TW200944230A - Anti-VEGF monoclonal antibody - Google Patents

Abstract

The present invention provides novel monoclonal antibodies with a high binding affinity to all five isoforms of humanVEGF.

Description

200944230 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to anti-VEGF monoclonal antibodies. [Prior Art] Angiogenesis (ie, formation of new blood vessels) is necessary during embryogenesis and occurs directly after vasculogenesis [Gilbert et al. (1997) Developmental Biology [developmental biology], Sunderland (ΜΑ): Sinauer]. It also plays an important role in a variety of pathological conditions, including neoplastic [Folkman et al. (1974) Xc/v Cimcer and 19: 331-58] and some non-neoplastic diseases such as age-related macular degeneration. (AMD), rheumatoid arthritis, and diabetic retinopathy [Ohno-Matsui et al. (2001) J Cell Physiol 189: 323-333; Aiello et al. (1994) N Engl J Med 331: 1480-1487; Boulton et al. (1998) Br J 82: 561-568] ° Vascular endothelial growth factor (VEGF) is the most potent positive regulator of angiogenesis [F err ara et al (2004) and ev25: 581-611]. In the mouse model, blocking the activity of VEGF derived from tumor cells with specific antibodies inhibits tumor growth [Kim et al_ (1993) iVaiwre 362: 841-844; Liang et al. (2006) ·/· 〇/. C/zem. 281: 951_961]. Bevacizumab, a specific human antibody against human VEGF, has been approved for the treatment of colorectal cancer patients [Hurwitz et al. (2004) C/z.«. Co/oreeia/ Cawcer 4: suppl .2, S62-S68], and Ranibizumab, a recombinant human anti-200944230 body against human VEGF, is also clinically effective in patients with neovascular AMD [R〇senfeM et al· (2006) TV V. Rong / JMed 5; 355(14): 1419-31]. Salt has demonstrated that in neovascular AMD, anti-angiogenic effects are a successful method of inhibiting tumor growth and improving vision via VEGF blockade. SUMMARY OF THE INVENTION The present invention is based on the identification of an antibody (i.e., VC300) that specifically binds to the amino acid residues Asn丨〇〇 to ❹ Lysm in human VEFG (SEQ ID NO: 9). Such an antibody has an unexpectedly low heart value (i.e., < 2 X 1〇-9 , which neutralizes VEGF activity. Thus, the present invention provides a human VEGF-specific monoclonal antibody that binds to human VEFG (SEQ ID NO: the amino acid residue Asni(R) in 9) to [ys 1 〇7, and having a Kd value of less than 2 X 10 9 。. In one example, such a single antibody has one or more SEQ ID NOs: heavy chain variable region of the amino acid sequence of 1, 2, and 3, and light chain variable region comprising one or more SEq m N〇s: amino acid sequences of 4, 5, and 6 Preferably, the antibody of the present invention has a heavy chain variable including all of the amino acid sequences SEQ ID NOs: 1, 2, and 3 (i.e., having the amino acid sequence of SEQ ID NO: 10) a region, and a light chain variable region comprising all of the amino acid sequences SEQ ID NOs: 4, 5, and 6 (i.e., having the amino acid sequence of SEQ ID NO: 11). Reference herein is directed to an antibody composition having a population of homologous antibodies (ie, a full-molecule immunoglobulin or a fragment thereof). In one example, the individual resistance system of the invention is In another example, the anti-system has a full-molecule immunoglobulin fragment with antigen-binding activity, such as a Fab, Fab', F(ab)2, or F(ab,) 2 fragment. 200944230 The monoclonal antibody may also be a non-naturally occurring molecule, such as a chimeric antibody, a humanized antibody, or a single chain antibody. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as , having a variable region derived from a murine corpus callosum and a human immunoglobulin constant region. The human anti-systematic non-human antibody, wherein the framework regions (FRs) of νΗ and VL are replaced by FRs of human antibodies. Single-stranded anti-system A single-chain polypeptide comprising both a heavy chain variable region and a light chain variable region.

Also within the scope of the invention includes the use of any of the above-described monoclonal antibodies to treat VEGF-related angiogenic diseases such as cancer, age-related macular degeneration (AMD), rheumatoid arthritis, and diabetic retinopathy. It is carried out by administering a therapeutically effective amount of the antibody to a subject in need of such treatment. "Delling Taiwan" refers to the administration of a composition comprising an active agent to a subject having a VEGF-associated vascular neoplasm, a disease, a symptom of a disease, or a tendency toward the disease, the purpose of which is to treat 'healing, Slow, change, remedy, improve, improve, or affect the disease, the symptoms of the 7th, or the tendency toward the disease. "Effective amount" in this article is the amount of each pure agent, which is required for the therapeutic effect of the subject in relation to - or multiple activities (IV); As is well known in the art I know that the effective amount will vary depending on the route of administration and the use of other active agents. The monoclonal antibodies of the present invention and the antibodies of the present invention can also be used as a medicament for the treatment of diseases. 4 VEGF Dependent Gray Tube New Details of the present invention - or a plurality of specific examples are shown in the following description. Other features, objects, and advantages of the invention will be apparent from the description and claims. [Examples] The following abbreviations are used throughout the present invention: AMD = age-related macular degeneration; CDR = complementarity determining region; CNV = choroidal neovascularization; HUVEC = human umbilical vein epithelial cells; mAb = monoclonal antibody; = vascular endothelial growth factor. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined. The term "Kd" is used herein to refer to the dissociation constant of a particular antibody-antigen effect or the "separation" rate divided by the "linkage" rate. For the purposes of the present invention, Kd was determined as shown in Example 4. As used herein, the term "VEGF" or "human VEGF" refers to the full length human vascular endothelial growth factor having the amino acid sequence of SEQ ID NO: 9 [Leung et al. (1989) 5c246: 1306-1309 ], or an isoform or fragment thereof, which has the effect of a platelet-derived growth factor. As mentioned in Ferrara et al., five isoforms can be produced from full-length VEGF by selective anterior [Ferrara (1999) / Mo/ Med 77: 527-543]. Based on structural and functional studies, it was confirmed that the receptor binding domain of VEGF is between the amino acid residues Gly8 to Asp1Q9 of full-length VEGF [Muller et al. (1997) Structure 5: 1325-1338; Fuh et al., (2006) J. Biol. Chem. 200944230 281: 6625-6631]. In the present invention, a recombinant VEGF3m (SEQ ID NO. 8, Figure 1A) is used for immunization of mice, and anti-monoclonal antibodies are obtained. The present invention provides an anti-VEGF monoclonal antibody which can bind to amino acid residues Asn(10) to Lys丨〇7 of human VEFG (SEQ m N〇:9) at a value of less than about 2 X 10 9 M2Kd (all five of which are A consensus epitope of human VEGF isoforms). While binding to VEGF, this anti-vegf antibody neutralizes 0 VEGF, thereby inhibiting angiogenesis. To prepare an antibody of the present invention, a peptide containing the epitope can be coupled to a carrier protein (e.g., KLH), mixed with an adjuvant, and injected into a host animal. The antibody prepared in the animal can then be purified by peptide affinity chromatography. Commonly used host animals include rabbits, mice, guinea pigs, and rats. Various adjuvants may be used depending on the host species to increase the immune response, and include Freund's adjuvant (complete and incomplete), mineral gel, such as aluminum hydroxide, CpG, interfacial activity. Substances such as hemolysis® lecithin, pluronic multi-fermentation, polyanions, peptides, oily lotions, keyhole limpet hemocyanin and dinitrophenol. Useful human adjuvants include BCG (Bacillus Calmette-Guerin) and C. rynebacterium parvum ° Asn (10) to LySl for human VEFG. 7 A multi-drug antibody having specificity is present in the serum of the immunized subject as described above. Individual antibodies can then be prepared using standard fusion techniques (see, for example, K〇h丨er et a丨.

Nature 256, 495; Kohler et al. (1976) Eur. J. Immunol. 6, 511, Kohler et al. (1976) V. W J Jiaqing (10) / 6,292; and 9 200944230

Hammerling et al. (1981) Monoclonal Antibodies and T Cell Hybridomas [single antibody and T cell fusion tumor], Elsevier, N.Y.). In particular, any technique for the preparation of antibody molecules can be provided by continuous cell lines in culture (such as those described in Kohler et al. (1975) 256, 495 and U.S. Patent No. 4,376,110); Human B cell fusion Tumor technology (Kosbor et al. (1983) Immunol Today 4, 72; Cole et al. (1983) 'House iASW 80, 2026); and £BV-fused tumor technique (Cole et al. (1983) Monoclonal Antibodies and Cancer Therapy [Single antibody and cancer therapy], Alan R Liss, Inc, pp. 77-96) Obtained monoclonal antibodies. Such antibodies can be of any immunoglobulin class, including IgG, IgM 'IgE, IgA, IgD, and any subtype thereof. The fusion tumor producing the monoclonal antibody of the present invention can be cultured in vitro or in vivo. The ability to produce high titers of monoclonal antibodies in vivo makes it a particularly useful method of preparation. Once the fusion cell line is established, the affinity of the monoclonal antibody produced by it for Asn(10) to Lys1〇7 of human VEFG can be determined by methods known in the art' and can be identified as having a value less than 2x 1〇-9M2Kd. A monoclonal antibody against systemic immunoglobulin molecules produced by a fusion tumor cell line. Such immunoglobulin molecules can, for example, be enzymatically cleaved to produce a functional fragment of the immunoglobulin, e.g., a fragment that retains antigen binding activity. Such functional fragments include Fab, Fabl, F(ab)2, or F(abt)2 fragments. The term "Fab fragment" as used herein refers to an antibody fragment comprising a strange domain of the 杈 chain and a first constant domain (CH1) of the heavy chain. "(4)·fragment" refers to an antibody fragment that differs from a Fab fragment in that it adds a number of residues at the carboxy terminus of the heavy chain (10) domain 200944230, including one or more vesicles taken from the hinge region of the antibody. Amino acid. The vocabulary "F(ab')2 fragment" refers to an antibody fragment which is prepared as a paired Fab' fragment with hinged cysteine in between. Such forms can be prepared by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant expression. The anti-VEGF antibodies of the invention can also be prepared by recombinant techniques. An example of this will be described later. The gene encoding the antibody heavy chain variable φ region (VH) and the light chain variable region (VL) is selected from the fusion tumor cell producing the antibody. Based on the amino acid sequences of VH and VL, the complementarity determining regions (CDRs) and framework regions (FRs) of both the heavy chain variable region and the light chain variable region can be determined. See www.bioinf.org.uk/abs. The polypeptide can then be expressed in a host cell and reconstituted in vivo or in vitro to form an antibody of the invention. • Techniques for making non-naturally occurring antibody molecules (e.g., chimeric antibodies, humanized antibodies, or single chain antibodies) are also well known in the art. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; ❹ Neuberger et al (1984) iVaiwre 3 1 2, 604; and Takeda et al. (1984) iVaiwre 314: 452. In one embodiment, a monoclonal antibody of the invention has a heavy chain variable region comprising one or more of the following VH-CDRs and a light chain variable region comprising one or more of the following VL-CDRs:

Vh-CDR1 : GYFMN (SEQ ID NO: 1);

Vh-CDR2: RINPYTGETLYNQKFKD (SEQ ID NO: 2); Vh-CDR3: RGYAGSGGIYSMDS (SEQ ID NO: 3); Vl-CDR1: TASSAVSSSYLH (SEQ ID NO: 4); 11 200944230 VL-CDR2: STSNLAS (SEQ ID NO : 5); VL-CDR3: HQYHRSPYT (SEQ ID NO: 6). In addition, it is possible to prepare variants of the antibodies of the invention having high affinity for VEGF using standard molecular biology techniques. A variant herein refers to a molecule whose amino acid sequence differs from the parent amine by the addition, deletion, and/or substitution of one or more amino acid residues in one or more CDRs of the parent antibody. Base acid sequence. The amino acid sequence of the variant CDR typically has at least 75% (e.g., < 80%, 85%, 90%, 95%, 98%, or 99%) sequence identity to the parent antibody CDR. The monoclonal antibodies of the present invention can be incorporated into a pharmaceutical composition suitable for administration to a subject. Generally, the pharmaceutical compositions comprise a therapeutically effective amount of a monoclonal antibody of the invention, together with a pharmaceutically acceptable diluent, carrier, and/or excipient. By "acceptable" is meant that the carrier must be compatible with the active ingredient of the composition (and preferably, the active ingredient is stable) and is not deleterious to the subject to be treated. The pharmaceutical composition for administration is designed to be suitable for the administration route of the drug, and a pharmaceutically acceptable diluent, carrier, and/or excipient such as a dispersing agent, a buffering agent, a surfactant, and a preservative. Agents, solubilizers, isotonic agents, stabilizers, and the like, are used as appropriate. ^ The monoclonal antibodies of the invention may also be embedded in microcapsules. For example, the monoclonal antibodies can be prepared by a coacervation technique or interfacial polymerization in a drug delivery system, such as 'liposome and nanoparticles, via standard or common techniques in any art. The above pharmaceutical composition can be used for the treatment of various VEGF-related gray tube neoplasias. The VEGF-associated angiogenic disease can include, but is not limited to, 12 200944230 AMD, rheumatoid arthritis, and diabetic retinopathy. It can be administered via a suitable route to a subject in need of such treatment, e.g., intravenous, intraperitoneal, subcutaneous, transpulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In a preferred embodiment of the invention, the monoclonal anti-system is formulated into a pharmaceutical composition, such as a solution suitable for intravenous injection. The effectiveness of the monoclonal antibodies of the present invention in preventing or treating diseases can be improved by administering the antibodies in series or in combination with another agent effective for such targets. These other agents may be present in the administered composition or may be administered separately. At the same time, the antibody may be administered in a series or in combination with radiation therapy, whether it involves radiation exposure or administration of radioactive material. Non-therapeutic uses of the monoclonal antibodies of the invention include, but are not limited to, affinity purification and diagnostic assays. The following specific examples are to be construed as illustrative only and not limiting in any way. In the absence of further elaboration, the skilled artisan can utilize the invention to its fullest extent in accordance with the teachings herein. All publications cited herein are hereby incorporated by reference in their entirety. Example 1 Production of anti-VEGF monoclonal antibody The cDNA encoding VEGF3_" 4 (SEQ ID NO: 7) was cloned and expressed in a prokaryotic system. The VEGFV^4 was purified to homogeneity for mouse immunization. Monoclonal antibodies were produced according to the method of Kohler [K〇hler (1975) 256: 495]. Briefly, six weeks old balb/c male 13 200944230 mice were immunized four times with the recombinant VEGF3.m. Lymphocytes were harvested and fused with osteoblastoma cells by polyethylene glycol 1500 (Roche Diagnostics GmbH, Mannheim, Germany) to produce fusion tumor cells. The fusion tumor cells are seeded and grown in a selection medium containing hypoxanthine, aminoguanidine, and thymidine (Invitrogen Corporation, Carlsbad, CA) to allow hypoxanthine 7 scutellaria ribosyltransferase - Growth of positive cells. The culture broth was collected and the presence of antibodies reactive with VEGF 3.114 antigen was screened by ELISA. Application 限制 Limit dilution to select positive production lines for antibody production. After several rounds of selection, a pure line producing a high level of anti-VEGF antibody was obtained and designated as VC300. Example 2 The epitope of VC3〇0 mAb was circled to elucidate the binding epitope of VC300 mAb. In the mapping, 'VEGF3.114 (SEQ ID NO: 8) and five VEGF fragments VEGF3-33, VEGF3_63, VEGF3_93 were used in the mapping. , VEGF 3.100, and VEGF3-107 (see Figure 1B). The nucleotide sequences of all such constructs were verified by DNA sequencing. The expression profile of the fusion protein was analyzed by Western blot analysis using 12.5% 10 SDS-PAGE and anti-five-His monoclonal antibody (Qiagen, Valencia, CA). These lysates were used in the validation of the binding epitope of the VC300 mAb. Peptide of various VEGF cDNAs containing expression vectors at 0.4 mM isopropyl-stone-D-thiogalactopyranoside (IPTG, Sigma-Aldrich, St. Louis, MO) at 37 ° C Fragmented E. coli (five co/i) cells were induced for 3 hours to express recombinant proteins, which were then centrifuged at 1 Torr, rpm for 2 minutes to harvest the cells. The cell pellets were suspended, lysed with protein sample buffer, boiled for 10 minutes, and analyzed by 12.5% SDS-PAGE. For the West 14 200944230 square point collapse analysis, the gel was electrolyzed onto the PVDF membrane and then incubated at room temperature in phosphate buffered saline (pH 7.4) containing 0.05% Tween 20 at 5% skim milk. The membranes were blocked for 1 hour and probed with a 1:1000 dilution of anti-five-His monoclonal antibody or VC300 mAb, followed by HRP-complex goat anti-mouse immunoglobulin (Chemicon Int., Temecula, CA). ) carry out the reaction. By adding diaminobenzidine (Sigma-Aidrich, St. Louis, MO) chromogenic reagent, 0.05% diaminobenzidine, 0.005% hydrogen peroxide in 0.1 φ M Tris-HCl (pH 7.4), The binding reaction is detected. It is expected that the epitope may span a contiguous region on the antigen, as the VC3 00 mAb recognizes VEGF3_114. Under this hypothesis, five VEGF cut-off fragments were used in the validation of binding epitopes. All of these VEGF fragments were well expressed in E. coli cells, such as by SDS-PAGE (Figure " 1C, left panel) and Western blot analysis using anti-five-His monoclonal antibodies (data not shown) Revealed. In Figure 1C (right panel), these results show that VC300 mAb does not react with the four fragments Φ vegf3_33, VEGF3.63, VEGF3-93, VEGF3-100, except for VEGF3.1〇7 and VEGF3.114. . These results indicate that the amino acid residues Asn100 to Lys1()7 of full length VEGF (SEQ ID NO: 9) provide a binding epitope on VEGF that can be recognized by the VC300 mAb. Example 3 Identification of VC300 mAb cDNA Total mRNA was extracted from 5 x 106 fusion tumor cells producing VC300 mAbs using an mRNA purification kit Oligotex Maxi Kit (Qiagen, Valencia, CA). cDNA was synthesized using RT-PCR according to the design of Amersdorfer et al. (1997) / «/eci 65(9): 3743-3752. The resulting cDNA of 15 200944230 was subcloned into a sputum vector (Invitrogen, CA, USA) and sequenced. The deduced amino acid sequence of VH and VL is shown in Figures 2A and 2B. In the VH sequence, it contains the heavy chain hypervariable region CDR1, CDR2, and CDR3, and the sequences thereof are GYFMN (SEQ ID NO: 1), RINPYTGETLYNQKFKD (SEQ ID NO: 2), and RGYAGSGGIYSMDS (SEQ ID NO: 3). In the VL sequence, the sequences of CDR1, CDR2, and CDR3 are TASSAVSSSYLH (SEQ ID NO: 4), STSNLAS (SEQ ID NO: ❹ 4), and HQYHRSPYT (SEQ ID NO: 6), respectively. BLAST searches were performed on the amino acid sequences (especially CDRs) of these VH& VL fragments from the NCBI database, and these data indicate that these sequences are novel CDRs in mouse monoclonal antibodies VH and VL. These data indicate that the VC300 mAb is a novel anti-VEGF monoclonal antibody and has a clinically useful value. Example 4 Preparation and Characterization of VC300 mAb The VC300 mAb was purified from mouse ascites by sulfuric acid precipitation and CM Sepharose chromatography (GE Healthcare, 〇 Piscataway, NJ). The purified VC300 mAb was analyzed by 7.5% SDS-PAGE and the protein concentration was quantified by absorbance at 280 nm. Isotype I of the VC300 mAb was determined by assay kit I of a single antibody isotype taken from PIERCE (Rockford, IL, USA). The separation constant (Kd) of antigen-antibody action was determined by a competitive ELISA [Lin et al., (1997) 254: 9-17]. Briefly, various concentrations of VEGF3_114 were incubated overnight at 4 °C with the antibody VC3 00 mAb to achieve equilibrium. The mixtures were transferred to wells coated with 3 μg/ml VEGF 14 and incubated for 1 hour at room temperature. By adding goat anti-mouse composite 16 200944230 HRP (horseradish peroxidase) followed by 2,2-carbazino-bis-3ethylbenzo-thiazoline-6 sulfonic acid (ABT, Sigma- Aldrich, St. Louis, MO) was incubated to show the amount of VC300 mAb attached to the well. The absorbance at 450 nm was measured and the Kd value was calculated as described above. The isoform of VC3 00 mAb was determined to be igGl (data not shown) and its Kd was approximately 2 X 10_9 Μ as shown in Figure 3. Because of its high affinity for VEGF, the VC300 mAb can effectively reduce VEGF-induced new blood vessel growth in vitro or in vivo. Example 5 VC3〇0 mAb neutralizing activity in vitro To evaluate the activity of VC300 mAb in vitro, HUVEC proliferation analysis was performed. Purchased 'HUVECs (H-UV001) from the Bioresource Conservation and Research Center (Hsinchu, Taiwan) in a gelatin-coated flask containing 20% (v/v) fetal bovine serum (FBS, Hyclone, Utah, USA), 3 μg/ml endothelial cell growth supplement (ECGS, Sigma-Aldrich, St. Louis, MO), 5 IU/ml heparin (Sigma-Aldrich, St. Louis, MO), 1 mM guanidinium pyruvate, The culture was carried out at 37 ° C and 5% C02 in M 199 medium (Invitrogen Corporation, Carlsbad, CA) of 2 mM glutamine, 100 U/ml penicillin, and 0.1 mg/ml of cinnamycin. For this proliferation assay, HUVECs were seeded at 1.5 cells (v/v) FBS, 5 IU/ml heparin, 1 mM pyruvate, 2 mM glutamine, 100 U at a density of 4000 cells/well. /ml penicillin, and 0.1 mg/ml streptomycin in Ml 99 medium, and incubated overnight at 37 °C. The HUVECs were challenged for 32 hours in the presence of increasing concentrations of VC300 mAb at 13.6 ng/ml of VEGF 3-114. Then BrdU was added and the 17 200944230 HUVECs were additionally incubated for an additional 16 hours. BrdU was included in the BrdU cell proliferation assay purchased from Calbiochem (San Diego, CA). The proliferation of HUVEC treated with VEGF at 13.6 ng/ml was increased approximately 3-fold compared to the non-VEGF control group at 48 h. The effect of VEGF was reduced to a baseline amount in the presence of about 2 nM anti-VEGF monoclonal antibody VC300 mAb. As shown in Figure 4, it plots the HUVEC proliferation curve with an IC50 value of approximately 1 nM. These data indicate that the VC300 mAb specifically binds to VEGF and neutralizes VEGF ❿ activity. Example 6 VC3〇0 mAb in vivo neutralizing activity CNV was generated by a previously described technique plus modification [Tobe et al., (1998)" / «乂 153: 1641-1646]. All experiments were conducted in accordance with the Declaration of Association for Research in Vision and Ophthalmology (ARVO) for the use of animals in ophthalmic and visual research. Briefly, 0.15 ml/kg of 2% lidocaine (Xylocaine; Astra, Astra Sodertalje, ❺ Sweden) and 50 mg/ml ketamine (Ketalar;

Intramuscular injection of an equal volume mixture of Parke-Davis, Morris Plains, NJ) Anesthetized brown Norwegian (BN) rats. After anesthesia, the right pupil was dilated with 1% tropicamide (1% Mydriacyl; Alcon Laboratories). A small piece of transparent sheet (3M, Minneapolis, MN) having a diameter of about 3 mm was adhered to the cornea as a contact lens by sodium hyaluronate (Healon; Pharmacia and Upjohn). The laser parameters used to transmit the krypton laser (Novus Omni; Coherent, Palo Alto, CA) via a slit lamp (Carl Zeiss, Oberkochen, Germany) are as follows: spot size = l 〇〇 mm, work 200944230 rate = 120 to 160 mW, and exposure period = 0, 1 second. Try to destroy the Bruch's membrane, as evidenced by the formation of central vacuoles, with or without retinal or intrachoroidal hemorrhage. Four lesions were created between the main retina vessels in the right fundus. These CNV lesions were examined on day 14 by ophthalmic microscopy, fundus photography, and conventional fluorescein angiography (FAG) using a digital fundus camera (Retinal Angiography; Heidelberg Engineering, Heidelberg, Germany). After the lesion was produced, the lesion was treated with saline or VC300 mAb and the rat retina was examined again by fluorescein angiography. Two weeks after induction of CNV by argon laser, fluorescein angiography of the rat retina was performed and fluorescence leakage at the initial laser lesion site was examined. As shown in Figures 5A and 5B, the intensity of the fluorescent leak indicates that the CNV lesion is successfully produced in the right and left eyes. Two weeks after induction of CNV, lesion sites were treated with or without VC300 mAb and fluorescein angiography of the rat retina was analyzed. These data show that the VC300 mAb effectively reduces the extent of fluorescence leakage as shown in Figure G 5C (VC300 mAb treatment group) and 5D (saline control group). These results indicate that the VC300 mAb neutralizes VEGF to reduce angiogenesis. Other Specific Examples All of the features disclosed in this patent specification can be combined in any combination. Each of the features disclosed in this patent specification may be substituted for other alternative features of the same, equivalent, or similar purpose. Therefore, unless expressly stated otherwise, the various features disclosed are merely examples of equivalent or similar features of the same series. From the above description, those skilled in the art can readily ascertain the invention of the present invention, and the present invention can be modified and modified to suit various uses and without departing from the spirit and scope thereof. condition. Therefore, other specific examples are also included in the scope of the following patent application. [Simplified description of the drawings] ° The contents of the invention and the foregoing embodiments will be read in conjunction with the attached drawings. Get a better understanding. For the purposes of the present invention, the presently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the exact arrangements and means shown. In these figures: Figure 1A shows the sequence of VEGF3·"4 (SEQ ID NO: 8), where α_helix and /5-strand according to Wiesmami et al [Wiesmann et al. (1998) • 扪07 37 :17765-17772] The bottom line is drawn and labeled, while the binding epitope of VC3〇〇mAb is shown in bold. Figure 1B shows various cut VEQF segments. Figure 1C shows the expression of these VEGF fragments in E. coli as determined by SDS-PAGE (left panel, ]V!=label), and binding epitope verification by Western blot analysis (right) Side panel). 2A and 2B show the amino acid sequence of the VC300 mAb in the heavy chain variable region (SEQ ID NO: 10) and the light chain variable region (SEq id NO: 11), respectively. These CDRs were developed according to Kabat et al. [(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 1991; 91-3234] The analysis method determines and draws the bottom line. Figure 3 shows the binding affinity of the VC3 00 mAb to human VEGF3_114. Figure 4 illustrates the effect of VC3 00 mAb on VEGF-induced HUVEC proliferation inhibition 200944. Figure 5 illustrates the effect of the VC300 mAb on reducing the extent of laser photocoagulation-induced CNV in rat mode. Rat retinal angiography after 2 weeks of CNV induction by argon laser is shown in A (right eye) and B (left eye), followed by another week of different treatment. The latter is shown in C (treated by VC300 mAb) ) and D (salt control group). The arrow represents the fluorescent leak area of the laser damage site. #

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Claims (1)

200944230 X. Application for Patent Park: 1. An anti-human antibody to endothelial growth factor (VEGF) monoclonal antibody, wherein the antibody can be less than about 2 X 1 〇-9 1^[Kd value binds to human VEFG (SEQ ID N〇) :9) comprising an epitope of an amino acid residue Asni(R) to LySl7, wherein the antibody has one or more amino acids selected from the group consisting of SEq ID NOs: 1, 2, and 3. A heavy chain variable region of the sequence, and a light bond variable region comprising one or more amino acid sequence φ columns selected from the group consisting of SEQ ID NOs: 4, 5, and 6. 2. The monoclonal antibody of claim 1, wherein the anti-stupid pain-containing amino acid "base acid sequence SEQ ID NOs: heavy chain variable region of 1, 2, and 3, and amino acid sequence SEQ ID NOs : Light chain variable regions of 4, 5, and 6. 3. The monoclonal antibody of claim 2, wherein the antibody has a heavy chain variable region comprising an amino acid sequence - SEQ ID NO: 10, and a light chain variable comprising the amino acid sequence SEq ID NO: 11 Area. 4. The monoclonal antibody of claim 1, wherein the anti-system is a functional fragment of a full-molecule immunoglobulin. 5. The monoclonal antibody of claim 4, wherein the fragment is Fab, Fab, F0b&gt;2, or F(ab丨)2. 6. The monoclonal antibody of claim 2, wherein the anti-system is selected from the group consisting of functional fragments of a full-molecule immunoglobulin consisting of Fab, Fab, F(ab)2, or F(ab')2. 7. The monoclonal antibody of claim 1, wherein the anti-system humanized antibody. 8. The monoclonal antibody of claim 2, wherein the anti-system humanized antibody. A method for treating a VEGF-associated angiogenic disease, comprising: 22, 2009, 430, </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; The _ related angiogenesis disease is selected from the group consisting of cancer, age-related macular degeneration (AMD), rheumatoid arthritis, and diabetic retinopathy. 11. The method of claim 10 wherein the VEGF-associated angiogenic disorder is age-related macular degeneration (AMD). The method of claim 9, wherein the anti-system is selected from the group consisting of Fab, Fab, F(ab)2, .F(ab')2. . The method of claim 9, wherein the antibody has a heavy chain variable region comprising an amino acid sequence. 〇s. 1, 2, and 3, and an amino acid sequence comprising SEQ I〇n〇s: 5. The light chain variable region of ό. The method of claim 13, wherein the antibody has a heavy chain variable region comprising an amino acid sequence seq 1〇, and a cyclic bond variable region comprising an amino acid sequence Seq1N〇:11. © 15. The method of claim 9, wherein the anti-system humanized antibody. 16. The method of claim 13, wherein the anti-system humanizes the antibody. The method of claim 13, wherein the VEGF-related angiogenic disease is a group consisting of free cancer, age-related macular degeneration (AMD), rheumatoid arthritis, and diabetic retinopathy. 18. The method of claim 17, wherein the VEGFjg angiogenic disease is age-related macular degeneration (AMD). 19. The method of claim 13, wherein the anti-system is selected from the group consisting of Fab, Fab', F(ab)2, or F(at)l)2. The method of claim 14, wherein the anti-system is selected from the group consisting of Fab, Fab1, F(ab)2, or F (ab%) of a full-molecular immunoglobulin fragment. twenty four