculated, no antibody sample and no VEGF-165). After incubation, 10 μl per well of WST-8 chromogenic solution was added and the said 96-well plate was incubated in CO ₂ incubator until color appeared, OD ₄₅₀ and OD ₆₃₀ were measured using a microplate reader after color appearance stabilized. For each well, the read value was (OD ₄₅₀ - OD ₆₃₀ ), and the neutralization rate for the indicated antibody, calculated as the OD value for each group, was determined as the group read value minus the blank well B read value, % neutralization rate = (negative control OD value M - sample OD value) / (negative control OD value M - OD value M') × 100%. A standard curve was calculated using the automatic analysis function of GraphPad Prism statistical software, selecting the sample concentration of the specified antibody as the horizontal coordinate and the degree of neutralization as the vertical coordinate, and a four-parameter logistic regression equation was used to fit the standard "S" curve to calculate half maximum effective concentration (EC50) of a sample of the specified antibody.

The results shown in Fig. 2 demonstrate that VEGF-R988 and VEGF-R613 effectively reduce the ability of VEGF165 to initiate HUVEC proliferation, with VEGF-R988 demonstrating the greatest degree of inhibition, VEGF-R812 having essentially no inhibitory ability, while VEGF-R859 has no inhibitory ability at all. inhibitory ability.

Example 2: Humanization, modification and production of rabbit antibody VEGF-R988

Based on the results of the functional rabbit antibody from Example 1, the VEGF-R988 antibody was selected for humanization and production.

2.1 Determination of the CDR sequences of the light and heavy chains of the rabbit antibody VEGF-R988

Based on the specified nucleotide sequence of the VEGF-R988 scFv antibody determined in Example 1.2, the amino acid sequence of the variable regions of the heavy chain and light chain of the VEGF-R988 scFv, see SEQ ID NO: 11/12, was obtained.

Taking into account the Kabat index and the IMGT numbering system, the amino acid sequence of each three CDRs of the light and heavy chains of the rabbit antibody VEGF-R988-scFv was determined, see Table 1. The above corresponding three CDRs of the indicated light chain and heavy chain were transferred into the humanized VEGF-R988-scFv antibody. R988-scFv in subsequent stages, see Example 2.2.

2.2 Transplantation of rabbit antibody CDR VEGF-R988

Humanization of the rabbit antibody was carried out using the classical method of humanization by CDR transplantation. The human light or heavy chain variable regions of the antibody whose sequences are most similar to the indicated rabbit light or heavy chain variable region sequences were selected as a template, and each of the three rabbit light or heavy chain CDRs (Table 1) was inserted into the indicated antibody variable regions human, resulting in humanized light chain variable region (VL) or heavy chain variable region (VH) sequences, respectively. The human template for the light chain variable region of VEGF-R988 is IGKV1-27*01, which is 65.30% homologous to the light chain of VEGF-R988, and the human template for the heavy chain variable region is IGHV4-4*08, which is 53.20% homologous % homologous to the heavy chain of VEGF-R988.

2.3 Back mutations in the humanized variable region framework

Because some key amino acids in the rabbit framework region are required to maintain CDR activity, key amino acids were backmutated into the corresponding rabbit antibody amino acid sequences, the following sites were backmutated: in the light chain, position 1 was backmutated to E, position 2 was backmutated to L, position 4 was backmutated to L, and position 63 was backmutated to K; while in the heavy chain, position 3 was back-mutated to V, position 37 was back-mutated to V, position 47 was back-mutated to Y, position 78 was back-mutated to V, position 79 was back-mutated to D, and position 91 was backmutated to F; all of the above sites were numbered according to the Kabat numbering scheme. The humanized antibody VEGF-H988-10 was produced by humanized CDR grafting and backbone mutations.

2.4 Production of humanized monoclonal antibody VEGF-H988-10 and affinity modification of CDR

The heavy chain variable region of VEGF-H988-10 (SEQ ID NO: 23) was obtained by complete gene synthesis and then introduced by infusion into the pre-cut Sea I + Nhe I (Fermentas) vector pSTEP2 vector carrying the nucleotide sequence encoding the signal peptide of the heavy chain chain (SEQ ID NO: 45) and the nucleotide sequence encoding the human IgG1 constant region (SEQ ID NO: 49), resulting in the heavy chain expression vector VEGF-H988-10 (SEQ ID NO: 19). The light chain variable region of VEGF-H988-10 (SEQ ID NO: 24) was obtained by complete gene synthesis and then introduced by infusion into the pre-cut SeaI + BsiW I (Fermentas) vector pSTEP2 vector carrying the nucleotide sequence encoding the light chain signal peptide (SEQ ID NO: 46) and the nucleotide sequence encoding the human kappa constant region (SEQ ID NO: 50), resulting in (SEQ ID NO: 20) the light chain expression vector VEGF-H988-10. Plasmids were extracted and co-transfected into HEK-293 cells, which were grown for 7 days. The culture supernatant was purified on a protein A column, resulting in highly purified antibodies.

Primers for the synthesis of the complete heavy chain variable region gene

Primers for the synthesis of the complete light chain variable region gene

To improve the affinity of VEGF-H988-10, SDM libraries of the heavy and light chain variable region CDR regions were constructed (including three libraries enriched with mutations LCDR1, LCDR3, and HCDR2); Meanwhile, to improve the chemical stability of the antibody, amino acid residues capable of undergoing deamidation or isomerization must be modified with other amino acid residues. Deamination of asparagine may occur, such as NG, NS, NA, NT, etc., resulting in the formation of isoaspartic acid residues affecting the stability or biological function of the antibody. The HCDR3 variable region of VEGF-H988 contains region(s) susceptible to deamidation, so SDM libraries were constructed to improve the chemical stability and biological function of the antibody. The above four mutant libraries were constructed in scFv form and cloned into a phage vector as a scFv-gIII fusion protein; for each CDR, CDR clones having optimal binding ability to the soluble VEGF antigen were screened, and finally the indicated VEGF-H988 antibody containing the optimized affinity and stability of the CDR was obtained. The indicated VEGF-H988 light and heavy chain CDR sequences are shown in Table 2.

2.5 Preparation of humanized monoclonal antibody VEGF-H988

The nucleotide sequence (SEQ ID NO: 44) encoding the light chain and signal peptide of the above antibody VEGF-H988, which in the following order contains the nucleotide sequence encoding the light chain signal peptide (SEQ ID NO: 46), the light chain variable region of the humanized antibody (SEQ ID NO: 48) and the human antibody light chain kappa constant region (SEQ ID NO: 50) were amplified by PCR and inserted into the self-developed vector pGS (Kpn I + Xba I) by infusion, and the correct sequence of the plasmid was verified by sequencing. A nucleotide sequence (SEQ ID NO: 43) encoding the heavy chain and signal peptide of the above antibody VEGF-H988, which in the following order contains a nucleotide sequence encoding the heavy chain signal peptide (SEQ ID NO: 45), the heavy chain variable region of the humanized antibody (SEQ ID NO: 47) and the human antibody heavy chain kappa constant region (SEQ ID NO: 49) were amplified by PCR and infused into the pGS (Nhe I + Not I) vector, which contains a light chain whose sequence is correct was verified and the correct sequences of the vectors expressing both the light and heavy chains of VEGF-R988 were verified by sequencing. These expression vectors are eukaryotic expression vectors containing GS genes as selection markers and light and heavy chain expression elements of the antibody. These expression vectors were transfected into CHO-K1-GS-deficient cells and cell lines with high VEGF-H988 expression were obtained from the MSX screen. Clones with high antibody expression were selected by ELISA, and these high expression cell lines were selected taking into account both cell growth status and key quality characteristics for the antibody drug product. Serum-free suspension cultures were used to grow the VEGF-H988-producing CHO cell line, resulting in VEGF-H988 antibodies of high purity and quality.

Example 3: Characterization Analysis of Humanized Antibody VEGF-H988

3.1 Characteristic analysis of humanized antibody VEGF-H988 binding to VEGF165

3.1.1 Humanized antibody VEGF-H988 specifically binds to VEGF165

Recombinant human VEGF165 protein (from SinoBiological, Inc.) at various concentrations (0.15 ng/ml, 0.46 ng/ml, 1.37 ng/ml, 4.12 ng/ml, 12.35 ng/ml, 37.04 ng/ml, 111.11 ng/ml, 333.33 ng/ml, 1000 ng/ml and 3000 ng/ml) were applied to a 96-well plate overnight at 4°C in an amount of 100 μl per well. . The plate was washed the next day and blocked at room temperature for 1 hour. After incubation with 100 μl of 1 mg/ml VEGF165-H988, Avastin (from Roche) or H7N9-R1 antibody as a negative control, respectively, the plate was washed to remove unbound antibodies, then incubated with goat anti-IgG F(ab')2 F (ab')2/HRP (from Jackson ImmunoResearch Laboratories, Inc.) and washed several times, and then chromogenic substrate solution was added until color appeared. OD ₄₅₀ was measured after color formation was complete. Using the concentration of the recombinant human VEGF165 protein as the horizontal coordinate and the OD ₄₅₀ value as the vertical coordinate, graphPad Prism 6.0 software was used to generate an “S” plot curve and the binding of the antibody to the recombinant human VEGF165 protein was analyzed.

The results shown in Fig. 3 show the EC ₅₀ value of the humanized VEGF165-H988 molecule specifically binding to recombinant human VEGF165 is 2.42 ng/ml, R ² =0.999; the EC ₅₀ value of Avastin binding to recombinant human VEGF165 is 2.77 ng/ml, R ² =1.000. This indicates that the ability of VEGF165-H988 to bind to recombinant human VEGF165 protein is slightly better than Avastin. Antibody H7N9-R1, used as a negative control, does not bind to recombinant human VEGF165 protein.

3.1.2 Binding affinity test of humanized antibody VEGF-H988 with recombinant VEGF165 protein

The affinities of VEGF165-H988 and Avastin (from Roche) were measured at various concentrations using a streptavidin-coated sensor and immobilized biotin-labeled VEGF165 protein.

First, recombinant human VEGF165 protein was labeled with biotin in a 1:2 molar ratio through the following process: buffer (20 mM Tris, 150 mM NaCl, pH 8.0) of recombinant VEGF protein was replaced with PBS by ultrafiltration in a 5000 MW ultrafiltration tube to obtain 567 .57 μg of protein measured by UV quantification, the resulting proteins were mixed with 20 mM biotin solution in a 1:2 molar ratio for incubation for 30 min at room temperature in the dark, then filtered again in an ultrafiltration tube by centrifugation at 5000 MW for removal of unbound biotin. After quantification by UV, biotin-labeled proteins were prepared by adding equal volumes of glycerol and BSA to a final concentration of 0.1%. The VEGF165 protein concentration determined by UV was 2.08 mg/ml.

The affinities of VEGF165-H988 and Avastin at various concentrations with biotinylated recombinant human VEGF proteins were then measured, and the resulting K _D values indicated the final affinities.

The results shown in Table 3 demonstrate that the binding affinity value _{KD of} VEGF165-H988 to recombinant human VEGF165 protein was 19.5 pM, the binding constant value k _on was 3.44E + 05 M ^-1 s ^-1 , and the value of the constant dissociation k _dis was 6.70E-06 s ^-1 ; the binding affinity value _KD of Avastin to VEGF protein was 29.2E-11 pM, the binding constant value kop was 1.87E+05 M ^-1 s ^-1 and the dissociation constant value _kdis was 5.46E-06 s ^-1 as shown in Table 3. Based on these results, it can be concluded that the affinity of VEGF165-H988 is slightly higher than that of Avastin, i.e. approximately 1.5 times higher affinity than Avastin.

3.1.3 Determination of species-specific cross-reactivity of the humanized antibody VEGF165-H988

Recombinant human VEGF165 protein or recombinant mouse mVEGF164 protein was diluted to 0.1 μg/ml, 1 μg/ml, and 10 μg/ml, respectively, and applied to a 96-well plate overnight at 4°C at 100 μl per well. . The plate was washed the next day, blocked at room temperature for 1 h. 100 μl of VEGF165-H988, Avastin (from Roche) or the H7N9-R1 negative control antibody were added at a concentration of 1 μg/ml, respectively, and incubated for 1 h. The plate was washed to remove unbound antibodies. The plate was incubated with goat anti-human IgG F(ab')2/HRP (Jackson ImmunoResearch Laboratories, Inc.) and then washed several times and chromogenic substrate solution added until color appeared. After color formation was complete, OD ₄₅₀ was measured. Selecting protein concentration as the horizontal coordinate and OD ₄₅₀ value as the vertical coordinate, graphPad Prism 6.0 software was used to plot the histogram.

The results shown in Fig. 4 demonstrate that VEGF165-H988 specifically binds to recombinant human VEGF165 protein and cross-links to recombinant mouse mVEGF164 protein.

3.2 Receptor blocking properties of the humanized antibody VEGF-H988

VEGF165 protein (from SinoBiological, Inc.) at a concentration of 1 μg/ml was applied to a 96-well plate in an amount of 100 μl/well overnight at 4°C. The next day, the plate was washed and blocked at room temperature for 1 hour 100 μl of 2 μg/ml VEGFR2-his protein (from SinoBiological, Inc.), various concentrations of VEGF-H988 antibody, EYLEA, Avastin (from Roche) or H7N9-R1 antibody as a negative control were added to each well, respectively, and were incubated together. The plate was washed to remove unbound antibodies. The plate was incubated with C-his-R023/HRP and then washed several times, and a chromogenic substrate solution was added until color appeared. For each group studied, OD ₄₅₀ was measured in duplicate after color appearance had stabilized. Using the concentration of the indicated antibody as the horizontal coordinate and the degree of inhibition P1% as the vertical coordinate, graphPad Prism 6.0 software was used to analyze the data and a curve graph was plotted to calculate the IC ₅₀ value.

Inhibition rate (%) = (OD _control - OD _sample )/OD _control × 100%, where OD _control denotes the OD value of the well with only added VEGFR2-his, but without the addition of humanized antibody, and OD _sample denotes the OD value of the well as with the added VEGFR2-his and with a humanized antibody.

As seen in Fit.5, the VEGFR2 protein can effectively bind to the VEGF165 protein on the support, and the VEGF-H988 antibody can effectively inhibit the binding of the VEGFR2 protein to the VEGFR165 protein, with a slightly weaker inhibitory ability than EYLEA, but stronger than Avastin, and the antibody used as a negative control has no inhibitory effect.

3.3 Inhibition of HUVEC cell proliferation using a humanized antibody

3.3.1 Neutralizing effect of VEGF165 at different concentrations of humanized antibody VEGF-H988

The effect of VEGF-H988 in neutralizing VEGF165-induced HUVEC cell proliferation was determined using the WST-8 method. HUVEC cells were inoculated into a 96-well plate at 4 x 10 ³ cells per well, grown in Ml99 medium containing 10% FBS and 5% L-Gln for 4 h, and then supplemented with VEGF-H988, EYLEA or Avastin (from Roche) at various concentrations in an amount of 50 μl per well, then VEGF-165 was added at final concentrations of 1000 ng/ml, 100 ng/ml or 10 ng/ml in an amount of 10 μl per well, the indicated 96-well plate was incubated at 37 ° C, 5% CO ₂ in a cell incubator for 3 days, and used control well B (without cells), negative control M (with added cells, without adding antibody sample, with added VEGF-165) and M' (with added cells , without adding antibody sample and without adding VEGF-165). After incubation, 10 μl per well of WST-8 chromogenic solution was added and the said 96-well plate was incubated in a CO ₂ incubator until color appeared, OD ₄₅₀ and OD ₆₃₀ were measured using a microplate reader after color appearance stabilized. For each well, the value (OD ₄₅₀ - OD ₆₃₀ ) was read, and the degree of neutralization of the indicated antibody was calculated as the OD value for each group, defined as the reading value of the indicated group minus the reading value of the blank well B, the degree of neutralization, % = (OD value of the negative control M - sample OD value) / (negative control OD value M - OD value M') × 100%. A standard curve was calculated using the automatic analysis function of GraphPad Prism statistical software, using the sample concentration of the specified antibody as the horizontal coordinate and the degree of neutralization as the vertical coordinate, and used a 4-parameter logistic regression equation to construct a standard "S" curve to calculate half maximum effective concentration (EC ₅₀ ) of a sample of said antibody.

As can be seen in FIG. 6 and 7, VEGF-H988 antibody can effectively reduce the ability of VEGF165 to initiate HUVEC proliferation. The neutralizing ability of VEGF-H988 was stronger than that of EYLEA and Avastin at various concentrations of recombinant human VEGF165; and differences in neutralizing capacity were greater with increasing concentrations of VEGF165. At high concentrations of VEGF165, VEGF-H988 still had a low neutralizing EC ₅₀ and had a maximum neutralizing level, while the neutralizing EC ₅₀ of EYLEA and Avastin gradually increased and was accompanied by a decrease in the maximum neutralizing level. The half maximum effective concentrations (EC ₅₀ ) and maximum neutralization levels of each VEGF165 neutralizing antibody at various concentrations are shown in Table 4.

3.3.2 Neutralizing effect of different VEGF subtypes using humanized antibody VEGF-H988

The WST-8 method was used to detect the effect of VEGF-H988 in neutralizing HUVEC cell proliferation caused by different VEGF subtypes (VEGF165, VEGFC and VEGFD). HUVEC cells were inoculated into a 96-well plate at 4 x 10 ³ cells per well, grown in M199 medium containing 10% FBS and 5% L-Gln for 4 h, and then added various concentrations of VEGF-H988 or Avastin (from Roche) in an amount of 50 μl per well, then a mixture of VEGF-165, VEGFC and VEGFD (final concentrations were 25 ng/ml, 1000 ng/ml, 6000 ng/ml, respectively) was added in an amount of 10 μl per well, indicated 96-well The plate was incubated at 37°C, 5% CO ₂ in a cell incubator for 3 days, and control well B (no cells), negative control M (with added cells, without added antibody sample, with added VEGF) and M'( with added cells, without added antibody sample and without VEGF mixture). After incubation, 10 μl per well of WST-8 chromogenic solution was added and the said 96-well plate was incubated in a CO ₂ incubator until color appeared, OD ₄₅₀ and OD ₆₃₀ were measured using a microplate reader after color appearance stabilized. For each well, the read value was (OD450 - OD ₆₃₀ ), and the degree of neutralization of the indicated antibody was calculated as the OD value for each group, defined as the read value for the indicated group minus the read value for blank well B, neutralization rate % = (OD value of the negative control M - sample OD value) / (negative control OD value M - OD value for M') × 100%. A standard curve was calculated using the automatic analysis function of GraphPad Prism statistical software, using the sample concentration of the specified antibody as the horizontal coordinate and the degree of neutralization as the vertical coordinate, and used a 4-parameter logistic regression equation to construct a standard "S" curve to calculate half maximum effective concentration (EC ₅₀ ) of a sample of said antibody.

As seen in Fit.8, in the case of different VEGF subtypes (VEGF165, VEGFC, VEGFD) acting simultaneously on HUVEC cells, the VEGF-H988 antibody has a stronger neutralizing effect than Avastin. In the concentration range of 0.016-4,000 nM, VEGF-H988 has a lower EC ₅₀ than Avastin, equal to 0.26 nM and 0.77 nM, respectively; and VEGF-H988 has a higher neutralization rate than Avastin, equal to 86.1% and 58.5%, respectively. The half maximum effective concentrations ( _EC50 ) and maximum neutralization levels of VEGF and Avastin for neutralization of various VEGF subtypes are given in Table 5.

Example 4: Multiple Dose Toxicity Study of VEGF-H988 Antibody in Mice

CD-1 mice were used, 8 animals in each group: aged group (35 weeks) and commonly used age group (9 weeks), half male and half female. Mice were divided into four groups (G1-G4) according to the complete randomization method, four mice in each group, half male and half female. The specific grouping was as follows: G1: aged mice group, vehicle control group; G2: experimental group of aged mice; G3: vehicle control group of normal aged mice; G4: experimental group of mice of normal age. The VEGF-H988 antibody was administered several times in the experimental groups, and the control groups received an equal volume of diluent. The dosage was 50 mg/kg, the volume of each injection was 5 ml/kg (from the 1st to the 8th dosage) and 10 ml/kg (from the 9th to the 16th dosage), respectively; method of administration - intraperitoneal (i.p.) administration, twice a week. Orbital blood samples were taken from all mice before the first injection and 0.5 hours, 2 hours, 4 hours, 6 hours, 24 hours, 48 hours, 72 hours after the first injection, as well as before the first injection and 1 hour after every second administration for pharmacokinetic and immunogenic assays. The body weight of the mice was measured and recorded before each dosage. The detailed dosage regimen is shown in Table 6.

The body weight of mice in the VEGF-H988 antibody groups was similar to the body weight of mice in the corresponding vehicle control groups, indicating that the VEGF-H988 antibody did not affect the body weight of the mice. During the study, clinical abnormalities were observed in the VEGF-H988 antibody groups. All mice were subjected to macroscopic necropsy, and no visible tissue or organ abnormalities were found. Histopathological examination showed that all mice had venous congestion in the liver, lungs and kidneys, but there was no significant difference between the control and study groups. In rare mice, pink serous or erythrocyte congestion in the spleen and inflammatory cell infiltration in the kidneys were observed, which was considered an individual difference and was not associated with the administration of the drug. The toxicokinetic parameters of mice after the first dose are summarized in Table 7.

As shown in the blood drug concentration versus time curve in FIG. 9-11, there was no significant difference in the trend of blood concentrations between male and female animals in each group. There was no significant change after repeated dosing in either group, which may be due to saturation levels being reached in the higher dose groups. Peak blood concentrations in the G2 (aged) group and in the G4 (regular-aged) group were essentially the same, and trough concentrations in the age group were slightly lower, which may be due to large inter-individual differences within the group.

Mice in each group did not show a significant increase or even a slight decrease in drug concentrations in the blood 1 hour after administration, which did not correlate well with immunogenicity (see Table 8 for immunogenicity test data). In addition, abnormal drug concentrations in the blood 1 hour after administration may also be related to the rate of drug distribution in vivo and individual characteristics.

Although the immunogenicity test results showed positive results in a subset of subjects, the difference between the SNR and the SCP cutoff value was small, in fact they are very close, thus a strong positive result is not shown and it is believed that the above positive results were likely false positives. Table 8 summarizes the immunogenicity results of the repeated dosing toxicity study of the VEGF-H988 antibody in mice.

A repeated dose toxicity study in mice showed that no significant drug-related toxic effects were observed when the VEGF-H988 antibody was administered multiple times at 50 mg/kg twice weekly by intraperitoneal injection to aged CD-1 mice. and CD-1 mice of normal age in the amount of 16 doses.

Example 5: Efficacy Study in Humans in a Xenograft Model of Colon and Rectal Cancer with the HCT-116 Cell Line

Female Balb/c-nu nude mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (animal use license No. SCXK (Beijing) 2016-0006, quality certificate No. 11400700373019). HCT116 tumor samples were obtained from mice inoculated with a proprietary HCT116 cell line. Female Balb/c-nu nude mice aged 6 weeks were inoculated subcutaneously with an HCT116 tumor measuring 2×2×2 ^mm3 , and when the tumor volume reached approximately 300 ^mm3 , the mice were injected intraperitoneally in groups with 1 mg/kg VEGF-H988 or Avastin (from SinoCelltech Co., Ltd.) or an appropriate solvent, respectively, twice a week. Tumor volumes were measured weekly to assess the antitumor efficacy of VEGF-H988 compared with Avastin.

As can be seen in FIG. 12, in the case of a larger initial tumor volume of approximately 300 mm ³ and a lower dose (1 mg/kg), the tumor growth rate gradually decreased in the VEGF-H988-treated group compared with the no-dose control group starting from the 7th days after administration. The inhibitory effect of Avastin on tumor growth began on day 11 after administration, later than in the group receiving an equivalent dose of VEGF-H988. Tumor inhibition studies in HCT-116 tumor xenograft models showed that the inhibitory effect of VEGF-H988 on tumor growth was better than Avastin.

Claims (24)

1. An isolated anti-VEGF antibody or antigen-binding fragment thereof, comprising a heavy chain variable region, comprising a heavy chain CDR1 region containing the amino acid sequence specified in SEQ ID NO: 30, and a heavy chain CDR2 region containing the amino acid sequence specified in SEQ ID NO: 31, and the heavy chain CDR3 region containing the amino acid sequence shown in SEQ ID NO: 32; and a light chain variable region comprising a light chain CDR1 region containing the amino acid sequence set forth in SEQ ID NO: 27, a light chain CDR2 region containing the amino acid sequence set forth in SEQ ID NO: 28, and a light chain CDR3 region containing the amino acid sequence specified in SEQ ID NO: 29, wherein said antigen-binding fragment is not a Fab fragment. 2. An anti-VEGF antibody or an antigen-binding fragment thereof according to claim 1, containing a heavy chain variable region containing the amino acid sequence specified in SEQ ID NO: 39, or an amino acid sequence identical to at least 90%, 92%, 95 %, 98% or 99% of the sequence specified in SEQ ID NO: 39, and a light chain variable region containing the amino acid sequence specified in SEQ ID NO: 40, or an amino acid sequence identical to at least 90%, 92%, 95%, 98%, or 99% of the sequence shown in SEQ ID NO: 40. 3. An anti-VEGF antibody or an antigen-binding fragment thereof according to claim 1 or 2, characterized in that said antibody further comprises a light chain constant region and a heavy chain constant region, preferably said light chain constant region contains the amino acid sequence specified in SEQ ID NO: 42, or an amino acid sequence that is at least 90%, 92%, 95%, 98% or 99% identical to the sequence specified in SEQ ID NO: 42, and/or the specified heavy chain constant region is a heavy chain constant region IgG1 and has the amino acid sequence set forth in SEQ ID NO:41, or an amino acid sequence that is at least 90%, 92%, 95%, 98%, or 99% identical to the sequence set forth in SEQ ID NO:41. 4. An anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-3, characterized in that said anti-VEGF antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody. 5. An anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-4, characterized in that said anti-VEGF antibody or antigen-binding fragment thereof is a monoclonal antibody. 6. An anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-5, characterized in that said anti-VEGF antibody or antigen-binding fragment thereof is an IgG antibody, preferably an IgG1 antibody. 7. An anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-6, characterized in that said anti-VEGF antibody or antigen-binding fragment thereof has a binding affinity KD of 1-100 pM, preferably 5-50 pM, more preferably 19.5 pM to recombinant human VEGF165 protein. 8. An anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-7, characterized in that said antigen-binding fragment is a fragment of Fv, Fab', Fab'-SH, F(ab')2, Fd, Fd', a single-chain antibody molecule or a single-domain antibody; wherein said single chain antibody molecule is preferably a scFV, di-scFV, tri-scFv, diabody or scFab antibody. 9. An isolated anti-VEGF antibody or antigen-binding fragment thereof, comprising a heavy chain variable region, comprising a heavy chain CDR1 region containing the amino acid sequence specified in SEQ ID NO: 16, a heavy chain CDR2 region containing the amino acid sequence specified in SEQ ID B NO: 17, and the heavy chain CDR3 region containing the amino acid sequence shown in SEQ ID NO: 18; and a light chain variable region comprising a light chain CDR1 region containing the amino acid sequence specified in SEQ ID NO: 13, a light chain CDR2 region containing the amino acid sequence specified in SEQ ID NO: 14, and a light chain CDR3 region containing the amino acid sequence , specified in SEQ ID NO: 15. 10. The anti-VEGF antibody or antigen-binding fragment thereof according to claim 9, containing a heavy chain variable region containing the amino acid sequence specified in SEQ ID NO: 25, or an amino acid sequence identical to at least 90%, 92%, 95 %, 98% or 99% of the sequence specified in SEQ ID NO: 25, and a light chain variable region containing the amino acid sequence specified in SEQ ID NO: 26, or an amino acid sequence identical to at least 90%, 92%, 95%, 98% or 99% of the sequence shown in SEQ ID NO: 26. 11. An anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 9, 10, characterized in that said anti-VEGF antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody. 12. An anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 9-11, characterized in that said anti-VEGF antibody or antigen-binding fragment thereof is a monoclonal antibody. 13. An anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 9-12, characterized in that the specified antigen-binding fragment is a fragment of Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fd', a single-chain antibody molecule or a single-domain antibody; wherein said single chain antibody molecule is preferably a scFV, di-scFv, tri-scFv, diabody or scFab. 14. Nucleic acid encoding an anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-13. 15. Nucleic acid according to claim 14, containing the nucleotide sequence specified in SEQ ID NO: 7, and/or the nucleotide sequence specified in SEQ ID NO: 8; or the nucleotide sequence specified in SEQ ID NO: 23 and/or the nucleotide sequence specified in SEQ ID NO: 24; or the nucleotide sequence set forth in SEQ ID NO: 47 and/or the nucleotide sequence set forth in SEQ ID NO: 48. 16. An expression vector containing the nucleic acid according to claim 14 or 15. 17. A host cell for producing an anti-VEGF antibody or an antigen-binding fragment thereof according to any one of claims. 1-13, containing the nucleic acid according to claim 14 or 15 or the expression vector according to claim 16. 18. A method for producing an antibody against VEGF or an antigen-binding fragment thereof according to any one of claims. 1-13, comprising growing the host cell of claim 17 under conditions suitable for expressing the antibody and isolating the expressed antibody from the culture fluid. 19. Pharmaceutical composition for the treatment of a disease associated with increased expression of VEGF, containing an antibody against VEGF or an antigen-binding fragment according to any one of paragraphs. 1-13, or a nucleic acid according to any one of paragraphs. 14, 15, or the expression vector according to claim 16, and a pharmaceutically acceptable carrier. 20. An anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-13, or a pharmaceutical composition according to claim 19 for use in the treatment and diagnosis of colon and rectal cancer. 21. A method of treating colon and rectal cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-VEGF antibody or an antigen-binding fragment thereof according to any one of claims. 1-13, or the pharmaceutical composition according to claim 19. 22. The use of an anti-VEGF antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-13, or the pharmaceutical composition according to claim 19 for the production of a medicinal product for the treatment of colon and rectal cancer. 23. Pharmaceutical combination for the treatment of a disease associated with increased expression of VEGF, containing an antibody against VEGF or its antigen-binding fragment according to any one of paragraphs. 1-13, or the pharmaceutical composition according to claim 19 with one or more therapeutic agents. 24. A kit for the treatment of a disease associated with increased expression of VEGF, containing: a) an antibody against VEGF or its antigen-binding fragment according to any one of paragraphs. 1-13, or a pharmaceutical composition according to claim 19, or a pharmaceutical combination according to claim 23 and b) a device for administration.