CN108148134B - anti-VEGF antibodies - Google Patents

Abstract

The present invention discloses antibodies, particularly heavy chain antibodies, more particularly single domain antibodies, that specifically bind Vascular Endothelial Growth Factor (VEGF). Methods of making the antibodies and therapeutic uses are also disclosed.

Description

anti-VEGF antibodies

The invention is a divisional application of an invention patent application with application number 201510005713.1, the original application date is 2015, 01, 06 and the publication date is 2016, 08, 03, and the invention is named as an "anti-VEGF antibody".

Technical Field

The present invention relates to antibodies and uses thereof, in particular, the invention relates to antibodies, particularly heavy chain antibodies, more particularly single domain antibodies, that specifically bind to Vascular Endothelial Growth Factor (VEGF); as well as methods for the preparation and therapeutic use of said antibodies.

Background

Angiogenesis refers to the development of new blood vessels from existing capillaries or postcapillary veins, and is a complex process involving multiple molecules of multiple cells. Angiogenesis is a complex process of the coordination of a pro-angiogenic factor and an inhibitory factor, and normally, the two are in equilibrium, and once the equilibrium is broken, the vascular system is activated, so that the angiogenesis is excessive or the vascular system is inhibited to degenerate the blood vessel.

Many diseases are known to be associated with deregulated angiogenesis and undesirable angiogenesis. These diseases include, but are not limited to, tumours such as so-called solid tumours and liquid (or blood) tumours (e.g. leukaemia and lymphoma), inflammation such as rheumatoid or rheumatic inflammation, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory conditions such as chronic asthma, arteriosclerosis or post-transplant arteriosclerosis, endometriosis, ocular neovascular diseases such as retinopathy (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis. Other diseases associated with deregulated angiogenesis and undesirable angiogenesis will be apparent to those skilled in the art.

The vascular endothelial growth factor is a heparin-binding growth factor with specificity to vascular endothelial cells, and can induce angiogenesis in vivo. Comprises VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placenta growth factor.

The main functions of VEGF-A are to promote vascular endothelial cell proliferation, migration and lumen formation, and also to increase vascular leakage, promote monocyte chemotaxis and B cell generation. The biological effects of VEGF-A are mediated by binding to its specific receptors, primarily to the specific receptors vascular endothelial growth factor receptor 1(VEGFR-1) and VEGFR-2. Among them, VEGFR-2 is considered to be the main VEGFR, which has an important effect on the proliferation of vascular endothelial cells. VEGFR-2 induces VEGF to bind dimers and receptors requiring autophosphorylation via intracellular kinases, thereby enhancing cell mitosis (Klettner A, Roider J. treating age-related cellular differentiation interaction of VEGF-antagnosts with the target. Mini Rev Med Chem,2009,9(9): 1127-1135). VEGF-a comprises 8 exons and 7 introns, which are transcriptionally spliced into multiple subtypes, mainly: VEGF121, VEGF145, VEGF206, VEGF165, VEGF189, these subtypes have different molecular masses, solubilities and heparin binding capacities, with VEGF165 being the most prominent subtype of VEGF-A (Ferrara N, Gerber HP, Le Couter J. the biology of VEGF and its receptors. Nat Med,2003,9(6): 669-. VEGF165 is a secreted soluble protein that acts directly on vascular endothelial cells to promote vascular endothelial cell proliferation, accelerate repair of vascular endothelial cell injury, increase vascular permeability, decrease intravascular thrombosis and thrombotic occlusion, and inhibit intimal hyperplasia (Huangchenxing, Shenzuguan, research on vascular endothelial growth factor and its use in tissue repair J. China J. repair and reconstruction surgery. 2002,160: 64-68).

The existing vascular endothelial growth factor drugs include Pegaptanib sodium (trade name Macugen), Ranibizumab (trade name Lucentis), Bevacizumab (trade name Avastin), VEGF Trap, and the like. The current focus of controversy for anti-VEGF agents is the potential to exacerbate tissue fibromembrane formation. The anti-VEGF drugs currently used clinically for treating various diseases (such as age-related macular degeneration) require frequent intraocular injections, resulting in the potential risk of endophthalmitis, which is a significant problem with anti-VEGF treatment. The effects of bevacizumab and Macugen on different subtypes of VEGF were observed by researchers using umbilical vein endothelial cells and fibroblasts from Tenon's capsules, and it was confirmed that VEGF-165, VEGF-121 mainly affected the growth of blood vessels, and VEGF-189 mainly affected the process of fibrosis formation. Bevacizumab and ranibizumab inhibit all active subtypes of VEGF-a (Van Bergen T, Vandewalle E, Van de Veire S, et a1. role of differential VEGF iso forms in the serum formation after filter glaucomatous permeability changer. exp Eye Res,2011,93: 689-.

At present, the anti-VEGF medicines need to be repeatedly treated every 4-6 weeks, the average annual injection amount of ranibizumab treatment in the 1 st year is about 6.9 times, and bevacizumab is about 7.7 times (Li X, Hu Y, Sun X, Zhang J, Zhang M.Bevacizumab for vascular-related vascular diagnosis in China.Ophthalmology.2012Oct, 119(10):2087-93), so that the potential risk of endophthalmitis exists in the frequent intraocular injection treatment, and a novel antibody medicine with long medicine effect and better retina permeation absorption is urgently needed to be developed so as to prolong the administration period and reduce the discomfort and risk brought by injection administration to patients.

In addition, the prior anti-VEGF preparation expression and purification process is complex, and the practical problems of high cost, poor stability, wide application range and the like generally exist.

A heavy chain antibody is an antibody isolated from the serum of camelids, and is composed of only a heavy chain, and its antigen binding region is only a single domain connected to an Fc region via a hinge region, and has a function of binding to an antigen after being isolated from the antibody, and thus is called a single-domain antibody (sdAb) or a nanobody (nanobody). Unlike conventional antibodies, single domain antibodies are peptide chains of about 110 amino acids with a molecular weight of about 1/10 that is similar to that of conventional antibodies, which provides a novel approach to the molecular construction of antibodies (Muylermans. Single domain peptide antibodies: current status. J Biotechnol 2001,74: 277-) -302). Such single domain antibodies have the characteristics of small molecule, good thermal stability, stability in detergent and high-concentration uric acid environments, good in vivo tissue permeability, good solubility (Stanfield R, Dooley H, Flajnik M, Wilson I.Crystal structure of a sharksingle-domain antibody V region in complex with lysom. science 2004,305(5691)), easy expression, favorable for prokaryotic system expression, low production cost, unique antigen recognition epitope, capability of recognizing hidden antigenic sites and the like, and gradually play an unexpectedly large role in immune experiments, diagnosis and treatment (DirkSaens, Gamreza Hassanzahde Ghassabah, Sege Muydermans. Single-domain antibodies building blocks 608 for biological applications, Occidality 600: 2008).

Accordingly, there is a need in the art for an antibody that overcomes the above-described deficiencies of existing anti-VEGF formulations, such as single domain antibodies that specifically bind to VEGF and inhibit its activity.

Disclosure of Invention

The present invention provides an anti-VEGF antibody, variant or derivative thereof, wherein the antibody comprises a heavy chain variable region comprising: (i) CDR1, CDR2 and CDR3 of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 or functionally active variants thereof; or (ii) the CDR1, CDR2 and CDR3 of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 or functionally active variants thereof; or (iii) the CDR1, CDR2 and CDR3 of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 or functionally active variants thereof; or (iv) the CDR1, CDR2 and CDR3 of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 or functionally active variants thereof; the functionally active variant is a functionally active variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs 1-12.

In a specific embodiment, the invention provides a heavy chain antibody, said antibody consisting of a heavy chain, the variable region of which comprises: (i) CDR1, CDR2 and CDR3 shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; or (ii) the CDR1, CDR2 and CDR3 of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 or functionally active variants thereof; or (iii) the CDR1, CDR2 and CDR3 of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 or functionally active variants thereof; or (iv) the CDR1, CDR2 and CDR3 shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 or functionally active variants thereof.

In one aspect, the heavy chain variable region of an antibody of the invention may comprise at least one amino acid addition, insertion, deletion and/or substitution. In another aspect, the antibodies of the invention can be monoclonal, chimeric or humanized antibodies, multispecific and/or bispecific antibodies and fragments thereof. In a specific embodiment, the antibody of the invention is a humanized antibody.

In a particular embodiment, the heavy chain of an antibody of the invention may further comprise a constant region. In another specific embodiment, the heavy chain of an antibody of the invention further comprises an Fc fragment.

In some specific embodiments, the antibody of the invention is a heavy chain antibody, i.e., consists only of heavy chains. In some specific embodiments, the antibodies of the invention are single domain antibodies.

In addition, the invention provides antibodies that compete for binding to VEGF with a reference antibody, which is any one of the antibodies described above.

The invention also relates to nucleic acid sequences encoding the above antibodies; vectors comprising these nucleic acid sequences; and host cells expressing the above antibodies, and/or comprising these nucleic acid sequences or vectors.

The present invention also provides a method for producing an antibody, comprising the steps of: culturing the above host cell under conditions that allow expression of the antibody; and purifying the antibody from the resulting culture product.

The invention also relates to a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable excipient. The pharmaceutical composition may also comprise one or more therapeutically active compounds, such as known anti-VEGF drugs or anti-tumor drugs.

In another aspect, the invention also relates to Antibody-conjugated drugs (ADCs) comprising an Antibody of the invention conjugated to another agent, such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

The antibody-conjugated drug may further comprise a linker unit between the drug unit and the antibody unit.

In addition, the invention relates to methods of modulating VEGF activity by administering an effective amount of an antibody of the invention. The present invention relates to methods of inhibiting angiogenesis by administering to a patient in need thereof an effective amount of an antibody of the present invention.

The invention also provides a method of treating a disease or disorder associated with VEGF, comprising administering to a patient in need thereof an effective amount of at least one antibody of the invention. The disease or disorder includes a tumor or cancer or an ophthalmic disease. The tumor or cancer comprises breast cancer, brain tumor, renal cancer, ovarian cancer, thyroid cancer, lung cancer, colorectal cancer, endometrial cancer, angiosarcoma, bladder cancer, embryo tissue cancer, neck tumor, malignant glioma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, etc. The ophthalmic diseases comprise macular edema caused by various reasons (including diabetic macular edema, macular edema caused by various diseases after cataract surgery or uveitis and the like), age-related macular degeneration, diabetic retinopathy, central retinal vein occlusion, neovascular glaucoma and other ophthalmic diseases involving new blood vessels.

In addition, the invention relates to the use of an antibody of the invention for the preparation of a medicament for modulating VEGF activity; the use of an antibody of the invention in the manufacture of a medicament for inhibiting angiogenesis; use of an antibody of the invention in the manufacture of a medicament for the treatment of a disease or disorder associated with VEGF.

The invention also provides a kit comprising a) an antibody of the invention, or the pharmaceutical composition; and b) instructions for use.

Drawings

FIG. 1 is a diagram showing the SDS-PAGE detection result of purified hVEGF165 protein. Wherein, Lane 1 is the standard protein Marker (Invitrogen, Cat. No.: LC 5677); lane 2 is 2. mu.g of non-reduced hVEGF 165; lane 3 is 5. mu.g of non-reduced hVEGF 165; lane 4 is 2. mu.g of reduced hVEGF 165; lane 5 is 5. mu.g of reduced hVEGF 165.

FIG. 2 is a graph showing the results of an immune response test, showing that animals produced a better immune response after injection of antigen and the serum titer was about 1:100 k.

FIG. 3 is the result of agarose gel electrophoresis detection of total RNA, which shows that the quality of the obtained RNA meets the requirement of library construction.

FIG. 4 shows the amplified V obtained by PCR after reverse transcription of the total RNA of FIG. 3 into cDNA_HAnd (5) agarose gel electrophoresis purification result of the H fragment.

FIG. 5 is a schematic view for connecting V_HPhagemid vector map of the H fragment.

FIG. 6 is a graph of the detection of the insertion rate of fragments of the phage display library. The PCR detection of 72 random clones, 69 of which had the insertion of the single domain antibody gene fragment, resulted in an insertion rate of 69/72-95.8%.

FIG. 7 is a sequence diversity test chart of the single domain antibody library obtained by sequencing the positive clone having the insert in FIG. 6, showing that the library diversity is good.

FIG. 8 is a map of a vector dedicated for FASEBA screening. The vector is ampicillin resistant, contains SASA and 6 × Histag, and can be used for secretion and expression of antibody.

FIG. 9 is an affinity ranking of antibodies after FASEBA screening; 9A, 9B, 9C are affinity ranking results for 3 different batches. The upper left diagram: sensorgrams of binding, dissociation of different clones; upper right panel: a matrix plot of binding, off-rates for different clones; lower left panel: normalized sensorgrams of different clones; right lower panel: and selecting a sensorgram of part of the antibody with higher affinity.

FIG. 10 is a graph of the results of a receptor competitive screen in which 15 single domain antibodies, preferably ranked by expression level and affinity, were used for the screen. Based on the competitive results and comparison with controls, 2 of them were selected for heavy chain antibody production for cell proliferation inhibition experiments.

FIG. 11 is a graph of a heavy chain antibody versus HUVEC cell proliferation inhibition assay. The inhibition function of 4 heavy chain antibodies is judged by the inhibition degree of different concentrations of antibodies on cell proliferation, wherein the inhibition function of A80887, A80723 and A69458 is strongest at the cellular level.

FIG. 12 is the variable region sequences of the 4 heavy chain antibodies of examples 8 and 9.

Fig. 13 is a schematic view of the sub-intestinal blood vessel of zebrafish. After a certain period of administration, 15 zebrafish per group were randomly photographed under a fluorescence microscope and the areas of the sub-intestinal plexuses (SIVs) were quantitatively analyzed. The T-test is adopted for comparison between two groups, the statistical analysis is carried out for comparison between the groups by adopting one-factor analysis of variance and Dunnett's T-test, p <0.05 shows that the statistical difference exists, and the calculation formula of the drug effect for inhibiting angiogenesis is as follows:

Detailed Description

The present invention relates to antibodies, variants or derivatives thereof, that specifically bind to VEGF; as well as methods for the preparation and therapeutic use of said antibodies. For example, the invention relates to heavy chain antibodies, more particularly single domain antibodies, that specifically bind VEGF. Meanwhile, the antibody of the present invention shows superior effects in inhibiting cell proliferation and angiogenesis over the prior art anti-VEGF monoclonal antibody (e.g., Avastin), as further described in the following examples.

The single domain antibody of the invention has smaller molecular weight than Fab fragment and full-length IgG antibody, generally 12-15kD, can be used for constructing multivalent antibody, and can improve affinity, prolong half-life period, prolong interval administration period and other characteristics through genetic engineering modification. Compared with the common antibody drug, the single domain antibody drug has more stable binding capacity with antigen under extreme conditions of high temperature, gastric acid, protease and the like, and has high conformational stability. Unlike whole antibody drugs that are prone to induce complement effector cytotoxic reactions, single domain antibody drugs lack the Fc fragment and do not cause complement effects. Meanwhile, because the single domain antibody has a small molecular weight, the antibody may have better permeability in administration to eye tissues and tumor tissues. Stability in protease, extreme temperature and pH environments, high affinity, making it feasible for oral and other routes of administration.

The single domain antibody can be expressed in prokaryotic or eukaryotic cells, such as escherichia coli or yeast cells in a large scale, and the expression amount is large, so that the mass production is greatly facilitated, the production cost is favorably controlled, and the market prospect of later-stage drug development is also favorably realized.

Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings that are understood by those of ordinary skill in the art to which this document pertains.

The term "antibody" is well understood in the biological and biomedical arts, and generally refers to intact antibodies and antibodiesAnd antibody fragments or single chains thereof. Antibodies are glycoproteins secreted by specialized B lymphocytes called plasma cells. It is also known as an immunoglobulin (Ig) because it contains common domains that are present in many proteins. Antibodies most likely comprise 2 heavy (H) chains and 2 light (L) chains, or antigen-binding portions thereof, typically linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (V)_H) And a heavy chain constant region. Each light chain is also composed of a variable region (V)_L) And constant region composition. The light chain constant region consists of one domain CL. V_HAnd V_LThe regions may be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). In some specific embodiments, an antibody of the invention consists only of a heavy chain. In some specific embodiments, the antibodies of the invention are single domain antibodies.

Complementarity Determining Regions (CDRs) and Framework Regions (FRs) of a given antibody may be determined using the method described by Kabat et al in Sequences of Proteins of Immunological Interest, 5 th edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No.91-3242,1991.

The invention includes "variants" of an antibody, e.g. the heavy chain variable region of an antibody of the invention may comprise at least one amino acid addition, insertion, deletion and/or substitution, e.g. 10, 20, 30, 40, 50, preferably e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid additions, insertions, deletions and/or substitutions.

The invention also includes "derivatives" of the antibodies. A "derivative" of an antibody is an antibody that is chemically modified, for example, by binding to other chemical moieties such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term "antibody" includes fragments, derivatives, variants thereof.

In one aspect, the invention provides an anti-VEGF antibody, variant or derivative thereof, wherein the antibody comprises a heavy chain variable region comprising: (i) CDR1, CDR2 and CDR3 of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 or functionally active variants thereof; or (ii) the CDR1, CDR2 and CDR3 of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 or functionally active variants thereof; or (iii) the CDR1, CDR2 and CDR3 of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 or functionally active variants thereof; or (iv) the CDR1, CDR2 and CDR3 shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 or functionally active variants thereof.

The functionally active variant is a functionally active variant having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequence of any one of SEQ ID NO 1-12.

In a specific embodiment, the invention provides a heavy chain antibody, said antibody consisting of a heavy chain, the variable region of which comprises: (i) CDR1, CDR2 and CDR3 of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 or functionally active variants thereof; or (ii) the CDR1, CDR2 and CDR3 of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 or functionally active variants thereof; or (iii) the CDR1, CDR2 and CDR3 of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 or functionally active variants thereof; or (iv) the CDR1, CDR2 and CDR3 shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 or functionally active variants thereof.

In a particular embodiment, the heavy chain of an antibody of the invention may further comprise a constant region. In another specific embodiment, the heavy chain of an antibody of the invention further comprises an Fc fragment.

In some specific embodiments, the antibody of the invention is a heavy chain antibody, i.e., consists only of heavy chains. In some specific embodiments, the antibodies of the invention are single domain antibodies.

In a more specific embodiment, the heavy chain variable region sequence of the antibody of the invention is set forth in SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16.

In addition, the invention also provides for the competition for binding of the VEGF antibody to a reference antibody, which is any one of the above antibodies.

The invention also relates to nucleic acid sequences encoding the above antibodies; vectors comprising these nucleic acid sequences; and host cells expressing the above antibodies, and/or comprising these nucleic acid sequences or vectors. A "host cell" is a cell for expressing a nucleic acid, e.g., a nucleic acid of the invention. The host cell may be a prokaryote, such as e.coli, or it may be a eukaryote, such as a unicellular eukaryote (e.g., yeast).

In specific embodiments, the nucleic acid sequence is shown as SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, as detailed in the examples below.

The present invention also provides a method for producing an antibody, comprising the steps of: culturing the above host cell under conditions that allow expression of the antibody; and purifying the antibody from the resulting culture product, as detailed in the examples below.

The invention also relates to a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable excipient. The pharmaceutical composition may also comprise one or more therapeutically active compounds, such as known anti-VEGF drugs or anti-tumor drugs.

The therapeutically active compound may be administered simultaneously or sequentially with the antibody of the invention.

The pharmaceutical compositions may be prepared according to techniques known in the art. The term "excipient" refers generally to any ingredient other than one or more active therapeutic ingredients. Excipients may be inert substances, inactive substances and/or pharmaceutically inactive substances. Excipients can serve a variety of purposes, for example, as carriers, vehicles, diluents, tablet adjuvants, and/or to improve administration and/or absorption of the active substance. The formulation of pharmaceutically active ingredients with various excipients is known in the art, see, e.g., Remington: the science and Practice of Pharmacy (e.g., 19 th edition (1995), and any later versions). Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity adjusting agents, chelating agents and stabilizers.

The antibodies of the invention may be administered in the form of a pharmaceutical composition. It can be prepared into liquid preparations such as injection, lyophilized preparation, spray, etc., and solid preparations such as capsule, etc. The route of administration may be, for example, intravenous injection, oral or topical administration, e.g., transdermal, conjunctival, and/or ocular, etc. In a particular embodiment, the route of administration is oral. In another specific embodiment, the route of administration is via the eye.

In another aspect, the invention also relates to antibody conjugate drugs comprising an antibody of the invention conjugated to another agent, such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

Local delivery of other agents using antibody conjugated drugs allows for targeted delivery of the agent to the tumor and intracellular accumulation in the tumor, and systemic administration of these unconjugated agents may result in unacceptable levels of cytotoxicity to normal cells as well as to tumor cells that need to be removed.

Antibody-conjugated drugs typically comprise a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker results in release of the drug unit from the antibody in an intracellular environment. The linker may be, for example, a peptidyl linker that is cleavable by an intracellular peptidase or protease, including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptidyl linker is at least two amino acids or at least 3 amino acids in length. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker.

In other embodiments, the linker unit is not cleavable and the drug is released, for example, by degradation of the antibody.

In addition, the invention relates to methods of modulating (preferably inhibiting) VEGF activity, inhibiting angiogenesis in a mammal by administering an effective amount of an antibody of the invention.

In addition, the invention relates to methods of modulating VEGF activity by administering an effective amount of an antibody of the invention. The present invention relates to methods of inhibiting angiogenesis by administering to a patient in need thereof an effective amount of an antibody of the present invention.

The invention also provides a method of treating a disease or disorder associated with VEGF, comprising administering to a patient in need thereof an effective amount of at least one antibody of the invention. The disease or disorder includes a tumor or cancer or an ophthalmic disease. The tumor or cancer comprises breast cancer, brain tumor, renal cancer, ovarian cancer, thyroid cancer, lung cancer, colorectal cancer, endometrial cancer, angiosarcoma, bladder cancer, embryo tissue cancer, neck tumor, malignant glioma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, etc. The ophthalmic diseases comprise macular edema caused by various reasons (including diabetic macular edema, macular edema caused by various diseases after cataract surgery or uveitis and the like), age-related macular degeneration, diabetic retinopathy, central retinal vein occlusion, neovascular glaucoma and other ophthalmic diseases involving new blood vessels.

By "patient in need thereof" is meant any mammal such as, but not limited to, humans, horses, cows, cats, mice, rabbits, rats, goats, and the like. Preferably, the mammal is a human.

In some therapeutic applications, a plurality of antibodies of the invention are administered in combination.

In addition, the invention relates to the use of an antibody of the invention for the preparation of a medicament for modulating VEGF activity; the use of an antibody of the invention in the manufacture of a medicament for inhibiting angiogenesis; use of an antibody of the invention in the manufacture of a medicament for the treatment of a disease or disorder associated with VEGF.

The invention also provides a kit comprising a) an antibody of the invention, or the pharmaceutical composition; and b) instructions for use.

The invention will be further described with reference to the following non-limiting examples.

Example 1 antigen preparation

The present example is directed to an antigen which is Human VEGF165(Human vascular endogenous growth factor 165, hVEGF165) molecules (Park JE, Keller GA, Ferrara N. the Vascular Endogenous Growth Factor (VEGF) antibodies: differential deposition in the molecular Biol cell 1993Dec.,4(12) 1317-26; Genegrouth S, Greenberg SM, Cohen T, Gitay-Goren H, Rockwell P, Maine TE, Levi BZ, New ferrite G. 4 genetic growth factor H-4 genetic growth factor H, Rockwell P, Rockwell TE, VEGF 165. 7. the molecular deposition of VEGF, VEGF 23. the biological assay of VEGF 23. the molecular biological growth factor J. the molecular biological assay of VEGF 23, growth protein J. the molecular growth factor 19, VEGF 23. the biological assay of VEGF 23. the molecular biological assay of VEGF 23. the molecular culture of the molecular culture J. the culture of VEGF 23. the molecular culture of the Human VEGF, the molecular assay of the VEGF 165. the molecular culture of the VEGF 23. the molecular culture of the Human VEGF, the molecular culture of the Human VEGF strain J. the culture of the Human VEGF165, the molecular culture of the Human VEGF165, VEGF of the Human VEGF165, the Human culture of the Human VEGF 23. the biological assay of the Human VEGF165, the Human VEGF of the Human culture of the biological assay of the Human VEGF 23. the biological assay of the molecular culture of the Human culture of the molecular culture of the VEGF165, the biological assay of the Human culture of the biological assay of the VEGF 23. the Human culture of the, wherein the nucleic acid sequence of the human VEGF165 antigen is shown in SEQ ID NO: 22; the amino acid sequence of the human VEGF165 antigen is shown in SEQ ID NO. 23.

According to the amino acid sequence, after codon optimization of mammalian expression, optimized DNA is obtained in a total synthesis mode and cloned into a eukaryotic expression vector pTT5 (authorized by the inventive organization NRC) for preparing transfection grade plasmids. HEK293E cells were transfected and cultured for 7 days, cells were harvested by centrifugation and supernatants were cultured for manually assembled Capto a Capto column and HiTrap^TMTwo-step ion exchange purification of Q HP, purification and endotoxin removal. Protein concentration detection adopts UV_280nmAnd (3) detecting a light absorption value, detecting the protein endotoxin level by adopting an LAL method, and determining the activity of the antigen by using a HUVEC cell proliferation experiment. The total concentration of hVEGF165 protein was 1.25mg/ml, the volume was 22ml, the total amount was 27.5mg, the endotoxin level was 0.537EU/ml (Table 1), the SDS-PAGE detection results are shown in FIG. 1, and the protein was stored at-80 ℃.

TABLE 1 hVEGF165 purified protein message

Example 2 animal immunization and immune response assays

1. Animal immunization

Alpaca (Lama pacos) was selected as the experimental animal and six-point injection immunization was performed on the scapula and back at 4 different time points, respectively. PBS was used as a diluent for the antigen, the volume of each immunization was 1ml, and the antigen amount and adjuvant information are shown in Table 2. The immunizing agent contains BSA with the final concentration of 1mg/ml, and the antigen and the adjuvant are prepared and mixed evenly and then are immunized before injection.

TABLE 2 alpaca immunoantigen information

The immunization schedule design (Table 3) was performed by collecting blood in jugular vein at four different times and adding anticoagulant during blood collection. Blood is collected for the first time by 5ml, and the rest three times are respectively 15 ml. After gradient centrifugation using Ficoll 1.077 reagent (Sangon, Cat. No.: F760014-100) and anticoagulation, peripheral blood lymphocytes were separated and counted for cell resuspension, and RNAlater (TIANGEN, Cat. No.: DP408-02) was added and stored at-20 ℃. Serum obtained by gradient centrifugation was also stored at-20 ℃.

TABLE 3 alpaca immunization time arrangement table

2. Immune response assay

And performing antigen-specific immunoreaction tests on the serum samples before, after and after the fourth immunization by adopting enzyme-linked immunosorbent assay (ELISA). The immunogen was diluted with NaHCO3(pH 9.6) solution and coated on a microplate (Corning, Cat. No.:9018) overnight at 4 ℃. After washing the plate four times in a plate washer with PBS-T solution, blocking was performed for 2h at 37 ℃ using 3% BSA blocking solution. After four washes with PBS-T, the gradient diluted sera were incubated overnight at 37 ℃. After four washes with PBS-T, horseradish peroxidase-labeled goat anti-llama secondary antibody (Novus Biologicals, Cat. No.: NB7242) was incubated. The color development was carried out for 10min using TMB and stopped by adding 1M HCl. After the reaction was terminated, the system was used for detecting the absorbance at 450nm using an MK3(Thermo) microplate reader. The results of ELISA reactions showed that the animals produced better immune responses after injection of protein antigens with serum titers of about 1:100k (FIG. 2).

Example 3 antibody phage library construction

RNA extraction 3.1

Adding a corresponding volume of TRIzol reagent to the fraction according to the number of cellsIn isolated peripheral blood lymphocytes, after cell lysis is complete, according to

Instructions for the Plus RNA purification System (Invitrogen, Cat. No.:12183-555) to complete the isolation of total RNA. The quality of total RNA was checked by agarose gel electrophoresis and the concentration of RNA was determined by absorbance. According to the measurement results, 105.6. mu.g of total RNA was obtained in total. RNA is morphologically intact on agarose gel electrophoresis (FIG. 3), and qualitatively meets the requirements of library construction.

3.2. Reverse transcription PCR

Total RNA was reverse transcribed into cDNA using oligo (dT)20 primers according to the SuperScriptTM III First-Strand Synthesis System Using the technical Instructions (Invitrogen, Cat. No.: 18080-. According to the sequence characteristics of camel antibodies, specific forward primers and reverse primers are selected for V_HAmplification of H (A. Bell et al, Differential tumor-targeting antibodies of three-dimensional-domain antibodies, Cancer Lett.2010Mar 1; 289(1): 81-90; and Honda Toshio, Akahori, Yasushi, Kurosawa Yoshikazu. methods of construction tumor antibodies Patent 2005/0037421A1), the specific sequences of the primers are shown in Table 4. Through the first PCR of cDNA pair, V containing about 600bp is separated and purified according to the molecular weight of PCR product_HH, and then obtaining V through second round PCR amplification_HH fragment and two Sfi I restriction sites with different recognition sequences are introduced at two ends of the DNA fragment simultaneously to obtain 101 mu g of gel purified V_HH fragment (fig. 4).

TABLE 4 primer sequence information and PCR amplification

3.3 library construction

Will be amplified with different batches of cells and different primersObtained V_HThe H fragments were mixed and then cleaved with the restriction enzyme Sfi I. Separating and purifying by 2% agarose gel electrophoresis to obtain enzyme-digested V_HH; meanwhile, the phagemid vector (FIG. 5) was digested with restriction enzyme Sfi I, separated by 1.5% agarose gel electrophoresis, purified and the digested vector was obtained. Determination of the V after cleavage by light absorption_HH and phagemid vector concentrations were mixed at vector/fragment molar ratios of 1:3,1:5, and 1:10, respectively, and T4 ligase (NEB, Cat. No.: M0202L) was added to prepare a ligation reaction system of the same volume, followed by ligation overnight at 16 ℃. And (3) sequentially performing phenol/chloroform extraction, chloroform extraction and ethanol precipitation on the connection system, and measuring the concentration of the connection product obtained by purification by a light absorption method. Products obtained from 3 different connection systems are all subjected to equal amount of DNA and TG1 electrotransformation competent electrotransformation, libraries of the 3 connection systems, namely the size of transformation efficiency, are calculated by a coating plate and a gradient dilution method, and positive clones are randomly picked to detect the diversity of the libraries. And selecting a system with the highest transformation efficiency for a large amount of connection and transformation, and counting the library capacity. The library capacity was approximately 1.8X 10 as shown by plate count⁸(Table 5).

TABLE 5 Single Domain antibody phage display library volume calculation

Random cloning PCR was performed on the library colonies, and the fragment insertion rate of the library was found to be 95.8% (fig. 6), and positive clones having the inserted fragment therein were sequenced, and the diversity of the library was found to be good by aligning the amino acid sequences of the CDR regions of the single domain antibodies (fig. 7). The coated overnight plates were harvested using 2YT medium containing 100. mu.g/ml ampicillin and 2% glucose, centrifuged at 5,000g to remove cell metabolites, and the cells were resuspended in the same medium and divided into two aliquots for library storage.

Example 4 phage display and screening

4.1. Preparation of antigenic biotinylation complexes

The hVEGF165 protein was biotinylated according to the protocol of the EZ-Link Sulfo-NHS-LC-Biotinylation kit (Pierce, Cat. No.: 21335). The extent of biotinylation of the protein was detected by HABA assay. The biotinylated hVEGF165 was mixed with 0.5ml of magnetic beads of M-280 streptavidin (Invitrogen, Cat. No.:112.06D), incubated overnight at 4 ℃, and then the magnetic beads were separated by magnetic stand and biotinylated proteins that failed to bind to the magnetic beads were eluted with PBS solution to prepare antigen-magnetic bead conjugate complexes. After biotinylation reaction and purification 0.52mg of hVEGF165 protein was obtained, HABA experiments showed biotin conjugation levels of 6 moles of biotin molecule per mole of protein.

4.2. Phage library resuscitation

Approximately 100. mu.l (MOI of approximately 20) of phage library stock was inoculated into 2YT medium, cultured at 225rpm at 30 ℃ and cultured to logarithmic growth phase (OD)₆₀₀0.5) M13KO7 helper phage (NEB, cat. No.: N0315S) was added and incubated at 225rpm, 30 ℃ overnight. And (3) centrifugally collecting the phage, mixing the culture supernatant with a PEG/NaCl solution, centrifugally precipitating the phage, centrifuging and re-suspending for multiple times, and finally suspending the recovered phage particles in 1-2ml of PBS. The titer of the recovered phage library was calculated by limiting gradient dilution to obtain 3.15X 10¹³Library of pfu/ml.

4.3. Phage screening for target proteins

Take 2X 10¹¹putting pfu phage as a first round, incubating the pfu phage with 10ul of antigen magnetic bead coupled complex at normal temperature, and gently mixing the pfu phage and the 10ul antigen magnetic bead coupled complex for 2 hours on a rotary instrument; separating the magnetic beads through a magnetic frame, washing away phage which is not combined with the magnetic beads, and carrying out resuspension on the magnetic beads and elution of nonspecific combination for 7 times; adding the final resuspended magnetic beads into a TEA solution, eluting and separating the phage combined with the antigen magnetic bead coupling compound, and quickly adding a Tris-HCl buffer solution for neutralization; calculating the output of the phage after the first round of screening by a limiting gradient dilution method, and simultaneously carrying out overnight culture and amplification on the phage obtained by the first round of elution, specific parameters and processes and the library beforeThe description is the same. The second round of screening will be at-10¹¹The library after phage amplification is produced in the first round of pfu and is used as input, and is incubated and screened with 1ul of antigen magnetic bead coupled complex, and the specific operation process and parameters are the same as those of the first round of screening.

4.4. Phage ELISA identification

Picking the monoclonal plaques on the overnight plate for the second round of phage production calculation, placing into a 96-well deep-well plate containing 500. mu.l 2YT medium per well, culturing at 225rpm and 30 deg.C, and culturing to logarithmic growth phase (OD)₆₀₀0.5) M13KO7 helper phage (NEB, cat. No.: N0315S) was added and incubated at 225rpm, 30 ℃ overnight. The cells were collected by centrifugation, the supernatant was collected and added to a well-blocked microplate previously coated with hVEGF165, and detected with HRP/anti-M13 monoclonal antibody (GEHealthcare, Cat. No.:27-9421-01) as the secondary antibody, and the other ELISA parameters were the same as those of the immunoreaction detection, and the positive rate of the product was evaluated based on the absorbance. V combining randomly picked fractions with antigen-recognized positive phage clones_HSequencing of the H fragment, by sequence alignment and analysis, deduces the clonal diversity produced by phage display. Whether more rounds of phage display screening are required is determined by the positive rate and the diversity of the sequences. The positive of the phage clone is more than 50%, and the diversity requirement is met. Therefore, the sdAb gene of the second round of phage production clone was selected to construct a fakeba library for further cloning screening.

TABLE 6 phage panning and ELISA detection

Example 5 FASEBA screening

FASEBA library construction

Extracting phage DNA generated in the last round of phage display, and amplifying the code V by PCR_HThe fragment of H is cloned into the FASEBA vector of the patent through connection, and all the constructed cloning structures are V_HH-linker-SASA-6 XHis (FIG. 8). The ligation product was transformed into TG1 cells.

FASEBA screening

5.2.1. Sample preparation and expression level assessment

Randomly picking single clone from the constructed FASEBA library, putting into 96-well deep-well plate containing 500. mu.l of 2YT medium per well, and culturing to OD₆₀₀When the expression rate was 0.6-0.8, IPTG was added overnight to induce expression. After collecting the cells by centrifugation, 100. mu.l of the clarified culture supernatant was removed, added to a microplate previously coated with BSA and blocked, and detected using an HPR-labeled mouse anti-His monoclonal antibody as a secondary antibody (GenScript, Cat. No.: A00186); meanwhile, an equal amount of culture supernatant was taken and added to a microplate previously coated with hVEGF165 protein and closed, and detected using an HPR-labeled mouse anti-His monoclonal antibody as a second antibody (GenScript, cat. No.: a00186), as OD₄₅₀Absorbance values were used to assess the expression levels of the different clones. More than 5000 monoclonal clones in different batches are used for screening expression quantity and antigen binding force, and 138 positive clones with higher expression quantity and high binding force with antigen are used for affinity sequencing and subsequent screening.

5.2.2. Chip preparation

BSA protein was immobilized on the surface of a CM5(GE healthcare, Cat. No.: BR-1006-68) chip by standard coupling protocols according to the BIAcore T200 apparatus instructions. The basic process is as follows: HBS-EP solution (0.01M HEPES [ pH7.4 ],0.15M NaCl,3mM EDTA and 0.005% [ v/v ] surfactant P20) was used as the apparatus running buffer at 25 ℃ at a flow rate of 10 ml/min. 0.4M 1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1M N-hydroxysuccinimide (NHS) (1:1) was first injected for more than 7 minutes to activate the carboxymethyl dextran surface, and then unbound activation sites were blocked by injection of a 20 μ g/ml BSA protein solution diluted in 10mM sodium acetate (pH4.5) for 7 minutes, and finally another injection of 7 minutes of 1M aminoethanol (pH 8.5). The above procedure resulted in a BSA coupling reaction level of 327 Reaction Units (RU).

5.2.3. Affinity ordering of anti-hVEGF 165 single domain antibodies

The expression supernatant of the above-mentioned sdAb-SASA clone was filtered through a 96-well filter plate (Pall, cat. No.: PN5045) at 4 ℃ for 5 minutes by centrifugation at 4000g to remove bacteria and other particles, and the assay sdAb antibody was diluted with HBS-EP solution and sequentially flowed over the BSA-coupled chip surface. The sequencing analysis process comprises the following four steps: a. capturing the SASA-conjugated single domain antibody using a BSA immobilized chip; b. injecting hVEGF165 to combine with the surface of the chip with the captured single domain antibody; c. injecting running buffer and monitoring the dissociation phase for 300 s; d. the BSA coupled chip surface was reconstituted by injection of 10mM glycine/HCl (pH 2.0), 30. mu.l/min, 30 s. The chip capture antibody, antigen binding, antigen dissociation, BSA chip surface of each round need regeneration. Purified SASA protein was flowed over the BSA chip surface at a concentration of 200nM as a control to examine the chip regeneration effect. 138 clones were sorted in 3 batches, with clone A10981 as reference, 6A 10981 duplicate clones were very consistent from batch to batch, with a 400s dissociation level of about-20%, of which 93 clones dissociated at a slower rate than A10981 (FIG. 9), and picked for sequencing. 53 of these single domain antibodies were selected for prokaryotic expression and tested in a cell proliferation inhibition assay, and 15 of these were used for further competitive screening.

5.2.4.hVEGFR2 competitive screening

The 15 single-domain antibodies, preferably selected by expression level screening and affinity ranking, were used for receptor competitive screening in order to obtain antibodies capable of blocking the antigen hVEGF165 and its receptor hVEGFR binding. The specific process is as follows:

a. the hVEGFR2 protein is fixed on the surface of a CM5 chip by an amino coupling fixing method (same as 5.2.2); b. injecting hVEGF165 protein, observing binding characteristics, and stopping injection when a binding curve is close to saturation; c. injecting different single-domain antibodies on the surface of the chip combined with hVEGF165 and observing the combination characteristics, and simultaneously injecting Avastin serving as a drug for resisting VEGF which is already marketed as a control; d. if the epitope to which the antibody binds VEGF165 is exactly the VEGF and VEGFR2 binding epitope, the antibody will no longer bind VEGF, or will compete for VEGF that has bound VEGFR2, the binding signal will be significantly less than VEGF itself; if the epitope to which the antibody binds VEGF165 is different or independent from the VEGF & VEGFR2 binding epitope, the antibody will bind VEGF165 already bound to the receptor, and the resulting binding signal will be significantly higher than for VEGF itself. Based on the competitive results and comparison with the control (fig. 10), 2 better clones were selected for heavy chain antibody preparation for cell proliferation inhibition experiments.

Example 6: preparation of Single Domain antibodies

Prokaryotic expression, purification and endotoxin removal of the single domain antibody were as follows.

6.1. Reagent preparation

6.1.1. Prokaryotic expression reagent

Tryptone,OXOID LP0042

Yeast extract,OXOID LP0021

Casein acid hydrolysate,Sigma C9386

KH2PO4, Chinese medicine AR CAS [7778-77-0]

Na2HPO4.12H2O, Chinese medicine AR 10020318

NH4Cl, Chinese medicine AR CAS [12125-02-9]

NaCl, Chinese medicine AR 10019318

MgCl2, Chinese medicine AR 7791-18-6

CaCl2, Chinese medicine AR 10043-52-4

Glucose, Chinese medicine AR 10010518

Glycerol, Sigma G5516-500ML

IPTG,Amresco 0487-100G

VB1, Alatin AR 1099302

Ampicillin, 100mg/ml, 0.22 μm filter treatment;

IPTG mother liquor: filtering with 0.22 μ M filter at 1M, packaging 1-2ml, and freezing at-20 deg.C (effective period of 3 months);

MgCl2 mother liquor: 1M, performing moist heat sterilization at 121 ℃ for 30min, subpackaging 1-2ml, and storing at 4 ℃ (the validity period is 6 months);

CaCl2 mother liquor: filtering with 0.22 μ M filter at 1M, packaging 1-2ml, and storing at 4 deg.C (effective period of 6 months);

VB1 mother liquor: filtering with 0.22 μm filter at 50mg/ml, packaging 1-2ml, and storing at 4 deg.C (effective period of 6 months);

glucose mother liquor: 20% (W/V), filtering with 0.22 μm filter, and storing at 4 deg.C (effective period of 3 months);

glycerol mother liquor: 50% (V/V), sterilizing with damp heat at 121 deg.C for 30min, and storing at 4 deg.C (effective period of 6 months);

casein acid hydrosate mother liquor: 4 percent, sterilizing for 30min at 121 ℃ by moist heat, and storing at room temperature (the validity period is 3 months);

10XM9 salt solution: 6% Na2HPO4(W/V), 3% KH2PO4(W/V), 1% NH4Cl (W/V), 0.5%;

NaCl (W/V), performing damp heat sterilization at 121 ℃ for 30min, and storing at room temperature (the validity period is 3 months);

2YT medium: 1.6% (W/V) Tryptone, 1.0% (W/V) yeast extract, 0.5% (W/V) NaCl, and performing moist heat sterilization at 121 ℃ for 30 min;

10XTB medium: 12% (W/V) Tryptone, 24% (W/V) yeast extract, 4% (V/V) glycerin, and moist heat sterilization at 121 ℃ for 30 min.

6.1.2. Protein purification reagent and equipment

10XBugBuster Protein Extraction Reagent(Novagen，70921-4)；

100mM PMSF: 1.74g PMSF in 100ml isopropanol solution (Biyunyan, ST 506);

10mg/ml nuclease (DNase I): usually 1. mu.l per g of wet weight bacteria (Life Science Product and service, DD 0099-1);

5mg/ml Lysozyme (Lysozyme): typically 100. mu.l per gram of wet weight bacteria (Shengxing Bio, L0005-10);

quick StartTM Bradford reagent (Bio-Rad, 500-;

High Affinity Ni-NTA Resin(GenScript，L00250)；

HiTrapTM Desalting，5ml，(GE Healthcare，17-1408-01)；

purifier 10(GE Healthcare)；

lysis buffer: 20mM HEPES, 150mM NaCl, 10% (V/V) glycerol, 40mM imidazole, pH 8.0;

binding buffer: 20mM Na2HPO4, 0.5M NaCl, 20mM imidazole, pH 7.4;

washing with a miscellaneous buffer solution: 20mM Na2HPO4, 0.5M NaCl, 40mM imidazole, pH 7.4;

elution buffer: 20mM Na2HPO4, 0.5M NaCl, 300mM imidazole, pH 7.4;

1XPBS：137mM NaCl，10mM Na2HPO4，2mM KH2PO4，pH 7.4。

6.1.3. endotoxin removing reagent and equipment

ToxinEraser TM Endotoxin Removal Resin 1.5ml Resin (GenScript, L00402);

PD-10 Columns；ToxinEraser TM Regeneration Buffer(GenScript，M01053)；

ToxinEraser TM Equilibration Buffer(GenScript M01054)；

endotoxin detection kit by gel method (GenScript, L00451);

limulus reagents (TAL sensitivity 0.25EU/ml for Tachypleus Amebocyte Lysate, Limulus reagents laboratory Co., Ltd., Xiamen);

0.1M NaOH (prepared in pyrogen-free water);

0.1M hydrochloric acid (made up of pyrogen-free water);

a constant temperature incubator at 37 ℃.

6.1.4. Filter sterilization reagent and equipment

Millex-GP Filter Unit，0.22μm(Millipore，Lot：R4AA43868)。

6.1.5. Protein concentration determination and detection reagent and equipment

Nanodrop 2000 spectrophotometer (Thermo);

ExpressPlus PAGE Gel：4-20％，12wells(GenScript，M42012)；

MOPS Running Buffer Powder(GenScript，M00138)；

loading Buffer (5X, reduction): 0.25M Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 7.8% DTT;

loading Buffer (5X, non-reducing): 0.25M Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol.

6.2. Method and process

6.2.1. Preparation of the Strain

a) And (3) transformation: transferring the prokaryotic expression plasmid constructed into the single domain antibody gene into a strain TG1 through chemical transformation or electric transformation, coating the strain on a 2YT ampicillin-resistant plate, and culturing at the constant temperature of 37 ℃ overnight;

b) picking a single clone: 10ml of 2YT medium was added with a final concentration of 200. mu.g/ml ampicillin and 2% (W/V) glucose; the forceps were fully cauterized on an alcohol burner, cooled and then the tip of a 10. mu.l sterilized pipette was grasped, 1 monoclonal was picked from the transformation plate and placed in the medium at 225rpm, 37 ℃ for incubation overnight at constant temperature.

6.2.2. Adaptation induction

a) Preparing an M9 culture medium: to a 1X M9 salt solution was added glucose at a final concentration of 0.2% (W/V), 1mM MgCl₂、0.1mM CaCl₂0.4% (W/V) Casein acid hydrosate, 5mg/L VB1, 200. mu.g/ml ampicillin, and preheating in a shaker at 37 ℃;

b) transferring: taking the overnight culture bacterial liquid out of the shaking table, transferring the overnight culture product according to a 1:100 transfer system, centrifuging the overnight culture product at 4000rpm for 10min, resuspending the overnight culture product with fresh 1X M9 salt solution, centrifuging the overnight culture product at 4000rpm for 10min, transferring the overnight culture product after resuspension to a preheated culture medium, then placing the overnight culture bacterial liquid on the shaking table at 37 ℃, and culturing the overnight culture product at 225rpm for 24 h;

c) induction: m9 medium was supplemented with 1 XTB medium at the final concentration and IPTG at the final concentration of 1mM and ampicillin at a final concentration of 200. mu.g/ml, and cultured at 225rpm for 48h at 25 ℃ (24h was supplemented once with ampicillin at a final concentration of 200. mu.g/ml);

d) collecting a sample: after the induction was completed, the overnight culture was dispensed into a centrifuge cup, centrifuged at 11,000rpm at 4 ℃ for 15min, and the cells were collected.

6.2.3. Protein purification

a) Sample preparation by BugBuster cracking method

i. Cracking: adding 5ml lysis buffer solution into each gram of wet bacteria for resuspension (lysis buffer solution: 10X BugBuster Protein Extraction Reagent is diluted to 1X by binding buffer solution, lysozyme, 2 mu g/ml nuclease and 1mM PMSF are added to the solution with final concentration of 100 mu g/ml), and incubating the solution at room temperature for 1h with medium speed oscillation;

preparation of crude extract of protein: the lysed sample was centrifuged at 12,000g for 30min at 4 ℃ and the supernatant was collected and filtered through a 0.22 μm filter.

b) Nickel column affinity purification

i. Balancing column materials: with ddH 5 column volumes₂Balancing High Affinity Ni-NTAResin by O and a balancing buffer solution;

combining: mixing the crude protein extract with a proper amount of High Affinity Ni-NTA Resin, and incubating for 1h with shaking. After the incubation is finished, adding the mixed solution of the crude extract and the column material into a PD-10 empty column to collect the column material, and collecting effluent liquid for further analysis;

washing impurities: eluting the hybrid protein with at least 50 column volumes of the wash buffer. (in the impurity washing process, the Bradford dye solution is used as an indicator, 5 mu l of the impurity washing solution is added into 200 mu l of the Bradford dye solution to observe whether the dye solution turns blue, if so, the elution of the impurity protein is continued until the dye solution does not substantially change the color, and the next step can be carried out);

elution: eluting the target protein with at least 10 column volumes of elution buffer. (elution was carried out using Bradford dye as indicator, as in step iii, to determine if elution was complete).

c) Desalination/buffer exchange

i. Balancing: in that

The purifier 10 system was charged with 5 column volumes of ddH at the appropriate flow rate (0.5ml/min)₂O and PBS 5ml HiTratpTM Desainting column;

loading: adding a proper amount (0.5ml) of protein solution purified by a nickel column into a HiTrAptM desaling column at a proper flow rate (0.5 ml/min);

elution: elution with at least 10 column volumes of PBS was continued at the appropriate flow rate (0.5ml/min) and the protein was collected at the target UV peak.

6.2.4. Endotoxin removal and detection

a) Endotoxin removal

i. Sample treatment: adjusting the ionic strength to 0.2 +/-0.5M by using 1M sodium chloride before purification, and adjusting the pH value to 7.4 +/-0.2 by using 0.1M sodium hydroxide or 0.1M hydrochloric acid;

activated resin: vertically fixing PD-10 Columns, removing a cover at the top of a pre-installed column, adding ToxinEraser TM endo toxin Removal resin, opening a flow rate controller to ensure that a protective solution runs dry under the action of gravity, adding 5ml of regeneration buffer solution, adjusting the flow rate controller, keeping the flow rate at 0.25ml/min (about 10 drops/min), adding 5ml of regeneration buffer solution after the regeneration buffer solution runs dry, repeating the operation twice to ensure that the system keeps pyrogen-free (namely no Endotoxin);

equilibrium resin: after the PD-10 Columns are activated, adding 6ml of balance buffer solution, adjusting a flow rate controller, keeping the flow rate at 0.5ml/min, draining the balance buffer solution, and repeating the operation twice according to the operation;

endotoxin removal: closing the flow rate controller, adding the sample by using the head of the heatless gun, opening the controller, controlling the flow rate to be not higher than 0.25ml/min, starting to use the heatless receiving tube to receive the sample after the volume of the effluent liquid reaches 1.5ml, adding 1.5ml-3.0ml of balance buffer solution for leaching after the sample is drained, and collecting the leacheate. The sample concentration and endotoxin level were measured (elution was done using Bradford stain as an indicator to determine if collection was complete).

b) Gel method for determining endotoxin

i. Diluting the sample: the sample was diluted to an appropriate concentration (0.005. mu.g/ml; 0.05. mu.g/ml; 0.5. mu.g/ml; 5. mu.g/ml) as required for the limulus reagent sensitivity (0.25 EU/ml);

diluting an endotoxin standard: preparing endotoxin standard substance, re-dissolving with bacterial endotoxin detection water, mixing for 15min in vortex oscillator, and diluting to appropriate concentration (0.5 EU/ml);

detecting: TAL reagent was taken, 100. mu.l of bacterial endotoxin test water was added and gently shaken for at least 30s until the reagent was completely dissolved, taking care not to cause air bubbles, 100. mu.l of each of the following samples were added: positive control (endotoxin standard 0.5EU/ml), negative control (no endotoxin water), sample to be tested after dilution: (1) sealing the pipe orifice of the four samples to be measured, shaking up gently, vertically placing in an incubator at 37 ℃ for incubation for 1 hour, and then taking out for observation;

results recording: taking the test tube out of the constant temperature incubator gently, slowly inverting the test tube for 180 degrees, wherein the content in the test tube is in real gel, does not deform and does not slide down from the tube wall to be positive; otherwise, it is negative. The positive tube is positive, the negative tube is negative, the state is the same in the same range, and the experiment is effective.

6.2.5. Filtration sterilization

The samples were filtered through a 0.22 μm filter under aseptic conditions in a biosafety cabinet and the appropriate amount was taken for subsequent testing.

6.2.6. Detection of concentration and purity

a) Protein concentration determination

i. Calculating the absorption coefficient of the protein according to the provided protein amino acid sequence

Measuring the UV absorbance of the protein solution (A280)

Calculating the protein concentration according to the formula: protein concentration ═ uv absorbance of protein solution (a)₂₈₀) Absorption coefficient of protein

b) Protein purity assay

According to the above-mentioned concentrations, a certain amount of protein (e.g., 2. mu.g) and an equivalent amount of standard protein (e.g., 2. mu.g BSA) were subjected to SDS-PAGE under reducing and non-reducing conditions to examine the purity and confirm the concentration.

Example 7 HUVEC cell proliferation inhibition assay for Single Domain antibodies

7.1. Cell preparation

Will be about 3 x 10⁵HUVEC (ATCC, Cat No. PCS-100-010) cells were thawed and inoculated in a10 cm culture dish and fresh medium was added every 3-4 days; when the cell fusion degree reaches 85% -95%, the cells are cultured in a dish and fresh culture medium is added, and the cells within 7 generations are used for a proliferation inhibition test and are generally fixed at 6 th generation.

VEGF-induced HUVEC proliferation inhibition assay

2 XVEGF and antibody samples to be tested at different concentrations were prepared separately from M199 buffer liquid (Medium-1991X Earle's Salts (Invitrogen #11150-059), 10% Fetal bone Serum (Gibco, Cat #10100139), heat inactivated; 10mM HEPES (Invitrogen, Cat #15630080), 100units/ml Penicillin 100. mu.g/ml Streptomyces (Invitrogen, Cat # 10378016)); premixing 50 mu L of 2 XVEGF and antibody samples to be detected with different concentrations, arranging multiple holes on a micropore plate, taking a cell culture medium as a blank control of reaction,avastin was used as a positive control; the trypsinized cells were collected, washed with M199 buffer and resuspended twice, the cells were resuspended to a cell density of 1X 10⁵After each cell/ml, 50 μ L of cell resuspension was added to each well of the plate; the microplate with cells added was placed in culture at 37 ℃ with 5% CO₂Culturing for 96 hours; after the culture is completed, the culture medium is used

The Luminescent Cell Viability Assay Kit (Promega, Cat No: G7571) detects Cell Viability, and simultaneously detects fluorescence intensity by PHERAStar Plus (BMG Labtech) and records relative luminescence units; the production inhibition rate was calculated by the following formula, where the growth inhibition rate is 100 × (1-relative luminescence unit/maximum production value), where the relevant luminescence units are measured for various samples, and the maximum production value is the relative luminescence unit when VEGF alone was added. Combining the data of maximum inhibition rate and EC50, 2 antibodies were selected from 53 single-domain antibodies with prokaryotic expression for constructing heavy-chain antibody and subsequent proliferation inhibition experiment.

Table 7: EC50 values for the HUVEC cell proliferation inhibition assay were as follows:

example 8 expression of heavy chain antibodies

2 single-domain antibodies which have certain proliferation inhibition function and are preliminarily screened in the example 7 and 2 single-domain antibodies which are screened by receptor competition are fused with Fc fragment (SEQ ID NO:21) of human IgG1 to construct a heavy chain antibody, and the heavy chain antibody is cloned into pTT5 vector and is expressed and purified in HEK293E and endotoxin is removed, and the specific process is as follows:

8.1. reagent preparation

The same as in example 6.

8.2. Method and process

8.2.1. Cell culture

a) Taking the HEK293 suspension cells out of a liquid nitrogen or a refrigerator at-86 ℃, quickly putting the HEK293 suspension cells into a water bath kettle at 37 ℃, and thawing the HEK293 suspension cells within 1-2 min;

b) adding the thawed cells to 10 volumes of preheated FreeStyle^TMIn 293Expression Medium, mixing by gentle inversion, centrifuging at low speed to collect cells and resuspending with appropriate amount of fresh preheated culture Medium;

c) the resuspended cells were transferred to a culture flask and incubated at 37 ℃ with 5% CO₂Culturing at 110 rpm.

8.2.2. Transfection

a) The day before transfection, HEK293 suspension cells are passaged at a proper density, and the cell density needs to reach 1.5-2.0 multiplied by 10 on the day of transfection⁶Individual cells/ml, cell viability needs to be greater than 95%;

b) an appropriate amount of DNA was mixed with PEI at an optimal ratio (e.g. 1: 3) preheating FreeStyle in right amount^TM293 expresson Medium, and standing for 10 minutes at room temperature;

c) adding the PEI-DNA mixture into a cell culture, gently rotating and uniformly mixing, and continuing to perform reaction at 37 ℃ and 5% CO₂Culturing at the rotation speed of 110 rpm;

d) adding Tryptone N1 with the final concentration of 0.5% (w/v) within 16-24 hours after transfection;

e) culture supernatants were harvested by centrifugation on day 6 post transfection and filtered through 0.22 μm filters for purification.

8.2.3. Protein purification

a) Protein A affinity purification

i. Balancing column materials: with ddH 5 column volumes₂O and Binding Buffer balance Protein A column material;

combining: mixing the filtered expression supernatant with a column material, incubating and combining for 1h, and filling the column material into a PD-10 manual purification column after the incubation is finished;

washing impurities: washing off the impurity protein by using Binding Buffer with at least 30 times of column material volume (the impurity washing process uses Bradford dye liquor as an indicator, 5 mu l of the impurity washing liquor is added into 200 mu l of the Bradford dye liquor to observe whether the dye liquor turns blue, if the dye liquor turns blue, the impurity washing process is continued until the dye liquor basically does not change color, and then the next step can be carried out);

elution: eluting the target protein with at least 10 column volumes of Elution Buffer and adjusting the pH to about 7.0 with Neutralization Buffer (Elution is completed by Bradford staining solution as indicator, and the method is the same as step iii).

b) Desalting/buffer exchange:

the protein obtained by affinity purification is treated with HiTrap^TMDesaling column in

The purifier 10 was replaced on the system in PBS buffer. The subsequent steps of endotoxin removal, filtration sterilization, concentration, purity determination and the like were the same as in example 6.

TABLE 8 correspondence between heavy chain antibody, single domain antibody clone number, and amino acid sequence and nucleotide sequence numbering of variable region thereof

Single domain antibody clone number	Heavy chain antibody clone number	Amino acid sequence numbering	Numbering of nucleotide sequences
				A14575	A69451	SEQ ID NO：16	SEQ ID NO：20
A14614	A69458	SEQ ID NO：15	SEQ ID NO：19
				A15578	A80723	SEQ ID NO：14	SEQ ID NO：18
A15775	A80887	SEQ ID NO：13	SEQ ID NO：17

Example 9 HUVEC cell proliferation inhibition assay for heavy chain antibodies

A total of 8 dilutions were made of 4 heavy chain antibodies (Table 8) starting at 20. mu.g/ml, diluted 1: 4 and controlled with Avastin (A68467) under exactly the same conditions as in example 7. The 4 heavy chain antibodies all have different degrees of inhibition functions (figure 11) judged by the inhibition degree of the cell proliferation by different concentrations of antibodies, wherein the inhibition functions of A80887, A80723 and A69458 are strongest at the cellular level. Specifically, for A69458, the antibody concentration was 10^-3When the concentration is about 1 mu g/ml, the inhibition effect is obviously better than that of a contrast; for A80723, at an antibody concentration of 10^-3～10^-1The inhibition effect is obviously better than that of a control when the concentration is mu g/ml; for A80887, at an antibody concentration of 10^-2When the concentration is about 1 mu g/ml, the inhibition effect is obviously better than that of a control.

TABLE 9 heavy chain antibody sample information

Example 10 evaluation of anti-neovascular Effect of the antibodies of the present invention on the Zebra Fish model

The sample to be tested (A80887) has a molecular weight of about 75kDa and a concentration of 5.1mg/ml, is stored in aliquots at-80 ℃, diluted with 1 XPBS (pH7.4) immediately before use, and stored on ice at the time of injection. The laboratory instruments and reagents used included: microinjector (IM300, Narishige); dissecting microscope (SMZ645, Nikon corporation); an electric focusing continuous variable-magnification fluorescent microscope (AZ100, Nikon corporation); 6-well plates (Nest Biotech); MESAB (Sigma); bevacizumab (trade name Avastin, Roche).

Propagation of transgenic angiofluorescent zebrafish embryos was performed in a natural pairwise mating fashion. 4-5 pairs of adult zebrafish are prepared for each mating, and 200-300 embryos are produced in each pair on average. Embryos were cleaned (dead embryos removed) at 6 hours (i.e. 6hpf) and 24hpf post fertilization and appropriate embryos selected based on their stage of development (Kimmel, 1995). Incubating embryo with fish culture water at 28 deg.C (water quality for fish culture: 200mg of instant sea salt is added into per 1L of reverse osmosis water, conductivity is 480-510 μ S/cm, pH is 6.9-7.2, and hardness is 53.7-71.6 mg/L CaCO)₃). Feeding is not required within 9 days post fertilization (9dpf) because the embryo can take nutrients from its yolk sac. After completion of the experiment, zebrafish at various developmental stages were subjected to overexposure treatment with tricaine methanesulfonic acid to anaesthetize the zebrafish. All experimental procedures were performed strictly in accordance with the international animal assessment and acceptance committee (AAALAC) standards.

Using a microinjector, the sample was injected into the blood circulation of transgenic vascular fluorescent zebrafish (equivalent to human intravenous administration) at the highest concentration, maximum injection volume, with no mortality and no overt abnormal phenotype. According to the experimental results, 1/10 maximum injection dose (highest concentration × maximum injection volume), 1/3 maximum injection dose and 3 maximum injection doses were selected for detection, and a positive control group (Avastin), a solvent control group (PBS) and a blank control group were set, and each group treated with 30-tailed zebrafish. After a certain period of administration, 15 zebrafish per group were randomly photographed under a fluorescence microscope and the areas of the sub-intestinal plexuses (SIVs) were quantitatively analyzed. The T-test is adopted for comparison between two groups, the statistical analysis is carried out for comparison between the groups by adopting one-factor analysis of variance and Dunnett's T-test, p <0.05 shows that the statistical difference exists, and the calculation formula of the drug effect for inhibiting angiogenesis is as follows:

no zebra fish death or abnormal phenotype appears in all experimental groups in the whole experimental process; the blank control group and the solvent control group have no statistical difference (p > 0.05); the positive control group (Avastin) was statistically different (p <0.05) compared to the solvent control group.

Through a dose groping experiment, the maximum concentration of the A80887 injection is 5.1mg/ml, and no death and obvious abnormal phenotype of zebra fish are seen. Thus, 40.8ng (0.544pmol), 136ng (1.81pmol), 408ng (5.44pmol) was selected as the total dose administered; .

The results of the experiments showed that the inhibition of neovascularization was 10.9% (p >0.05), 18.5% (p <0.05) and 23.2% (p <0.001) at doses of A80887 of 20.4ng (0.272pmol), 68ng (0.907pmol) and 204ng (2.72pmol), respectively. The angiogenesis inhibition rates for Avastin doses of 400ng (2.68pmol) and 1 μ g (6.7pmol) were 6.9% (p >0.05) and 19.5% (p <0.01), respectively, and p <0.05 for both comparisons. At equimolar masses (both about 2.7pmol), the A80887 inhibited neovascularization with an efficiency of 23.2% and was significantly better than Avastin 6.9% (compare the two, p < 0.001). However, when Avastin 1. mu.g (6.7pmol) was compared with A8088720.4 ng (0.272pmol), 68ng (0.907pmol) and 204ng (2.72pmol), the inhibition rate of the new blood vessels was not statistically different. The raw data are shown in the following table.

TABLE 10 inhibition of blood vessels by A80887

The zebra fish angiogenesis model is widely accepted for pharmacodynamic evaluation and drug new target point verification. At present, anticancer drugs (including drugs which have been approved to be on the market by FDA) entering the stage of preclinical experiment (Pre-Clinical Trial) or Clinical Trial (Clinical Trial), such as Vatalanib (Novartis) (Chan, 2002), Thalidomide (cell) (Yabu, 2005), Compound 6(TargeGen) (Murphy, 2010), Rosuvastatin (Wang2010), Solenopsin (Eli Lilly) (Arbiser, 2007) and the like, are successfully and effectively verified by using a zebra fish angiogenesis inhibition model. The influence of the compound on the formation of new blood vessels is generally evaluated by selecting the vascular plexus under the intestines or the internode blood vessels of the transgenic vascular fluorescent zebra fish, and the influence of the sample A80887 on the formation of new blood vessels is evaluated by selecting a method for quantifying the area of the vascular plexus under the intestines.

Avastin is a recombinant human monoclonal IgG1 antibody that binds to Vascular Endothelial Growth Factor (VEGF) and prevents it from binding to endothelial cell surface receptors (Flt-1 and KDR). Avastin (with a molecular weight of 149kDa) is selected as a macromolecular positive control drug for inhibiting angiogenesis. The experimental result shows that the Avastin has a better angiogenesis inhibition effect, so that the model is reliably evaluated by taking the Avastin as a positive control drug.

In conclusion, ① A80887 and Avastin both have obvious inhibition effect on the neovascularization of zebra fish. ② at the equivalent molar mass (about 2.7pmol), the effect of A80887 on inhibiting the neovascularization is obviously better than that of Avastin. ③ A80887 at the dosage of 20.4ng, 40.8ng, 68ng, 136ng, 204ng and 408ng respectively, and the dosage is about 81.6, 163.2, 272, 544, 816 and 1632 mug/kg body weight respectively relative to the reference dosage of human administration.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Sequence listing

<110> Zhuhaisheng biopharmaceutical Co., Ltd

<120> anti-VEGF antibody

<160>32

<170>SIPOSequenceListing 1.0

<210>1

<211>5

<212>PRT

<213> alpaca (Lama pacos)

<400>1

Ser Tyr Ala Met Gly

1 5

<210>2

<211>21

<212>PRT

<213> alpaca (Lama pacos)

<400>2

Ala Ile Ser Trp Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Ala Val

1 5 10 15

Asp Ser Val Arg Gly

20

<210>3

<211>15

<212>PRT

<213> alpaca (Lama pacos)

<400>3

Asp Phe Gly Thr Arg Leu Arg Phe Thr Thr Asn Asp Tyr Gln Tyr

1 5 10 15

<210>4

<211>5

<212>PRT

<213> alpaca (Lama pacos)

<400>4

Asn Asn Val Met Gly

1 5

<210>5

<211>17

<212>PRT

<213> alpaca (Lama pacos)

<400>5

Ala Phe Asn Gly Trp Ser Ser Val Thr Glu Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>6

<211>14

<212>PRT

<213> alpaca (Lama pacos)

<400>6

Gly Arg Arg Trp Arg Ala Asn Arg Glu Thr His Tyr Asp Tyr

1 5 10

<210>7

<211>5

<212>PRT

<213> alpaca (Lama pacos)

<400>7

Tyr Tyr Ala Val Ser

1 5

<210>8

<211>17

<212>PRT

<213> alpaca (Lama pacos)

<400>8

Gly Ile Ser Arg Ser Gly Gly Ser Val Asn Phe Ala Gly Phe Val Lys

1 5 10 15

Gly

<210>9

<211>15

<212>PRT

<213> alpaca (Lama pacos)

<400>9

Asp Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Asn Tyr Ala Tyr

1 5 10 15

<210>10

<211>5

<212>PRT

<213> alpaca (Lama pacos)

<400>10

Ser Tyr Arg Leu Gly

1 5

<210>11

<211>17

<212>PRT

<213> alpaca (Lama pacos)

<400>11

Ala Ile Ser Trp Lys Asp Asp Thr Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>12

<211>15

<212>PRT

<213> alpaca (Lama pacos)

<400>12

Arg Gly Tyr Ser Arg Ser Trp Asn Pro Trp Ser Glu Tyr Asp Tyr

1 5 10 15

<210>13

<211>128

<212>PRT

<213> Artificial sequence (RenGongXuLie)

<400>13

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Trp Ser Gly Gly His Thr Tyr Tyr Ala Asp Ser Ala

50 55 60

Val Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Gly Asn Ala Lys

65 70 75 80

Asn Thr Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ala Ala Asp Phe Gly Thr Arg Leu Arg Phe Thr Thr

100 105 110

Asn Asp Tyr Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>14

<211>123

<212>PRT

<213> Artificial sequence (RenGongXuLie)

<400>14

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Tyr Ser Gly Ala Thr Phe Ser Asn Asn

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Arg Ala Arg Asp Phe Val

35 40 45

Ala Ala Phe Asn Gly Trp Ser Ser Val Thr Glu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Phe Val Ser Arg Asp Asn Asp Lys Ser Thr Met Tyr

65 70 75 80

Leu Gln Met Ile Asn Leu Lys Pro Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ala Gly Arg Arg Trp Arg Ala Asn Arg Glu Thr His Tyr Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>15

<211>124

<212>PRT

<213> Artificial sequence (RenGongXuLie)

<400>15

Asp Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Val Leu Ser Gly Arg Pro Phe Ser Tyr Tyr

20 25 30

Ala Val Ser Trp Phe Arg Gln Ala Pro Gly Gly Glu Arg Glu Phe Val

3540 45

Ala Gly Ile Ser Arg Ser Gly Gly Ser Val Asn Phe Ala Gly Phe Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Ser Ala Val Asn

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Arg Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Asn Tyr Ala

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>16

<211>124

<212>PRT

<213> Artificial sequence (RenGongXuLie)

<400>16

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Arg Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Trp Lys Asp Asp Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Thr Pro Glu Asp Ser Ala Val Tyr Ser Cys

85 90 95

Ala Ala Arg Gly Tyr Ser Arg Ser Trp Asn Pro Trp Ser Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Arg Val Thr Val Ser Ser

115 120

<210>17

<211>384

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>17

caggtaaagc tggaggagtc tgggggagga ttggtgcaga ctgggggctc tctgagactc 60

tcctgtgcag cctctggacg caccttcagt tcctatgcca tgggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtagcagct attagctgga gtggtggtca cacatactat 180

gcagactcag ctgttgactc cgtgaggggc cgattcacca tctccagagg caacgccaag 240

aacacggtat atctgcaaat gaacaatctg aaacctgagg acacggccgt ttactactgt 300

gcagccgact tcggtactag actacggttt acaactaatg actatcagta ctggggccag 360

gggacccagg tcaccgtctc ctca 384

<210>18

<211>369

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>18

gatgtacagc tggtggagtc tgggggagga ttggtgcagg ctggggactc tctgagactc 60

tcctgtgcgt actctggcgc aaccttcagt aacaatgtca tgggctggtt ccgccaggct 120

ccagggaggg cgcgtgactt tgtagcagca tttaacggtt ggagtagtgt tacagagtat 180

gcagactccg tgaagggccg attcttcgtc tccagagaca acgacaagag cacgatgtat 240

ctgcaaatga tcaacctcaa acccgacgac acggccgttt atttttgtgc agcagggagg 300

cgttggcgtg caaataggga gactcactat gactactggg gccaggggac ccaggtcacc 360

gtctcctca 369

<210>19

<211>372

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>19

gatgtacagc tggtggattc tgggggagga ttggtgcagc ctgggggctc tctgaccctc 60

tcctgtgtgc tctctggacg tccctttagt tactatgccg tgagctggtt ccgccaggct 120

ccaggggggg agcgcgagtt cgtagcagga atttcgagga gtggtggaag tgtaaacttt 180

gcaggcttcg tgaagggccg attcaccgtc tccagagaca acgccaagag cgcggtgaat 240

ctccaaatga acagcctgaa acgcgaggac acggccgttt attactgtgc agccgatact 300

aatgtctatg cctccgcgac gttgtccaat tatgcctatt ggggccaggg gacccaggtc 360

accgtctcct ca 372

<210>20

<211>372

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>20

caggtaaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggacg caccttcagt agttatcgct tgggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtagcagct attagctgga aagatgatac cacatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240

ctacaaatga acagcctgac acctgaggac tcggccgttt attcttgtgc agccaggggt 300

tatagtagat cttggaaccc gtggagcgag tatgactact ggggtcaggg gacccgggtc 360

accgtctcct ca 372

<210>21

<211>232

<212>PRT

<213> human (Homo sapiens)

<400>21

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 5560

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210>22

<211>498

<212>DNA

<213> human (Homo sapiens)

<400>22

gcacccatgg cagaaggagg agggcagaat catcacgaag tggtgaagtt catggatgtc 60

tatcagcgca gctactgcca tccaatcgag accctggtgg acatcttcca ggagtaccct 120

gatgagatcg agtacatctt caagccatcc tgtgtgcccc tgatgcgatg cgggggctgc 180

tgcaatgacg agggcctgga gtgtgtgccc actgaggagt ccaacatcac catgcagatt 240

atgcggatca aacctcacca aggccagcac ataggagaga tgagcttcct acagcacaac 300

aaatgtgaat gcagaccaaa gaaagataga gcaagacaag aaaatccctg tgggccttgc 360

tcagagcgga gaaagcattt gtttgtacaa gatccgcaga cgtgtaaatg ttcctgcaaa 420

aacacagact cgcgttgcaa ggcgaggcag cttgagttaa acgaacgtac ttgcagatgt 480

gacaagccga ggcggtga 498

<210>23

<211>165

<212>PRT

<213> human (Homo sapiens)

<400>23

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1 5 10 15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

20 25 30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

35 40 45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

50 55 60

Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile

65 70 75 80

Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe

85 90 95

Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg

100 105 110

Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe

115 120 125

Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser

130 135 140

Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys

145 150 155 160

Asp Lys Pro Arg Arg

165

<210>24

<211>45

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>24

gcccagccgg ccatggccsm bgtrcagctg gtggaktctg gggga 45

<210>25

<211>45

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>25

gcccagccgg ccatggccca ggtaaagctg gaggagtctg gggga 45

<210>26

<211>45

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>26

gcccagccgg ccatggccca ggctcaggta cagctggtgg agtct 45

<210>27

<211>41

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>27

gcccagccgg ccatggccga ggtgcagctg gtggagtgtg g 41

<210>28

<211>21

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>28

cgccatcaag gtaccagttg a 21

<210>29

<211>29

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>29

ggggtacctg tcatccacgg accagctga 29

<210>30

<211>34

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>30

catgtgtaga ctcgcggccc agccggccat ggcc 34

<210>31

<211>47

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>31

catgtgtaga ttcctggccg gcctggcctg aggagacggt gacctgg 47

<210>32

<211>42

<212>DNA

<213> Artificial sequence (RenGongXuLie)

<400>32

catgtgtaga ttcctgcggc cgctgaggag acggtgacct gg 42

Claims (5)

1. An anti-VEGF antibody, characterized by: the Fc fragment consists of a heavy chain variable region and an Fc fragment, wherein the amino acid sequence of the Fc fragment is shown as SEQ ID NO:21, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: as shown at 14.

2. A nucleic acid encoding the heavy chain variable region of claim 1, having the sequence set forth in SEQ ID NO:18, respectively.

3. A vector comprising the nucleic acid of claim 2.

4. A host cell expressing the anti-VEGF antibody of claim 1, or comprising the nucleic acid of claim 2 or the vector of claim 3.

5. A pharmaceutical composition comprising the anti-VEGF antibody of claim 1 and a pharmaceutically acceptable excipient.

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