Throughout this application, various publications are referenced within parentheses. The disclosures of these publications are hereby incorporated by reference herein in their entireties.
BACKGROUND OF THE INVENTION
Age-related macular degeneration (AMD), is the leading cause of vision loss and blindness among people with age 60 and above. AMD is diagnosed as either dry (non-neovascular) or wet (neovascular) AMD. About 85 to 90 percent of AMD patients are diagnosed with dry AMD. It is an early stage of disease which may result from the aging and thinning of macular tissues, resulting in deposition of pigment characterized by yellow spots called drusen in macula. Gradual central vision loss may occur with dry macular degeneration but usually is not nearly as severe as wet AMD.

In a significant proportion of cases, dry AMD progresses to the more advanced and damaging form of the disease, wet AMD, leading to serious vision loss. Wet AMD is characterized by the process of choroidal neovascularization which involves the formation of new blood vessels (angiogenesis) beneath the retina resulting in leakage of blood and fluid. This leakage causes permanent damage to light-sensitive retinal cells, resulting in vision loss. Vascular endothelial growth factor (VEGF) appears to play a pivotal role in the pathogenesis of choroidal neovascularization.

Treatment methods for wet AMD include laser photocoagulation, photodynamic laser therapy with Visudyne® (Valeant Pharmaceuticals International) and anti-VEGF drugs. The discovery of anti-VEGF agents has revolutionized treatment of the condition. Currently, the four anti-VEGF agents either approved or in common use include pegaptanib (Macugen®, Valeant Pharmaceuticals International), ranibizumab (Lucentis®, Novartis and Roche), aflibercept or

VEGF Trap-Eye (EYLEA®, Bayer and Regeneron Pharmaceuticals) and bevacizumab (Avastin®, Roche).

Anti-VEGF therapy may be used for treating wet AMD. However, current treatment involves giving frequent injections of the anti-VEGF drug into the affected eye once a month for three months and then, monitoring the treatment to give further injections. The frequent administration of injections leads to complications such as macular edema and stress in the eyes of patients. If macular edema is prolonged, retinal thinning, scarring or retinal hole can eventually form. As such there remains an urgent need for improved drugs and methods for treating diseases associated with abnormal vascularization, such as wet AMD.

SUMMARY OF THE INVENTION

This invention encompasses novel anti-VEGF antibodies; and polynucleotides comprising sequences encoding such novel anti-VEGF antibodies and compositions, including pharmaceutical compositions, comprising them and uses thereof.

The present invention also provides using the novel anti-VEGF antibodies, polynucleotides comprising sequences encoding such novel anti-VEGF antibodies and compositions, including pharmaceutical compositions in methods for inhibiting or treating diseases such as wet age related macular degeneration (AMD), diabetic maculopathy, proliferative diabetic retinopathy, macular edema in retinal vein occlusion (RVO), iris neovascularization, choroidal neovascularisation caused by pathological myopia, retinopathy of maturity, neovascular glaucoma and cancer.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTING

FIGURE 1. Standard curve for determining Fab protein concentration based on bovine serum albumin (BSA) as a reference standard and absorbance at S9S nm using the Bradford protein assay showing linearity from 50 μg/mL to 500 μg/mL of BSA.

FIGURE 2. Standard curve for quantifying amount of Fab with anti-VEGF activity relative to known concentrations of anti-VEGF Fab (Lucentis* Novartis and Roche) in an ELISA format with immobilized human VEGF (hVEGF) 121 isoform and horse radish peroxidase (HRP)-conjugated anti-kappa light chain antibody to detect soluble anti-VEGF Fab bound to immobilized hVEGF and generate a colorimetric product with absorbance at 450 nm.

FIGURE 3. Competitive binding assay to determine the half maximal inhibitory concentration (IC50) of ranibizumab and Fab Clone #201 (Fab 201) for hVEGF-121. The Fabs were incubated in two separate experiments with various concentration of hVEGF-121 reported in the x-axis. The unbound fraction of each Fab was captured with a hVEGF-121 coated plate and quantified with anti-Kappa-light-chain-HRP-conjugated antibody, as shown on the y-axis. ΙΟΟρΜ of each Fab was incubated with serial dilutions of hVEGF-121. Unbound Fabs were captured with immobilized hVEGF-121 and detected with anti-Kappa-light-chain-HRP-conjugated antibody. Kd estimated from the ICS0 calculated from the data fitted with a 5 parameter asymmetric model with Prism software are shown in Table 3.

FIGURE 4a-b. CDR sequences of Fabs obtained from screening a naive synthetic antibody fragment (Fab) phage display library with recombinant hVEGF 121 isoform (A), and analysis of binding affinity using immobilized hVEGF and serial dilutions of soluble selected Fabs from the naive phage display library in a Fab-ELISA format (B). Amino acid sequences corresponding to the CDR according to Kabat are underlined (A) and titration by Fab-ELISA performed as follows: increasing concentration of Fabs were incubated on wells coated with 2ug/mL of hVEGF-121. The bound Fabs were detected with anti-Kappa-light-chain-HRP-conjugated antibody (B). The dissociation constant (Kd) was estimated from the 50% effective maximal concentration (EC50) and reported in the table below the line graph.

FIGURE S. A bar graph showing inhibition of binding of selected Fabs to immobilized hVEGF in the presence (+) or the absence (-) of FLT, a VEGF natural receptor, as a competitor in a competitive Fab-ELISA format, consistent with the 6 Fabs and FLT having overlapping binding sites on hVEGF. Wells of a microtiter plate were coated with 2ug/mL of hVEGF. Washed wells were pre-incubated with either 500nM FLT (R&D systems, catalog number 321-FL-250) in PBT (+) or PBT alone (-

) for 1 hour. Sub-saturating amounts of Fabs were then added to either the FLT- or the FLT+ wells and plates were incubated for IS minutes and washed. The identity of the Fab added to the well is identified on the y-axis. Bound Fabs were then detected with an anti-Kappa-light-chain-HRP antibody. The background subtracted OD 450nm measurement is reported on the y-axis.

FIGURE 6a-c. (A) CDR sequences of Fabs issued from the selection of libraries based on FFO 124-1 Fab sequence. Fabs are ranked by residual binding observed in the competition-phage-ELISA when ΙΟηΜ of hVEGF was preincubated with the Fab-phage. (B and C) Competitive Phage-ELISA results for different clones with the sequence listed in panel A. The residual binding of the Fab-phage to immobilized hVEGF after preincubation with InM (B) or ΙΟηΜ (C) of hVEGF is shown in percentage of binding without hVEGF preincubation. (*) CDR-H3 (defined by Kabat) also had "HAWYYGWAFDY" sequences in this group of clones. (**) CDR-H3 (defined by Kabat) also had "HAWYYGWALDY" and "HAWYYGWAFDY" sequences in this group of clones. The sequence and competitive phage ELISA for the parental Fab, FFO 124-1, is shown for comparison. Amino acid sequences corresponding to the CDR accordingly to Kabat are underlined. Amino acid sequences that are different from the parental Fab (FFO 124-1) are highlighted in bold.

FIGURE 7a-b. (A) CDR sequences of Fabs issued from the selection of libraries based on FF01S8-C4 Fab sequence, yields in shake flasks and affinity measurements by single point competitive ELISA and/or competitive ELISA (Kd - data and fit shown in B). Amino acid sequences corresponding to the CDR accordingly to Kabat are underlined. Amino acid sequences different from the parental Fab (FF0158-C4) are highlighted in bold.

FIGURE 8. CDR sequences of Fabs issued from the selection of libraries based on FF0188-H5 Fab sequence, production yields in shake flasks, affinity measurements by single point competitive ELISA and/or competitive ELISA and melting temperature (Tm). Amino acid sequences corresponding to the CDR accordingly to Kabat are underlined. Amino acid sequences different from the parental Fab (FF0188-H5) are highlighted in bold.

FIGURE 9a-c. Fabs CDR sequences, production yield and hVEGF binding kinetic parameters are shown. (A) Table with CDR sequences, yields in shake flasks, hVEGF-121 binding off rates measured by SPR and melting temperature (Tm) - data shown in B. Amino acid sequences corresponding to the CDR accordingly to abat are underlined Amino acid sequences different from the parental Fab (FFO 117-A5) are highlighted in bold. (B) Thermal shift assay. (C) Example of full length sequences for Fab FF03046-2.

FIGURE 10. Inhibition of hVEGF-dependent human vascular endothelium cell (HUVEC) proliferation by anti-VEGF Fabs, FF03046-1, FF03046-2 and Ranibizumab (Lucentis®; Novartis and Roche) in which recombinant human VEGF 165 isoform is used to stimulate proliferation of HUVEC cells. 2x10s cells/ml of log-phase Huvec cells were added to serial dilutions of Fabs pre-incubated with 260pM of rhVEGF-165. Cells were then incubated at 37°C for 3 days before. The number of live cells was assessed by resazurin staining.

FIGURE 11. Competitive Fab-ELISA. 5pM of hVEGF-165 was incubated with serial dilutions of anti-VEGF antibody as described in the figure legend. Pre-formed h VEGF- 165/anti- VEGF antibody complexes along with any unbound hVEGF-165 in the binding solution are transferred into a 96-well polystyrene microplate coated with a monoclonal antibody specific for human VEGF, as provided in the Quantikine® ELISA Human VEGF Immunoassay (R&D Catalog Number: SVE00). hVEGF-165 bound to the plate is detected using a polyclonal anti-hVEGF antibody conjugated to horseradish peroxidase and tetramethylbenzidine as a chromagen to quantify hVEGF-165 bound to the plate as provided in the Quantikine® assay kit. Kd calculated from the data fitted with a 5 parameter asymmetric model with Prism software are shown in the table at the bottom of the figure.

FIGURE 12. DNA and protein sequence of FF03092-1 , the anti-VEGF Fc-Fab fusion.

FIGURE 13. DNA and protein sequence of FF03077-4, the anti-VEGF Fc-scFv fusion. Single chain nucleic acid sequence: the scFv fusion sequence is underlined; the Fc sequence is in italic; the linker between the Fc and the scFv is underlined and in bold DNA coding sequences and amino acid sequences corresponding to the CDRs defined in accordance with IMGT® are shown

in bold; DNA coding sequences and amino acid sequences corresponding to CDR sequences of Fab FF03046-2 defined in accordance with abat in the scFv are double underlined.

FIGURE 14. Competitive ELISA. 5pM of hVEGF-165 was incubated with serial dilutions of anti-VEGF antibody as described in the figure legend. Pre-formed hVEGF-165/anti-VEGF antibody complexes along with any unbound hVEGF-165 in the binding solution are transferred into a 96-well polystyrene microplate coated with a monoclonal antibody specific for human VEGF, as provided in the Quantikine® ELISA Human VEGF Immunoassay (R&D Catalog Number: SVE00). hVEGF-165 bound to the plate is detected using a polyclonal anti-hVEGF antibody conjugated to horseradish peroxidase and tetramethylbenzidine as a chromagen to quantify hVEGF-165 bound to the plate as provided in the Quantikine® assay kit. Kd calculated from the data fitted with a 5 parameter asymmetric model with Prism software are shown in the table at the bottom of the figure.

FIGURE 15. DNA and protein sequence of FF03092-3, the anti-VEGF full length mature IgGl.

FIGURE 16. DNA and protein sequence of anti-VEGF Fab #216 with an alternative embodiment coding sequence from Example 3 indicated by a double underline.

FIGURE 17. Schematic representation of expression plasmid for anti-VEGF Fab 201.

FIGURE 18. Schematic representation of expression plasmid for anti-VEGF Fab 216.

FIGURE 19. Competitive binding assay to determine the half maximal inhibitory concentration (IC50) of Lucentis (Ranibizumab, Novartis and Roche) and Fab Clone #216 (Fab 216) for hVEGF-121. The Fabs were incubated in two separate experiments with various concentration of hVEGF-121 (Peprotech, Cat-100-20A) reported in the x-axis. The unbound fraction of each Fab was captured with a hVEGF-121 coated plate and quantified with anti-Kappa-light-chain-HRP-conjugated antibody, as shown on the y-axis. 100pM of each Fab #216 was incubated with serial dilutions of hVEGF-121. Unbound Fabs were captured with immobilized hVEGF-121 and detected with anti- appa-light-chain-HRP-conjugated antibody. Kd estimated from the IC50 calculated from the data fitted with a 5 parameter asymmetric model with Prism software are shown in the table at the bottom of the figure

FIGURE 20. Competitive -ELISA. ΙΟρΜ of hVEGF-165 was incubated with serial dilutions of anti-VEGF antibody, Fab 216. Pre-formed hVEGF- 165/anti-VEGF antibody complexes along with any unbound hVEGF-165 in the binding solution are transferred into a 96-well polystyrene microplate coated with a monoclonal antibody specific for human VEGF, as provided in the Quantikine® ELISA Human VEGF Immunoassay (R&D Catalog Number: SVE00). hVEGF-165 bound to the plate is detected using a polyclonal anti-hVEGF antibody conjugated to horseradish peroxidase and tetramethylbenzidine as a chromagen to quantify hVEGF-165 bound to the plate as provided in the Quantikine® assay kit. Kd was estimated using IC50 calculated from the data fitted with a S parameter asymmetric model with Prism software are shown in the table at the bottom of the figure.

FIGURE 21. Fermentation data and expression yield for anti-VEGF Fab.

FIGURE 22. Stability of drug product Fab 201 at 4°C.

FIGURE 23. Alignment of immunoglobulin heavy chain variable region of Fab 201 and Lucentis® (ranibizumab). Lines above and below the aligned immunoglobulin heavy chain variable region sequences indicate the location of the CDRs of Fab 201 as defined by Kabat and IMGT® methods, respectively, and given in SEQ ID NO: 16. Sequence of Lucentis® (ranibizumab) is obtained from GenBank Accession Number APZ76728.1 and APZ76729.1.

FIGURE 24. Alignment of immunoglobulin kappa ligh chain variable region of Fab 201 and Lucentis® (ranibizumab). Lines above and below the aligned kappa light chain variable region sequences indicate the location of the CDRs of Fab 201 as defined by Kabat and IMGT® methods, respectively, and given in SEQ ID NO: 16. Sequence of Lucentis® (ranibizumab) is obtained from GenBank Accession Number APZ76728.1 and APZ76729.1.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which relevant embodiments of the invention belong.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "an antibody" includes a plurality of such antibodies. Also, as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative ("or").

As used herein, "at least one" is intended to mean "one or more" of the listed elements.

As used herein, the term "substantially free" includes being free of a given substance or cell type or nearly free of that substance or cell type, e.g. having less than about 1 % of the given substance or cell type.

As used herein, the term vascular endothelial growth factor "VEGF" refers, unless specifically or contextually indicated otherwise, to any native, variant or synthetic VEGF polypeptide, or fragment thereof, of the VEGF family which comprises seven members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-G and PLGF, wherein all members have a common VEGF homology domain composed of a cysteine knot motif with eight invariant cysteine residues involved in inter- and intramolecular disulfide bonds at one end of a conserved central four stranded β-sheet within each monomer, which dimerize in an antiparallel, side-by-side orientation, and participate in angiogenesis (reviewed in Hoeben, A., et al. (2004) Vascular endothelial growth factor and angiogenesis. Pharmacol. Rev. 56, 549-580; see Figure 1 of Hoeben et al. (2004) for a 3D structure of cysteine knot motif with inter- and intramolecular disulfide bonds). Each member within the VEGF family is encoded by a distinct gene; however, each member may comprise a number of different isoforms due to alternative splicing or proteolysis.

In a preferred embodiment, VEGF is VEGF-A and its isoforms that play a role in promoting or maintaining angiogenesis. For example, alternative splicing of the primary transcript of the VEGF-A gene, also referred to in the literature as VEGF without reference to "-A", produces different VEGF-A mRNAs encoding different VEGF-A isoforms, indicated often simply as VEGF followed by a number, with different isoforms having a pro- angiogenic or anti-angiogenic property depending on the presence of an alternatively spliced exon 8a or 8b of the VEGF-A gene. Of particular interests are VEGF-A isoforms obtained from translation of the alternative spliced exon 8a, which are generally pro-angiogenic, in contrast to those with exon 8b, which are generally anti-angiogenic. In humans, the exon 8a-containing VEGF-A isoforms from alternative

splicing comprise: VEGF-A121 (also, often called VEGFm), VEGF-A145 (also, often called VEGFMS), VEGF-Ai48 (also, often called VEGFHS), VEGF-Aies (also, often called VEGFies), VEGF-A183 (also, often called VEGF|83), VEGF-A|89 (also, often called VEGF189) and VEGF-A206 (also, often called VEGF206) (Nowak, D. G., et al. (2008) Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors. J. Cell Sci. 121, 3487-3495). The number following VEGF in each isoform of VEGF- A refers to the number of amino acids, after signal sequence cleavage following secretion; this number may be a subscript or inline, such as VEGF121 or VEGF121, respectively. In rodents, such as mouse, orthologs of these VEGF isoforms contain one less amino acid. VEGF is normally a homodimeric glycoprotein with its isoforms differing in their abilities to bind heparin. The larger highly basic VEGF189 and VEGF206 isoforms bind tightly to cell-surface heparin-containing proteoglycans in the extracellular matrix (ECM); whereas, the acidic VEGF121 isoform lacks ability to bind heparin and is freely diffusible. VEGF 165 isoform has intermediate properties with a significant fraction bound to heparin and ECM. In addition to VEGF isoforms produced by alternative splicing, the ECM-bound VEGF isoforms may undergo proteolysis to generate a bioactive fragment with high mitogenic activity such as VEGFuo or VEGF100 from proteolysis of VEGF 165 or VEGF 189 of human VEGF-A isoforms (Ferrara, N., Gerber, H.-P., and LeCouter, J. (2003) The biology of VEGF and its receptors. Nature Medicine 9, 669-676; also, see Hoeben, A., et al. (2004) above). In addition to VEGF 100, any fragment of VEGF protein derived from the VEGF family of genes, either through natural proteolysis or man made, may be considered as VEGF so long as an antibody may be raised against or be directed to it, and the VEGF protein fragment has a role in promoting or maintaining angiogenesis, macular degeneration, tumor or human disease.

In addition to isolating VEGF protein from a subject or cultured cells, VEGF protein may be produced by recombinant DNA methods. For example, VEGF protein for any VEGF isoform or fragment or variant may be produced in a bacterium, a yeast, an insect cell or a mammalian cell or be produced in vitro in cell-free translation extract or coupled transcription-translation extract, as is known in the art. In the case of production in a bacterial cell, the resulting recombinant VEGF protein produced in bacterium can have the same primary amino acid sequence but may lack post translational modification such as glycosylation unlike VEGF protein isolated from mammalian cells. Nevertheless, the present disclosure encompasses VEGF produced in a bacterial cell,

produced in vitro, such as using an in vitro cell-free translation system or coupled transcription-translation system, or produced by recombinant methods, in addition to VEGF isolated from a natural source. A VEGF antigen may be naturally occurring, recombinant or synthetic VEGF molecule. The VEGF antigen may be present on an intact VEGF protein, VEGF isoform, a fragment of VEGF or a peptide with a sequence derived from a portion of VEGF.

As used herein, "wild type VEGF sequence" generally refers to a primary amino acid sequence found in a naturally occurring VEGF isoform or derived from translation of the VEGF mRNA following processing of the primary VEGF gene transcript in a mammalian cell or derived from the translation of any VEGF cDNA. The VEGF cDNA may be obtained, for example, by reverse transcription of polysomal VEGF mRNA from a mammalian cell, such as a mouse or human cell.

As used herein, "antibody" includes monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies (e.g., antibodies with more than one antigen binding site of the same specificity so as to permit association with more than one antigen depending on the valency and increased avidity observed for example for bivalent IgG antibody versus its monovalent Fab antibody fragment), multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). An antibody can be human, humanized, affinity matured and/or synthetic. An antibody may be produced by any method known in the art, such as, for example in vivo in an animal, in tissue or cell culture, or in vitro protein synthesis systems by enzymatic and/or chemical systhesis methods. Recombinant DNA methods may be used in the production of an antibody, and the resulting antibody may be considered to be a recombinant antibody. A recombinant antibody may be produced free of animal products such as produced in a bacterium. An antibody or its fragment may be displayed on the surface of a bacteriophage.

"Antibody fragment" or "antibody fragments" are only a portion of an antibody, wherein the portion retains at least one, preferably many or all, of the functions normally associated with that portion when present in an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules including single-chain Fv (scFv) antibody molecule; multivalent antibodies formed from multiple

copies of the same antibody fragment with the same specificity; and multispecific antibodies formed from antibody fragments.

A synthetic human anti-VEGF antibody includes an "affinity matured" antibody which includes one or more changes in one or more hypervariable regions or complementarity determining regions (CDRs) thereof, which result in an improvement in the affinity of the antibody for VEGF, compared to a parent antibody which does not possess those changes. CDRs found in the variable region of an antibody may be defined by Kabat method or IMGT method (Martin, A. C. R. (1996) Accessing the Kabat Antibody Sequence Database by Computer PROTEINS: Structure, Function and Genetics 25: 130-133; Johnson, G. and Wu, T. T. (2004) The Kabat Database and a Bioinformatics Example. Methods in Molecular Biology 248: 1 1-25; Kabat, E. A.,Wu, T. T., Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, MD.; MacCallum, R. M., Martin, A. C. R. and Thornton, J. T. (1996) Antibody-antigen interactions: Contact analysis and binding site topography. J. Mol. Biol. 262, 732-745; Lefranc, M.-P., Pommii, C, Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, G. (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev. Comp. Immunol. 27: 55-77; and Lefranc, M.-P. (2005) IMGT, the international ImMunoGeneTics information system®. Nucleic Acids Res. 33: D593-D597; Lefranc, M.-P. et al. (2009) IMGT®, the international ImMunoGeneTics information system®. Nucleic Acids Res. 37: D1006-D1012). The specificity of an antibody may be defined or described by a set of CDRs which may be defined by a number of methods including Kabat or IMGT® method.

The antigen-binding region of an antibody may include an antigen-binding site formed by the CDRs of the light chain variable region and heavy chain variable region.

A disorder or disease is a condition that would benefit from treatment with anti-VEGF antibodies/compositions or method of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cell proliferative disorders. The cell proliferative disorder may include a disease such as wet age related macular degeneration, diabetic maculopathy, proliferative diabetic retinopathy, macular edema in retinal vein occlusion (RVO), iris neovascularization, choroidal neovascularisation caused by pathological myopia, retinopathy of maturity, neovascular glaucoma, diabetic retinopathy, retinal neovascularization, pars plana vitrectomy (PPV), diabetic macular edema (DME) or cancer.

As used herein, "treating" means using a therapy to ameliorate a disease or disorder or one or more of the biological manifestations of the disease or disorder; to directly or indirectly interfere with (a) one or more points in the biological cascade that leads to, or is responsible for, the disease or disorder or (b) one or more of the biological manifestations of the disease or disorder; to alleviate one or more of the symptoms, effects or side effects associated with the disease or disorder or one or more of the symptoms or disorder or treatment thereof; or to slow the progression of the disease or disorder or one or more of the biological manifestations of the disease or disorder. Treatment includes eliciting a clinically significant response. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or disorder. For example, treatment of an eye condition may improve the symptoms of the condition, reduce the severity of a condition, alter the course of condition's progression and/or improve the basic condition. Treatment may also include improving quality of life for a subject afflicted with the disease or disorder (e.g., a subject afflicted with a cancer may receive a lower dose of an anti-cancer drug that cause side-effects when the subject is immunized with a composition of the invention described herein). Throughout the specification, compositions of the invention and methods for the use thereof are provided and are chosen to provide suitable treatment for subjects in need thereof.

A "subject" may be a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, farm animals (such as cows, sheeps, and goats), sport animals, pets (such as cats, dogs and horses), primates (such as, monkeys, gorillas and chimpanzees), mice and rats.

As used herein, an "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

In order that the invention herein described may be more fully understood the following description is set forth.

Compositions of the Invention and Methods of Making Same

This invention encompasses novel anti-VEGF antibodies; and polynucleotides comprising sequences encoding such novel anti-VEGF antibodies and compositions, including pharmaceutical compositions, comprising them. The terms "pharmaceutical formulations", "pharmaceutical compositions" and "dosage forms" are used interchangeably herein and refer to a composition containing the active ingredient(s) of the invention in a form suitable for administration to a subject.

As used herein, compositions comprise one or more antibodies of the invention that bind to VEGF, and/or one or more polynucleotides comprising sequences encoding one or more antibodies that bind to VEGF. These compositions may further comprise suitable carriers, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art.

Merely by way of example, an antibody is a protein that may take the shape of a Y which may be created by joining two immunoglobulin light chains and two immunoglobulin heavy chains, wherein one light chain associates with one heavy chain to form one arm of the protein. The two arms join through the association of one heavy chain to the other. Typically, each arm of the antibody recognizes the same antigen and binds an epitope on the antigen through the antigen binding site formed by the variable region of the associated light and heavy chain. The portion of the variable region of the light and heavy chain that bind to the epitope are called hypervariable regions or "complementarity determining regions (CDRs)." An antibody's specificity for a particular antigen depends on the amino acid sequences of the hypervariable region or CDRs, which show greatest variability between antibodies of different binding specificities. The

remaining non-hypervariable regions or non-CDRs called the framework regions (FRs). For example, there may be three CDRs from amino-to-carboxyl terminal of a variable region called CDRl-3 for each light chain and each heavy chain, which are separated by four FRs, namely, FR1-4. To distinguish the light chain CDR from heavy chain CDR, the light chain CDRl-3 are designated CDRL1-CDRL3, or alternatively CDR-L1 to CDR-L3. Similarly, the heavy chain CDRl-3 are designated CDRH1-CDRH3 or CDR-H1 to CDR-H3. The four FRs in the light chain variable region are designated FRL1-FRL4 or FR-L1 to FR-L4. Further, the four FRs in the heavy chain variable region are designed FRH1-FRH4 or FR-H1 to FR-H4. Accordingly, the typical variable region is formed by attaching from the amino-to-carboxyl terminus for the light chain in the order FRL 1 -CDRL 1 -FRL2-CDRL2-FRL3-CDRL3-FRL4. Similarly, the heavy chain is formed in the order FRH 1 -CDRH 1 -FRH2-CDRH2-FRH3-CDRH3-FRH4. The light chain variable region is followed by a kappa or lambda light chain constant region, which completes the light chain. Whereas, the heavy chain variable region is joined at its C-terminus to constant regions, CHI, CH2 and CH3 along with a hinge region between CHI and CH2 with cysteine residue(s) that participate in an interstrand crosslink with the hinge region of the second heavy chain. Sequence of the heavy chain constant region determines the class of immunoglobulin (IgA, IgD, IgE, IgG, or IgM) and within each class determines the subclass (e.g., for IgG class, the IgGl, IgG2, IgG3 or IgG4 subclass).

The complementarity determining region (CDR) or hypervariable region of an immunoglobulin light and heavy chains may be defined based on sequence comparison as by Elvin A. Kabat and colleagues (Kabat, E.A., Wu, T. T., Perry, H. M., Gottesman, K. S. and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Volumes 1-3. 5th edition. NIH Publication No. 91-3242 (ISBN: 094137565X, 9780941375658), with non-CDR sequences designed as specific FRs, depending on the relationship to each CDR. Alternatively, another method for identification of CDRs in immunoglobulin was developed by Marie-Paule Lefranc and colleagues to produce the IMGT® charts and methods for identifying CDRs (Lefranc, M.-P., et al. (1999) IMGT, the international ImMunoGeneTics database. Nucl. Acids Res. 27, 209-212; Lefranc, M.-P., et al. (2015) IMGT®, the international ImMunoGeneTics® information system 25 years on. Nucl. Acids Res. 43, D413-D422). Both the Kabat method and IMGT method for identifying CDRs are used herein. Both methods produce either identical CDR sequences or overlaps between CDR

sequences or one CDR sequence contained in its entirety with the CDR sequence determined for the corresponding CDR by the other method. In cases where the CDR sequences identified by both methods failed to yield same length or are overlapping, the framework regions (by default the region outside of the CDR or between CDRs in the variable region) are not identical in size or similarly will have overlapping sequences.

In one embodiment, the invention provides an isolated anti-VEGF antibody or portion or variant thereof having a heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences: CDRHl comprising histidine at amino acid position 31 to histidine at amino acid position 35 (SEQ ID NO: 1), CDRH2 comprising tyrosine at amino acid position 50 to glycine at amino acid position 66 (SEQ ID NO: 1) and CDRH3 comprising histidine at amino acid position 99 to tyrosine at amino acid position 109 (SEQ ID NO: 1).

A variant of an anti-VEGF antibody of the invention may include antibodies that comprise an amino acid sequence wherein one or more amino acid residues are different relative to the amino acid sequences of any of the Y variants disclosed herein, (e.g., see Example 3 and Figures 9C and 16) e.g., changes due to amino acid residues that are inserted into, deleted from and/or substituted in the framework region but no amino acid residues are inserted into, deleted from and or substituted in the the CDR regions (e.g. either as defined by abat numbering or IMGT numbering; or any other CDR identification system such as Chothia nomenclature). Variants include antibody fragments, such as Fab fragment and single chain Fv (scFv) fragment. Variants may further include fusion proteins, such as e.g., in Figures 12 and 13. Variants and antibodies of disclosed herein may be produced by recombination DNA methods.

A portion of an isolated antibody of the invention includes useful immunologically functional fragments or provides specificity of binding an antigen or epitope on an antigen (e.g. recognizes and binds a VEGF) such as a CDR region, a variable domain of a heavy and/or light chain or a portion of an antibody chain that binds a VEGF. It would be clear to one skilled in the art that the antibodies of the invention can be camelized.

For example, an isolated antibody or portion or variant thereof is one which has been separated and/or recovered from its production environment. The production environment may be cell-based, e.g., cells, or may be cell free, e.g., in vitro translation or coupled transcription-translation cell-free system. In preferred embodiments, the antibody will be purified (a) to greater than about 95% by weight of antibody as determined by, e.g., the Lowry method, and most preferably more than about 99% by weight or (b) to homogeneity by SDS-PAGE under reducing or nonreducing conditions. As used herein, the term "about" when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or (-) 10 %, 5 % or 1 %.

In another embodiment, the invention provides an isolated anti-VEGF antibody or portion or variant thereof having a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences: CDRL1 comprising arginine at amino acid position 24 to alanine at amino acid position 34 (SEQ ID NO: 3), CDRL2 comprising lysine at amino acid position 50 to alanine at amino acid position 56 (SEQ ID NO: 3), and CDRL3 comprising glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 3).

In yet an additional embodiment, the anti-VEGF antibody or portion thereof may further comprise a heavy chain variable domain comprising the following CDR amino acid sequences defined in accordance with IMGT numbering shown in bold in Figure 16: CDRH1 comprising amino acid sequences GFDLDHYS, CDRH2 comprising amino acid sequences IYPSYGYT and CDRH3 comprising amino acid sequences ARHAWYYGWGLDY; and a light chain variable domain comprising the following CDR amino acid sequences defined in accordance with IMGT numbering shown in bold in Figure 16: CDRL1 comprising amino acid sequences QAAYGR, CDRL2 comprising amino acid sequences KAS and CDRL3 comprising amino acid sequences QQRGWYLFT

An additional embodiment provides the anti-VEGF antibody or portion thereof further comprising a heavy chain variable domain comprising the following CDR amino acid sequences: CDRH1 comprising histidine at amino acid position 31 to histidine at amino acid position 35 (SEQ ID NO:

1), CDRH2 comprising tyrosine at amino acid position 50 to glycine at amino acid position 66 (SEQ ID NO: 1) and CDRH3 comprising histidine at amino acid position 99 to tyrosine at amino acid position 109 (SEQ ID NO: 1).

The invention provides a humanized anti-VEGF antibody or portion thereof. The anti-VEGF antibody of the invention inhibits VEGF-induced angiogenesis in vivo.

Also, the invention provides synthetic human anti-VEGF antibodies or portion or variant thereof. In one embodiment, it comprises a light chain variable domain having the following hypervariable region or complementarity determining region (CDR) amino acid sequences: a CDRL1 which comprises arginine at amino acid position 24 to alanine at amino acid position 34 (SEQ ID NO: 3) or a portion thereof, a CDRL2 which comprises lysine at amino acid position 50 to alanine at amino acid position 56 (SEQ ID NO: 3) or a portion thereof, and a CDRL3 which comprises glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 3) or a portion thereof; and a heavy chain variable domain having the following hypervariable region or complementarity determining region (CDR) amino acid sequences: a CDRH1 which comprises histidine at amino acid position 31 to histidine at amino acid position 35 (SEQ ID NO: 1) or a portion thereof, a CDRH2 which comprises tyrosine at amino acid position 50 to glycine at amino acid position 66 (SEQ ID NO: 1) or a portion thereof and a CDRH3 which comprises histidine at amino acid position 99 to tyrosine at amino acid position 109 (SEQ ID NO: 1 ) or a portion thereof.

Examples of anti-VEGF antibodies of the invention include, but are not limited to, variants 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219 and 220 as shown in SEQ ID NOS: 21-28, respectively.

In yet an additional embodiment, the anti-VEGF antibody may further comprise a heavy chain framework region (FR) 1 , FRH 1 , amino sequence comprising glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1).

In yet an additional embodiment, the anti-VEGF antibody may further comprise a heavy chain framework region (FR) 2, FRH2, amino sequence comprising tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1).

In yet an additional embodiment, the anti-VEGF antibody may further comprise a heavy chain framework region (FR) 3, FRH3, amino sequence comprising arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1).

In yet an additional embodiment, the anti-VEGF antibody may further comprise a heavy chain framework region (FR) 4, FRH4, amino sequence comprising tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1).

In yet an additional embodiment, the anti-VEGF antibody may further comprise the following heavy chain framework region (FR) amino acid sequences: FRH1 comprising glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1), FRH2 comprising tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1), FRH3 comprising arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1) and FRH4 comprising tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1).

In yet an additional embodiment, the anti-VEGF antibody may further comprise a light chain framework region (FR) 1, FRL1, amino sequence comprising aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3).

In yet an additional embodiment, the anti-VEGF antibody may further comprise a light chain framework region (FR) 2, FRL2, amino sequence comprising tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3).

In yet an additional embodiment, the anti-VEGF antibody may further comprise a light chain framework region (FR) 3, FRL3, amino sequence comprising glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3).

In yet an additional embodiment, the anti-VEGF antibody may further comprise a light chain framework region (FR) 4, FRL4, amino sequence comprising phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3).

In yet an additional embodiment, the anti-VEGF antibody may further comprise the following light chain framework region (FR) amino acid sequences: FRL1 comprising aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3), FRL2 comprising tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3), FRL3 comprising glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3) and FRL4 comprising phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3).

In yet an additional embodiment, the anti-VEGF antibody or portion or variant thereof may further comprise: (c) a light chain variable domain having the following framework region amino acid sequences: a FRL1 which comprises aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3) or a portion thereof, FRL2 which comprises tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3) or a portion thereof, FRL3 which comprises glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3) or a portion thereof, and FRL4 which comprises phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3) or a portion thereof; and (d) a heavy chain variable domain having the following framework region amino acid sequences: a FRH1 which comprises glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1) or a portion thereof, FRH2 which comprises tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1) or a portion thereof, FRH3 which comprises arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1) or a portion thereof, and FRH4 which comprises tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1) or a portion thereof.

The invention also encompasses isolated anti-VEGF antibody and polynucleotide embodiments. The invention also encompasses substantially pure antibody and polynucleotide embodiments.

The anti-VEGF antibodies of the invention may be monoclonal (e.g., full length or intact monoclonal antibodies). In an embodiment of the invention, the anti-VEGF antibody of the invention is a full length antibody. Further, in one example, the full length antibody comprises a light chain comprising the amino acid sequence shown in SEQ ID NO:3 and a heavy chain shown in SEQ ID NO:l. In another embodiment, the full length antibody comprises a light chain comprising the amino acid sequence shown in SEQ ID NO:3 and a heavy chain shown in SEQ ID NO:l, a light chain comprising the aspartic acid at amino acid position 24 to cysteine at amino acid position 237 (SEQ ID NO: 17) and a heavy chain comprising the glutamic acid at amino acid position 24 to threonine at amino acid position 251 (SEQ ID NO: 18) (e.g. heavy chain Fab 216; Fig. 16), or a light chain comprising the aspartic acid at amino acid position 24 to cysteine at amino acid position 237 (SEQ ID NO:76) (e.g., light chain Fab 216; Fig. 16) and a heavy chain comprising the glutamic acid at amino acid position 24 to threonine at amino acid position 251 (SEQ ID NO:78) (e.g., heavy chain Fab 216; Fig. 16)

Also encompassed within the scope of the invention are Fab, Fab', Fab'-SH and F(ab')2 fragments of the anti-VEGF antibodies provided herein. These antibody fragments can be created by traditional means, such as enzymatic digestion, or may be generated by recombinant techniques, as well as through the use of chemical methods. For example, Fab'-SH, Fab' with reduced -SH group such as at the cysteine that plays a role in interstrand cross link between two heavy chains, may be produced by recombinant techniques or by chemical methods by reducing the disulfide bonds of F(ab')2 antibody fragment. Such antibody fragments may be chimeric or humanized. Fab and scFv fragments may be engineered to form dimers, trimers or tetramers by chemical or genetic crosslinks to improve retention and internalization properites as compared with the parent IgG. These fragments are useful for the diagnostic and therapeutic purposes set forth below.

A Fv fragment contains a complete antigen-recognition and -binding site. In a single-chain Fv species, a single heavy- and a single light-chain variable domain may be covalently linked by a peptide linker such that the light and heavy chains may associate in a "dimeric" structure. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv

comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

A Fab fragment contains a single antigen-binding site, and a residual "Fc" fragment. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH 1 ) of the heavy chain. Fab' fragments are encompassed herein and differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments are encompassed herein and are a pair of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are known and encompassed herein.

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. For example, the antibodies of the invention may be any of immunoglobulin class: IgA, IgD, IgE, IgG, and IgM. Moreover, within these classes, they can be further divided into subclasses (isotypes) including any of, e.g., IgGi, Ig(j2, IgG3, IgG4, IgAi, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins may be any of α, δ, ε, γ, and μ, respectively.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

The anti-VEGF monoclonal antibodies of the invention can be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods.

Additionally, the anti-VEGF antibodies of the invention can be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities. In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage

libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution. Any of the anti-VEGF antibodies of the invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-VEGF antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

Chimeric antibodies of the invention are immunoglobulin molecules that comprise a human and non-human portion. The antigen combining region (variable region) of a chimeric antibody can be derived from a non-human source (e.g. murine) and the constant region of the chimeric antibody which confers biological effector function to the immunoglobulin can be derived from a human source. The chimeric antibody should have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule.

In general, the procedures used to produce chimeric antibodies can involve the following steps: a) identifying and cloning the correct gene segment encoding the antigen binding portion of the antibody molecule; this gene segment (known as the VDJ, variable, diversity and joining regions for heavy chains or VJ, variable, joining regions for light chains or simply as the V or variable region) may be in either the cDNA or genomic form;

b) cloning the gene segments encoding the constant region or desired part thereof;

c) ligating the variable region with the constant region so that the complete chimeric antibody is encoded in a form that can be transcribed and translated;

d) ligating this construct into a vector containing a selectable marker and gene control regions such as promoters, enhancers and poly(A) addition signals;

e) amplifying this construct in bacteria;

f) introducing this DNA into eukaryotic cells (transfection) most often mammalian lymphocytes;

g) selecting for cells expressing the selectable marker;

h) screening for cells expressing the desired chimeric antibody; and

k) testing the antibody for appropriate binding specificity and effector functions.

The invention encompasses multiple types of vectors. The term vector is any molecule or entity used to transfer protein coding information into a host cell including, but not limited to, plasmids, bacteriophage, virus, cosmids, and phagemids. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as recombinant expression vectors or expression vectors. In general, expression vectors for use in recombinant DNA techniques may be in the form of plasmids.

Antibodies of several distinct antigen binding specificities have been manipulated by these protocols to produce chimeric proteins [e.g. anti-TNP: Boulianne et al., Nature 312:643 (1984); and anti-tumor antigens: Sahagan et al., J. Immunol. 137:1066 (1986)]. Likewise, several different effector functions have been achieved by linking new sequences to those encoding the antigen binding region. Some of these include enzymes [Neuberger et al., Nature 312:604 (1984)], immunoglobulin constant regions from another species and constant regions of another immunoglobulin chain [Sharon et al., Nature 309:364 (1984); Tan et al., J. Immunol. 135:3565-3567 (1985)]. Additionally, procedures for modifying antibody molecules and for producing chimeric antibody molecules using homologous recombination to target gene modification have been described (Fell et al., Proc. Natl. Acad. Sci. USA 86:8507-8511 (1989)).

The antigen-binding domain of an antibody is formed from two variable (V) regions of about 110 amino acids, one each from the light (VL) and heavy (VH) chains, that both present three hypervariable loops or complementarity-determining regions (CDRs). Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covaiently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently. "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen

binding. As used herein, scFv encoding phage clones and Fab encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones".

"Hypervariable regions" are the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. In general, antibodies may comprise six hypervariable regions. These include three regions in the variable heavy chain (VH) which are designated HI, H2, H3. Additionally, there are three in the variable light chain (VL) which are designated LI, L2, L3. A number of hypervariable region delineations are in use and are encompassed herein. For example, the abat Complementarity Determining Regions (CDRs) are based on sequence variability and may be used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR). Then the clones may be recombined randomly in phage libraries and searched for antigen-binding clones. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization. Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro.

Filamentous phage may be used to display antibody fragments. The antibody fragments then can be displayed as single chain Fv fragments. The VH and VL domains may be connected on the same polypeptide chain by a flexible polypeptide spacer, or as Fab fragments, in which one chain is fused to pill of the filamentous phage and the other is secreted into the bacterial host cell periplasm where assembly of a Fab-coat protein structure which becomes displayed on the phage surface by displacing some of the wild type coat proteins.

In general, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If a library biased in favor of anti-VEGF clones is desired, the subject is immunized with VEGF to generate an antibody response, and spleen cells and/or circulating B cells other peripheral blood lymphocytes (PBLs) are recovered for library construction. In a preferred embodiment, a human antibody gene fragment library biased in favor of anti-VEGF clones is obtained by generating an anti-VEGF antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that VEGF immunization gives rise to B cells producing human antibodies against VEGF. The generation of human antibody-producing transgenic mice is described below.

Nucleic acids (also referred to herein as polynucleotides) refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be ribonucleotides, deoxyribonucleotides, modified nucleotides or bases, and/or their analogs. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, including, for example, via conjugation with a label.

Percent amino acid sequence identity in connection with a peptide or polypeptide sequence of the invention means the percentage of amino acid residues in a candidate sequence that shares identity with the amino acid residues in the specific peptide or polypeptide sequence of the invention, after aligning the sequences and, if necessary, including gaps to obtain the maximum percent sequence identity.

In another embodiment, the invention embodies an amino acid sequence with a percent identity of at least 91%. In another embodiment, the invention embodies an amino acid sequence with a percent identity of a range of about 92-98%. In another embodiment, the invention embodies an amino acid sequence with a percent identity of at least 99%. In one embodiment, the amino acid sequence of the invention comprises only a portion of the entire sequence as provided in the invention, e.g., a deletion. Such a deletion may be internal or at an end. In one embodiment, the amino acid sequence of the invention comprises additional amino acids wherein the additional

amino acids are inserted within the amino acid sequence of the invention or are attached to the end. In one embodiment, the invention embodies an amino acid substitution wherein the substitution does not interfere with the binding to VEGF or neutralizing VEGF function. Such substitutions are often conservative amino acid substitution wherein the substituting amino acid has functionally equivalent physiochemical properties, such as aliphatic (glycine, alanine, valine, leucine, isoleucine), hydroxyl or sulfur/selenium-containing (serine cysteine, selenocysteine, threonine, methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, arginine) and acidic and their amide (aspartate, glutamate, asparagine, and glutamine).

Nucleic acid encoding antibody variable gene segments (including VH and VL segments) may be recovered from the cells of interest and amplified. For example, the antibodies produced by naive libraries (either natural or synthetic) can be of moderate affinity ( d of about 106 to 107 M"1), but affinity maturation can also be mimicked in vitro by constructing and reselecting from secondary libraries. Additionally, affinity maturation can be performed by randomly mutating one or more CDRs, e.g. using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual Fv clones and screening for higher affinity clones. Another effective approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling. This technique allows the production of antibodies and antibody fragments with higher affinities with d in the about 10'9 M range.

For example, an anti-VEGF antibody, portion or variant thereof of the invention may bind human VEGF with a K(d) value of no more than about 50 nM. In an another embodiment, the anti-VEGF antibody, portion or variant thereof binds human VEGF with a K(d) value of no more than about 10 nM. In yet another embodiment, the anti-VEGF antibody, portion or variant thereof binds human VEGF with a K(d) value of no more than about 2.5 nM. In yet a further embodiment, the anti-VEGF antibody, portion or variant thereof binds human VEGF with a K(d) value of no more than about 0.5 nM. In an additional embodiment, the anti-VEGF antibody, portion or variant thereof binds human VEGF with a K(d) value of no more than about 0.15 nM. In yet additional

embodiment, the anti-VEGF antibody, portion or variant thereof binds human VEGF with a K(d) value between 5 pM and 150 pM. In a further embodiment, the anti-VEGF antibody, portion or variant thereof binds human VEGF with a K(d) value of about 0.15 nM. In another further embodiment, the anti-VEGF antibody, protion or variant thereof of the invention binds human VEGF with a K(d)value of 90 pM + 20 pM. In still a further embodiment, the anti-VEGF antibody, portion or variant thereof binds human VEGF with a K(d) value of about 25 pM. In an embodiment, the anti-VEGF antibody, portion or variant thereof binds human VEGF with a K(d) value of about 10 pM. In yet another embodiment, the anti-VEGF antibody, portion or variant thereof of the invention has a higher affinity for human VEGF than ranibizumab (Lucentis®; Novartis and Roche) or bevacizumab (Avastin®; Roche) with more than 3-fold lower K(d) value than that for ranibizumab or bevacizumab. In yet a further embodiment, the antibody or portion or variant thereof has a relative (d) value for human VEGF that is more than 4-fold lower than ranibizumab (Lucentis®; Novartis and Roche). In another embodiment, the antibody or portion or variant thereof has a relative K(d) value for human VEGF that is more than 10-fold lower than ranibizumab (Lucentis®; Novartis and Roche). In another embodiment, the antibody or portion or variant thereof has a relative K(d) value for human VEGF that is more than 50-fold lower than ranibizumab (Lucentis®; Novartis and Roche). In another embodiment, the antibody or portion or variant thereof has a relative K(d) value for human VEGF that is about 55-fold lower than ranibizumab (Lucentis®; Novartis and Roche).In still another embodiment, the antibody or portion or variant thereof has a relative K(d) value for human VEGF that is more than 10-fold lower than bevacizumab (Avastin®; Roche). In another embodiment, the antibody or portion or variant thereof has a relative K(d) value for human VEGF that is more than 50-fold lower than bevacizumab (Avastin®; Roche). In another embodiment, the antibody or portion or variant thereof has a relative K(d) value for human VEGF that is more than 100-fold lower than bevacizumab (Avastin®; Roche). In another embodiment, the antibody or portion or variant thereof has a relative K(d) value for human VEGF that is about 110-fold lower than bevacizumab (Avastin®; Roche).

In yet a further embodiment, the antibody or portion or variant thereof has an IC50 value of no more than about 200 pM for inhibiting VEGF-induced proliferation of endothelial cells in vitro. Further, an additional embodiment, the antibody or portion or variant thereof is more effective at inhibiting VEGF-induced proliferation of endothelial cells in vitro than ranibizumab (Lucentis®;

Novartis and Roche). In a further embodiment the relative IC50 value for inhibiting VEGF- induced proliferation of endothelial cells in vitro is about 1.5-fold lower than ranibizumab (Lucentis®; Novartis and Roche).

In an embodiment, the invention provides antibodies which have increased stability. In one embodiment, the anti-VEGF antibody or portion of variant thereof, has greater stability than ranibizumab (Lucentis®; Novartis and Roche). In another embodiment, the anti-VEGF antibody or portion of variant thereof, has greater storage life than ranibizumab (Lucentis®; Novartis and Roche).

In an embodiment, the invention provides antibodies which have increased thermal stability than ranibizumab (Lucentis®; Novartis and Roche). For example, the melting temperature of the humanized anti-VEGF antibody of the invention may be about 1.5 °C higher than that of ranibizumab (Lucentis®; Novartis and Roche).

In one embodiment, an anti-VEGF antibody of the invention, or portion or variant thereof, does not bind BSA or Fc.

Following construction of the DNA molecule encoding the antibodies against VEGF of the invention, the DNA molecule is operably linked to an expression control sequence in an expression vector, such as a plasmid, wherein the control sequence is recognized by a host cell transformed with the vector. In general, plasmid vectors contain replication and control sequences which are derived from species compatible with the host cell. The vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells. Suitable vectors for expression in prokaryotic and eukaryotic host cells are known in the art and some are further described herein. Eukaryotic organisms, such as yeasts, or cells derived from multicellular organisms, such as mammals, may be used.

Host cells are transfected and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as

appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaP04 precipitation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell. Methods for transfection are well known in the art.

Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. Methods for transformation are well known in the art, and some are further described herein.

Prokaryotic host cells used to produce the antibodies against VEGF of the invention can be cultured as described generally in Sambrook et al., supra.

Mammalian host cells may be used to produce the antibodies against VEGF and may be cultured in a variety of media, which is well known in the art and some of which is described herein.

The host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal. Purification of antibodies against VEGF of the invention may be accomplished using art-recognized methods, some of which are described herein. The purified antibodies can be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like, for use in the affinity chromatographic separation of phage display clones.

Phage library samples may be contacted with immobilized VEGF under conditions suitable for binding of at least a portion of the phage particles with a solid phase. Normally, the conditions, including pH, ionic strength, temperature and the like are selected to mimic physiological

conditions. The phages bound to the solid phase may be washed and then eluted. Phages can be enriched in a single round of selection; and if enriched, can be grown in bacterial culture and subjected to further rounds of selection. It is possible to select between phage antibodies of different affinities, even with affinities that differ slightly, for VEGF.

Reactivity of anti-VEGF mAbs against the target antigen may be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, VEGF proteins, peptides, VEGF-expressing cells or extracts thereof. Examples of such assays are presented in Example 1, infra.

The antibody or fragment thereof of the invention may be cytostatic to the cell, to which it binds. As used herein, "cytostatic" means that the antibody can inhibit growth, but not necessarily kill, VEGF-positive cells.

DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy chain and or light chain constant regions to form clones encoding full or partial length heavy and/or light chains. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species. In a preferred embodiment, a Fv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence(s) for all human, full or partial length heavy and/or light chains.

Antibody Fragments

The present invention encompasses antibody fragments. The smaller size of the antibody fragments compared to whole antibodies have certain advantages. For example, fragments may allow for more rapid clearance, and may lead to improved access to sites of interest when compared with whole antibodies.

For example, in one embodiment, the antibody fragment is a Fab which comprises a light chain variable domain comprising the amino acid sequence beginning at aspartic acid at position 1 and ending at lysine at position 107 of SEQ ID NO:3 and a heavy chain variable domain comprising the amino acid sequence beginning at glutamic acid at position 1 and ending at serine at position 120 of SEQ ID NO: 1.

In another embodiment, the Fab fragment is joined to a Fc region which comprises the amino acid shown in SEQ ID NO:5.

In yet another embodiment, the variant of the antibody is a recombinant protein comprising the antigen-binding region of the antibody of the invention. For example, the variant may be a scFv which comprises the amino acid sequence beginning at aspartic acid at position 241 and ending at serine at position 483 of SEQ ID NO:9. In a further embodiment of the invention, the scFv is joined to a Fc region. Merely by way of example, the scFv joined to a Fc region may comprise the amino acid shown in SEQ ID NO:9.

There are various techniques for the production of antibody fragments. The antibodies or fragments may be produced by recombinant means. For example, these fragments can be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from, e.g., E. coli, thus allowing the relatively easy production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed herein. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments. According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv.

Synthetic Human Anti-VEGF Antibodies

The invention further provides antibodies (e.g., polyclonal, monoclonal, chimeric, synthetic and humanized antibodies) that bind to VEGF. The most preferred antibodies will selectively bind to VEGF and will not bind (or will bind weakly) to non-VEGF proteins. The most preferred antibodies will specifically bind to VEGF. It is intended that the term "specifically bind" means that the antibody predominantly binds to VEGF. Anti-VEGF antibodies that are particularly contemplated include monoclonal and polyclonal antibodies as well as fragments thereof (e.g., recombinant proteins) containing the antigen binding domain and/or one or more complement determining regions of these antibodies. These antibodies can be from any source, e.g., rat, dog, cat, pig, horse, mouse or human.

In one embodiment, the anti-VEGF antibodies of the invention may be anti-VEGF neutralizing antibodies. In one embodiment, the anti-VEGF antibodies of the invention specifically bind to the VEGF protein and, e.g., inhibits VEGF-induced angiogenesis in vivo. As will be understood by those skilled in the art, the regions or epitopes of a VEGF protein to which an antibody of the invention is directed may vary with the intended application.

For example, antibodies intended for use in an immunoassay for the detection of membrane-bound VEGF on viable cancer cells or ocular cells should be directed to an accessible epitope on membrane-bound VEGF. Different VEGF isoforms may have different potential for cell membrane binding. For example, the larger highly basic VEGF 189 and VEGF206 isoforms (isoforms of VEGF-A) bind tightly to cell-surface heparin-containing proteoglycans in the extracellular matrix (ECM) and hence is found tightly bound to cells; whereas, the acidic VEGF121 isoform (VEGF-A m) lacks ability to bind heparin and is freely diffusible. VEGFies isoform *VEGF-Ai65) has intermediate properties with a significant fraction bound to heparin and ECM. Examples of such antibodies are described in the Examples which follow. Antibodies that recognize other epitopes may be useful for the identification of VEGF within damaged or dying cells, for the detection of secreted VEGF proteins or f agments thereof.

Anti-VEGF antibodies of the invention may be particularly useful in diagnostic assays, imaging methodologies, treatment of eye disease, treatment of vision impairment, prevention of blindness and therapeutic methods in the management of cancer. The invention provides various

immunological assays useful for the detection of VEGF proteins and for the diagnosis of cancer. Such assays generally comprise one or more anti-VEGF antibodies capable of recognizing and binding a VEGF protein, and include various immunological assay formats well known in the art, including but not limited to various types of precipitation, agglutination, complement fixation, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA) (H. Liu et al. Cancer Research 58: 4055-4060 (1998), immunohistochemical analysis and the like.

In one embodiment, anti-VEGF antibodies of the invention and fragments thereof (e.g., Fv, Fab', F(ab')2) and variants thereof (e.g., scFv) are used therapeutically to treat a disease selected from retinal disorder, age-related macular degeneration (AMD), macular degeneration, wet age-related macular degeneration, diabetic retinopathy, diabetic maculopathy, proliferative diabetic retinopathy, macular edema in retinal vein occulusion (RVO), macular edema secondary to Retinal vein Occlusion (RVO), iris neovascularization, retinal neovascularization, choroidal neovascularization caused by pathological myopia, macular edema, retinopathy of prematurity (ROP), retinopathy of maturity, pars plana vitrectomy (PPV), neovascular glaucoma, diabetic macular edema (DME) and eye disease associated with angiogenesis. The anti-VEGF antibodies of the invention and fragments thereof and variants thereof may be used to prevent or stop progression of the forementioned diseases or conditions.

A therapeutically effective amount of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

In one embodiment, anti-VEGF antibodies of the invention and fragments thereof (e.g., Fv, Fab', F(ab')2) are used for detecting the presence of a cancer. The anti-VEGF antibodies of the invention and f agments thereof may be used to detect the presence of an ocular cell- or endothelial cell-expressing VEGF, -responding to VEGF, or -participating in VEGF-dependent angiogenesis. The presence of such VEGF positive (+) cells within various biological samples, including serum, vitreous humor, eye, prostate and other tissue biopsy specimens, other tissues such as bone, urine, etc., may be detected with VEGF antibodies. In addition, anti-VEGF antibodies may be used in various imaging methodologies, such as immunoscintigraphy with Induim-111 (or other isotope) conjugated antibody.

Anti-VEGF antibodies may also be used in methods for purifying VEGF proteins and peptides and for isolating VEGF homologues and related molecules. For example, in one embodiment, the method of purifying a VEGF protein comprises incubating an anti-VEGF antibody, which has been coupled to a solid matrix, with a lysate or other solution containing VEGF under conditions which permit the anti-VEGF antibody to bind to VEGF; washing the solid matrix to eliminate impurities; and eluting the VEGF from the coupled antibody. Additionally, anti-VEGF antibodies may be used to isolate VEGF positive cells using cell sorting and purification techniques.

In one embodiment, an isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Tables 5a and 5c. In another embodiment, an isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising a heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Tables 5b and 5d.

In one embodiment, an isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising any of the light chain of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 comprising amino acid sequence as provided in Table 5a or 5c. In another embodiment, an isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising any of the heavy chain of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 comprising amino acid sequence as provided in Table 5b or 5d.

In one embodiment, an isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising: a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Table 5a, and a corresponding heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Table 5b.

In one embodiment, an isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Table 5c, and a corresponding heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Table 5d.

Humanized and Human Antibodies

The present invention encompasses novel humanized anti-VEGF antibodies. Humanized antibodies are chimeric antibodies that contain sequence derived from non-human immunoglobulin. For example, humanized antibodies are human immunoglobulins (recipient antibody) wherein residues from a hypervariable region of a recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as a nonhuman primate, mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin may be replaced by corresponding non-human residues. Further, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody in order to further refine antibody performance. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Chimeric antibodies may have a portion of the heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

Methods for producing fully human anti-VEGF monoclonal antibodies of the invention, include phage display and transgenic methods. For example, fully human anti-VEGF monoclonal antibodies of the invention may be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display). Fully human anti-VEGF monoclonal antibodies of the invention may also be produced using transgenic mice engineered to contain a human immunoglobulin gene.

Bispecific Antibodies

Bispecific antibodies are anti-VEGF monoclonal, preferably human or humanized, antibodies of the invention that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for VEGF and the other is for any other antigen. Exemplary bispecific antibodies may bind to two different epitopes of the VEGF protein.

In one embodiment of the invention, the bispecific antibody has a binding specificity for two different antigens, one of the antigens being that with which the antibody of invention binds. For example, the bispecific antibody may comprises an amino acid sequence comprising the amino acid sequence for CDRH1 (e.g., amino acid position 31 to histidine at amino acid position 35

(SEQ ID NO: 1)) or a portion thereof, CDRH2 (e.g., tyrosine at amino acid position 50 to glycine at amino acid position 66 (SEQ ID NO: 1)) or a portion thereof, CDRH3 (e.g., histidine at amino acid position 99 to tyrosine at amino acid position 109 (SEQ ID NO: 1)) or a portion thereof, CDRL1 (e.g., arginine at amino acid position 24 to alanine at amino acid position 34 (SEQ ID NO: 3)) or a portion thereof, CDRL2 (e.g., lysine at amino acid position 50 to alanine at amino acid position 56 (SEQ ID NO: 3)) or a portion thereof, or CDRL3 (e.g., glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 3)) or a portion thereof, or a combination thereof.

Bispecific antibodies may also be used to localize cytotoxic agents to cells which express VEGF. These antibodies possess an VEGF-binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon-.alpha., vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Methods for making bispecific antibodies are known in the art. Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of the anti-VEGF antibodies described herein are contemplated. Amino acid sequence variants of the antibody may be prepared by, e.g., introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.

Further, the contemplates Fc region variants that may be obtained by introducing one or more amino acid modifications in an Fc region of the immunoglobulin polypeptides of the invention. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or

more amino acid positions including that of a hinge cysteine. In accordance with this description and the teachings of the art, it is contemplated that in some embodiments, an antibody used in methods of the invention may comprise one or more alterations as compared to the wild type counterpart antibody, e.g. in the Fc region.

Nucleic Acids. Vectors. Host Cells and Recombinant Methods

Another aspect of the invention provides various nucleic acid molecules encoding anti-VEGF antibody and fragments thereof, preferably in isolated form, including DNA, RNA, DNA RNA hybrid, and related molecules, nucleic acid molecules complementary to the anti-VEGF antibody-coding sequence or a part thereof, and those which hybridize to the anti-VEGF antibody -encoding nucleic acids. Particularly preferred nucleic acid molecules will have a nucleotide sequence substantially identical to or complementary to the human or murine DNA sequences herein disclosed. Specifically contemplated are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acids based on an alternative backbone or including alternative bases, whether derived from natural sources or synthesized.

The invention further provides fragments of the anti-VEGF antibody-encoding nucleic acid molecules of the present invention. As used herein, a fragment of an anti-VEGF antibody-encoding nucleic acid molecule refers to a small portion of the entire anti-VEGF antibody-encoding sequence. The size of the fragment will be determined by its intended use.

In one embodiment, the invention provides an isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF. In an embodiment of the invention, said specific binding member comprises an antigen-binding portion of an antibody, wherein the immunoglobulin heavy chain or a portion thereof, of the antigen-binding portion of the antibody comprises an immunoglobulin heavy chain CDRH1 comprising the nucleic acid sequence beginning at cytosine at position 91 and ending at cytosinc at position 10S (SEQ ID NO: 1) or a portion thereof, an heavy chain CDRH2 comprising the nucleic acid sequence beginning at thymine at position 148 and ending at cytosine at position 198 (SEQ ID NO: 1) or a portion

thereof, and an heavy chain CDRH3 comprising the nucleic acid sequence beginning at cytosine at position 295 and ending at cytosine at position 327 (SEQ ID NO: 1) or a portion thereof; and/or wherein the immunoglobulin light chain or portion thereof, of the antigen-binding portion of the antibody comprises a light chain CDRL1 comprising the nucleic acid sequence beginning at cytosine at position 70 and ending at cytosine at position 102 (SEQ ID NO: 3) or a portion thereof, a light chain CDRL2 comprising the nucleic acid sequence beginning at adenine at position 148 to cytosine at position 168 (SEQ ID NO: 3) or a portion thereof, and a light chain CDRL3 comprising the nucleic acid sequence beginning at cytosine at position 265 and ending at guanine at position 291 (SEQ ID NO: 3) or a portion thereof.

For example, a nucleic acid molecule encoding an anti-VEGF antibody of the invention may be considered isolated when the nucleic acid molecule is substantially separated from contaminant nucleic acid molecules that encode polypeptides other than a VEGF antibody. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated VEGF antibody-encoding nucleic acid molecule. While achieving a high degree of purity is desirable, isolated nucleic acid need not be pure or achieve absolute purity in order to be an isolated nucleic acid.

In another embodiment, any of the nucleic acid sequence as provided in (a) or (b) above may be replaced with an equivalent nucleic acid sequence encoding for same amino acid sequence. In yet an additional embodiment, the antibody further comprises a human or humanized constant region. For example, the human or humanized constant region may be an IgGl constant region. In another example, the human or humanized constant region is an IgG4 constant region.

In one embodiment of the invention, the isolated nucleic acid comprises the sequence shown in SEQ ID NO: 16.

In still another embodiment, the isolated nucleic acid sequence encodes a specific binding member which is a single chain Fv molecule comprising the nucleic acid sequence beginning at guanine at position 721 to thymine at position 1449 as shown in SEQ ID NO: 9 or a portion thereof. In still another additional embodiment, the specific binding member is a Fab comprising the nucleic acid sequences shown in SEQ ID NOS: 1 and 3 or a portion(s) thereof. In a further embodiment, the heavy chain of the antibody comprises a FR amino acid sequence comprising the amino acid sequence beginning at guanine at position 70 to thymine at position 711 as shown in SEQ ID NO: 16 and the nucleic acid sequence beginning at guanine at position 897 to adenine at position 1580 in SEQ ID NO: 16 or a portion(s) thereof. In another embodiment, the specific binding member is a Fab comprising the nucleic acid sequence beginning at adenine at position 1 to thymine at position 711 as shown in SEQ ID NO: 16 and the nucleic acid sequence beginning at adenine at position 828 to adenine at position 1580 in SEQ ID NO: 16 or a portion(s) thereof.

In still another embodiment, the specific binding member is a Fab comprising the nucleic acid sequences shown in SEQ ID NO: 16 or a portion thereof.

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises an immunoglobulin heavy chain framework region (FR) 1 , FRH1, comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 90 (SEQ ID NO: 1) encoding an amino acid sequence glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1).

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises an immunoglobulin heavy chain framework region (FR) 2, FRH2, comprising the nucleic acid sequence beginning at thymine at position 106 and ending at adenine at position 147 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1).

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises an immunoglobulin heavy chain framework region 3,

FRH3, comprising the nucleic acid sequence beginning at cytosine at position 199 and ending at cytosine at position 294 (SEQ ID NO: 1) encoding an amino acid sequence arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1).

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises an immunoglobulin heavy chain framework region 4, FRH4, comprising the nucleic acid sequence beginning at thymine at position 328 and ending at guanine at position 360 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1).

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises a heavy chain framework region FRH1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 90 (SEQ ID NO: 1) encoding an amino acid sequence glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1), a heavy chain framework region FRH2 comprising the nucleic acid sequence beginning at thymine at position 106 and ending at adenine at position 147 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1), a heavy chain framework region FRH3 comprising the nucleic acid sequence beginning at cytosine at position 199 and ending at cytosine at position 294 (SEQ ID NO: 1) encoding an amino acid sequence arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1), and a heavy chain framework region FRH4 comprising the nucleic acid sequence beginning at thymine at position 328 and ending at guanine at position 360 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1), wherein the nucleic acid sequences encoding the heavy chain framework regions, FRH1 to FRH4, and the heavy chain complementarity determining regions, CDRH1 to CDRH3, are joined in the order FRH 1 -CDRH 1 -FRH2-CDRH2-FRH3-CDRH3-FRH4.

In one embodiment, the isolated nucleic acid comprises the nucleic acid sequence for a heavy chain variable region beginning at guanine at position 1 and ending at guanine at position 360 (SEQ ID NO: 1) encoding an amino acid sequence glutamic acid at amino acid position 1 to serine at amino acid position 120 (SEQ ID NO: 1 ).

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises an immunoglobulin light chain framework region 1 , FRL 1 , comprising the nucleic acid sequence beginning at guanine at position 1 and ending at cytosine at position 69 (SEQ ID NO: 3) encoding an amino acid sequence aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3).

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises an immunoglobulin light chain framework region 2, FRL2, comprising the nucleic acid sequence beginning at thymine at position 103 and ending at cytosine at position 147 (SEQ ID NO: 3) encoding an amino acid sequence tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3).

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises an immunoglobulin light chain framework region 3, FRL3, comprising the nucleic acid sequence beginning at guanine at position 169 and ending at thymine at position 264 (SEQ ID NO: 3) encoding an amino acid sequence glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3).

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises an immunoglobulin light chain framework region 4, FRL4, comprising the nucleic acid sequence beginning at thymine at position 292 and ending at adenine at position 321 (SEQ ID NO: 3) encoding an amino acid sequence phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3).

In an embodiment, the isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody additionally comprises a light chain framework region FRL1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at cytosine at position 69 (SEQ ID NO: 3) encoding an amino acid sequence aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3), a light chain framework FRL2 comprising the nucleic acid sequence beginning at thymine at position 103 and ending at cytosine at position 147 (SEQ ID NO: 3) encoding an amino acid sequence tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3), a light chain framework FRL3 comprising the nucleic acid sequence beginning at guanine at position 169 and ending at thymine at position 264 (SEQ ID NO: 3) encoding an amino acid sequence glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3), a light chain framework FRL4 comprising the nucleic acid sequence beginning at thymine at position 292 and ending at adenine at position 321 (SEQ ID NO: 3) encoding an amino acid sequence phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3), wherein the nucleic acid sequences encoding the light chain framework regions FRL1 to FRL4 and the light chain complementarity determining regions CDRL1 to CDRL3 are joined in the order FRL 1 -CDRL 1 -FRL2-CDRL2-FRL3-CDRL3-FRL4.

In one embodiment, the isolated nucleic acid of claim 4 or 68, wherein the nucleic acid encoding for each CDR and each FR amino acid sequence is defined by IMGT method are the following:

IMGT-defined CDRH1 comprising the nucleic acid sequence beginning at guanine at position 76 and ending at thymine at position 99 (SEQ ID NO: 79); IMGT-defined CDRH2 comprising the nucleic acid sequence beginning at adenine at position 151 and ending at thymine at position 174 (SEQ ID NO: 79); IMGT-defined CDRH3 comprising the nucleic acid sequence beginning at guanine at position 289 and ending at cytosine at position 327 (SEQ ID NO: 79); IMGT-defined CDRLl comprising the nucleic acid sequence beginning at cytosine at position 79 and ending at cytosine at position 96 (SEQ ID IMGT-defined CDRL2 comprising the nucleic acid sequence beginning at adenine at position 148 and ending at cytosine at position 156 (SEQ ID NO: 77); IMGT-defined CDRL3 comprising the nucleic acid sequence beginning at cytosine at position 265 and ending at guanine at position 291 (SEQ ID NO: 77); IMGT-defined FRHl comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 75 (SEQ ID NO: 79) encoding an amino acid sequence glutamic acid at amino acid position 1 to serine at amino acid position 25 (SEQ ID NO: 79); IMGT-defined FRH2 comprising the nucleic acid sequence beginning at adenine at position 100 and ending at cytosine at position 150 (SEQ ID NO: 79) encoding an amino acid sequence isoleucine at amino acid position 34 to tyrosine at amino acid position 50 (SEQ ID NO: 79); IMGT-defined FRH3 comprising the nucleic acid sequence beginning at thymine at position 175 and ending at thymine at position 288 (SEQ ID NO: 79) encoding an amino acid sequence tyrosine at amino acid position 59 to cysteine at amino acid position 96 (SEQ ID NO: 79); IMGT-dewfined FRH4 comprising the nucleic acid sequence beginning at thymine at position 328 and ending at guanine at position 360 (SEQ ID NO: 79) encoding an amino acid sequence tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 79); IMGT-defined FRL1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 78 (SEQ ID NO: 77) encoding an amino acid sequence aspartic acid at amino acid position 1 to serine at amino acid position 26 (SEQ ID NO: 77); IMGT-defined FRL2 comprising the nucleic acid sequence beginning at guanine at position 97 and ending at cytosine at position 147 (SEQ ID NO: 77) encoding an amino acid sequence valine at amino acid position 33 to tyrosine at amino acid position 49 (SEQ ID NO: 77); IMGT-defined FRL3 comprising the nucleic acid sequence beginning at guanine at position 157 and ending at thymine at position 264 (SEQ ID NO:77) encoding an amino acid sequence glutamic acid at amino acid position 53 to cysteine at amino acid position 88 (SEQ ID NO: 77); and/or IMGT-defined FRL4 comprising the nucleic acid sequence beginning at thymine at

position 292 and ending at adenine at position 321 (SEQ ID NO: 77) encoding an amino acid sequence phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 77).

In one embodiment, the isolated nucleic acid comprises the nucleic acid sequence for a light chain variable region beginning at guanine at position 1 and ending at adenine at position 321 (SEQ ID NO: 3) encoding an amino acid sequence aspartic acid at amino acid position 1 to lysine at amino acid position 107 (SEQ ID NO: 3).

In an additional embodiment of the invention, the nucleic acid encodes a specific binding member which is an antibody.

In yet another embodiment, the heavy chain of the antibody comprises the FF03046-2 VH domain from glutamic acid at amino acid position 1 to serine at amino acid position 120 (SEQ ID NO: 1) and a CHI domain of a human IgGl constant region rom alanine at amino acid position 121 to valine at amino acid position 21S (SEQ ID NO: 1), and wherein the light chain of the antibody comprises the FF03046-2 VL domain from aspartic acid at amino acid position 1 to lysine at amino acid position 107 (SEQ ID NO: 3) and a human kappa light chain constant region from arginine at amino acid position 108 to cysteine at amino acid position 214 (SEQ ID NO: 3).

In yet another embodiment, the heavy chain of the antibody comprises the FF03046-2 VH domain from glutamic acid at amino acid position 1 to serine at amino acid position 120 (SEQ ID NO: 1), a CHI domain of a human IgGl constant region from alanine at amino acid position 121 to valine at amino acid position 218 (SEQ ID NO: 1), and a portion of a hinge region of a human IgGl constant region from glutamic acid at amino acid position 219 to threonine at amino acid position 228 (SEQ ID NO: 1), and wherein the light chain of the antibody comprises the FF03046-2 VL domain from aspartic acid at amino acid position 1 to lysine at amino acid position 107 (SEQ ID NO: 3) and a human kappa light chain constant region from arginine at amino acid position 108 to cysteine at amino acid position 214 (SEQ ID NO: 3).

Also, the invention provides an embodiment wherein the heavy chain of the antibody comprises the FF03046-2 VH domain from glutamic acid at amino acid position 261 to serine at amino acid position 380 (SEQ ID NO: 16) and a CHI domain of a human IgGl constant region from alanine at amino acid position 381 to valine at amino acid position 478 (SEQ ID NO: 16), and wherein the light chain of the antibody comprises the FF03046-2 VL domain from aspartic acid at amino acid position 24 to lysine at amino acid position 130 (SEQ ID NO: 16) and a human kappa light chain constant region from arginine at amino acid position 131 to cysteine at amino acid position 237 (SEQ ID NO: 16).

The invention provides an embodiment wherein the heavy chain of the antibody comprises the FF03046-2 VH domain from glutamic acid at amino acid position 261 to serine at amino acid position 380 (SEQ ID NO: 16), a CHI domain of a human IgGl constant region from alanine at amino acid position 381 to valine at amino acid position 478 (SEQ ID NO: 16), and a portion of a hinge region of a human IgGl constant region from glutamic acid at amino acid position 479 threonine at amino acid position 488 (SEQ ID NO: 16), and wherein the light chain of the antibody comprises the FF03046-2 VL domain from aspartic acid at amino acid position 24 to lysine at amino acid position 130 (SEQ ID NO: 16) and a human kappa light chain constant region from arginine at amino acid position 131 to cysteine at amino acid position 237 (SEQ ID NO: 16).

The invention provides an isolated nucleic acid having a sequence as provided in SEQ ID NO: 16.

In one embodiment, a constitutive promoter is used to the control the expression of the antibody, portion or variant thereof of the invention. In a separate embodiment, a regulatible or an inducible promoter is use to express the antibody, portion or variant thereof of the invention. In one embodiment, the regulatible or an inducible promoter is a pho A promoter. The invention provide an isolated nucleic acid, wherein the pho A promoter comprises a nucleic acid sequence as provided in SEQ ID NO: 15 or a portion thereof.

The invention provides an isolated nucleic acid sequence, wherein termination of transcription of the nucleic acid is under the control of a transcriptional terminator. In one embodiment, the

transcriptional terminator is a ribosomal RNA gene terminator. In one embodiment, the ribosomal RNA gene terminator comprises a nucleic acid sequence as provided in SEQ ID NO: 19 or a portion thereof.

The invention provides an isolated nucleic acid sequence wherein the isolated nucleic acid sequence is in a vector. In one embodiment, the vector comprises an origin of replication. In another embodiment, the vector comprises a colEl origin of replication.

In one embodiment, the vector comprises a selectable marker or a screening marker. In one embodiment, the selectable marker or screening marker is selected from the group consisting of a drug selectable marker, a fluorescent protein, a cell surface marker, an enzyme, a luminescent protein, a metabolic marker, a growth factor and a resistance factor. In one embodiment, the vector is pBR322 deleted of tetracycline resistance gene as provided in SEQ ID NO: 20.

The invention provides an isolated nucleic acid, wherein the nucleic acid encoding the antibody of the invention does not bind BSA or Fc.

For recombinant production of an antibody of the invention, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

The isolated nucleic acid encoding an anti-VEGF antibody of the invention may have a silent mutation or mutations. Additionally, the isolated nucleic acid of the invention may have a missense mutation or mutations, wherein the nucleotide substitution results in a change in the identity of the amino acid but not in a prematurely truncated polypeptide due to the substitution leading to specification of a translation termination codon. The isolated nucleic acid of the invention may have an inframe deletion or insertion, wherein the deletion or insertion occurs as a multiple of 3 bases or basepairs.

Also provided are recombinant DNA molecules (rDNAs) that contain an anti-VEGF antibody- encoding sequences as herein described, or a fragment thereof. As used herein, a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in vitro. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning (1989). In the preferred rDNA molecules of the present invention, an anti-VEGF-antibody encoding DNA sequence that encodes an anti-VEGF antibody or a fragment thereof, is operably linked to one or more expression control sequences and/or vector sequences. The rDNA molecule can encode either the entire VEGF antibody, or can encode a fragment of the VEGF antibody.

The choice of vector and/or expression control sequences to which the VEGF antibody -encoding sequence is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed. A vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the VEGF antibody -encoding sequence included in the rDNA molecule.

What is claimed is:
1. An isolated anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising a heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences: CDRH1 comprising histidine at amino acid position 31 to histidine at amino acid position 35 (SEQ ID NO: 1), CDRH2 comprising tyrosine at amino acid position 50 to glycine at amino acid position 66 (SEQ ID NO: 1) and CDRH3 comprising histidine at amino acid position 99 to tyrosine at amino acid position 109 (SEQ ID NO: 1).

2. An isolated anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences: CDRL1 comprising arginine at amino acid position 24 to alanine at amino acid position 34 (SEQ ID NO: 3), CDRL2 comprising lysine at amino acid position 50 to alanine at amino acid position 56 (SEQ ID NO: 3), and CDRL3 comprising glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 3).

3. An isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising

a. a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences as shown in Figure 9C, or as described in SEQ ID NO: 3 and

b. a heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences as shown in Figure 9C, or as described in SEQ ID NO: 1.

4. An isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising

a. a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences as shown in Figure 16 and

b. a heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences as shown in Figure 16.

5. The anti-VEGF antibody or portion thereof of claim 1 further comprising a light chain variable domain comprising the following CDR amino acid sequences: CDRL1 comprising arginine at amino acid position 24 to alanine at amino acid position 34 (SEQ ID NO: 3), CDRL2 comprising lysine at amino acid position 50 to alanine at amino acid position 56 (SEQ ID NO: 3), and CDRL3 comprising glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 3).

6. An isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising

a. a heavy chain variable domain comprising the following CDR amino acid sequences defined in accordance with IMGT numbering shown in bold in Figure 16: CDRH1 comprising amino acid sequences GFDLDHYS, CDRH2 comprising amino acid sequences IYPSYGYT and CDRH3 comprising amino acid sequences ARHAWYYGWGLDY; and

b. a light chain variable domain comprising the following CDR amino acid sequences defined in accordance with IMGT numbering shown in bold in Figure 16: CDRL1 comprising amino acid sequences QAAYGR, CDRL2 comprising amino acid sequences KAS and CDRL3 comprising amino acid sequences QQRGWYLFT.

7. A synthetic human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising

a) a light chain variable domain having the following hypervariable region or complementarity determining region (CDR) amino acid sequences: a CDRL1 which comprises arginine at amino acid position 24 to alanine at amino acid position 34 (SEQ ID NO: 3) or a portion thereof, a CDRL2 which comprises lysine at amino acid position 50 to alanine at amino acid position 56 (SEQ ID NO: 3) or a portion thereof, and a CDRL3 which comprises glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 3) or a portion thereof; and b) a heavy chain variable domain having the following hypervariable region or complementarity determining region (CDR) amino acid sequences: a CDRH1 which comprises histidine at amino acid position 31 to histidine at amino acid position 35 (SEQ ID NO: 1) or a portion thereof, a CDRH2 which comprises tyrosine at amino acid position 50 to glycine at amino acid position 66 (SEQ ID NO: 1) or a portion thereof and a CDRH3 which comprises histidine at amino acid position 99 to tyrosine at amino acid position 109 (SEQ ID NO: 1) or a portion thereof.

8. The anti-VEGF antibody or portion or variant thereof of claim 6 further comprising: c) a light chain variable domain having the following framework region amino acid sequences: a FRL1 or a portion thereof, FRL2 or a portion thereof, FRL3 or a portion thereof, and FRL4 or a portion thereof; and

d) a heavy chain variable domain having the following framework region amino acid sequences: a FRH1 or a portion thereof, FRH2 or a portion thereof, FRH3 or a portion thereof, and FRH4 or a portion thereof.

9. The anti-VEGF antibody of claim 1 or 7 further comprising a heavy chain framework region (FR) 1, FRH1, amino sequence comprising glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1).

10. The anti-VEGF antibody of claim 1 or 7 further comprising a heavy chain framework region (FR) 2, FRH2, amino sequence comprising tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1).

1 1. The anti-VEGF antibody of claim 1 or 7 further comprising a heavy chain framework region (FR) 3, FRH3, amino sequence comprising arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1).

12. The anti-VEGF antibody of claim 1 or 7 further comprising a heavy chain framework region (FR) 4, FRH4, amino sequence comprising tryptophan at amino acid position 1 10 to serine at amino acid position 120 (SEQ ID NO: 1).

13. The anti-VEGF antibody of claim 1 or 7 further comprising the following heavy chain framework region (FR) amino acid sequences: FRH1 comprising glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1), FRH2 comprising tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1), FRH3 comprising arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1) and FRH4 comprising tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1).

14. The anti-VEGF antibody of claim 2 or 7 further comprising a light chain framework region (FR) 1, FRL1, amino sequence comprising aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3).

15. The anti-VEGF antibody of claim 2 or 7 further comprising a light chain framework region (FR) 2, FRL2, amino sequence comprising tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3).

16. The anti-VEGF antibody of claim 2 or 7 further comprising a light chain framework region (FR) 3, FRL3, amino sequence comprising glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3).

17. The anti-VEGF antibody of claim 2 or 7 further comprising a light chain framework region (FR) 4, FRL4, amino sequence comprising phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3).

18. The anti-VEGF antibody of claim 2 or 7 further comprising the following light chain framework region (FR) amino acid sequences: FRL1 comprising aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3), FRL2 comprising tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3), FRL3 comprising glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3) and FRL4 comprising phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3).

19. The anti-VEGF antibody or portion or variant thereof of claim 7 further comprising: c) a light chain variable domain having the following framework region amino acid sequences: a FRL1 which comprises aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3) or a portion thereof, FRL2 which comprises tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3) or a portion thereof, FRL3 which comprises glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3) or a portion thereof, and FRL4 which comprises phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3) or a portion thereof; and d) a heavy chain variable domain having the following framework region amino acid sequences: a FRHl which comprises glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1) or a portion thereof, FRH2 which comprises tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1) or a portion thereof, FRH3 which comprises arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1) or a portion thereof, and FRH4 which comprises tryptophan at amino acid position 1 10 to serine at amino acid position 120 (SEQ ID NO: 1) or a portion thereof.

20. The anti-VEGF antibody of claim 1, 2, 3, 4, 5, 6 or 7 which is a full length antibody.

21. The anti-VEGF antibody of claim 20, wherein the full length antibody comprises a light chain comprising the amino acid sequence shown in SEQ ID NO:3 and a heavy chain shown in SEQ ID NO:l, a light chain comprising the aspartic acid at amino acid position 24 to cysteine at amino acid position 237 (SEQ ID NO: 17) and a heavy chain comprising the glutamic acid at amino acid position 24 to threonine at amino acid position 251 (SEQ

ID NO: 18), or a light chain comprising the aspartic acid at amino acid position 24 to cysteine at amino acid position 237 (SEQ ID NO:76) and a heavy chain comprising the glutamic acid at amino acid position 24 to threonine at amino acid position 251 (SEQ ID NO:78).

22. The anti-VEGF antibody of claim 1, 2, 3, 4, 5, 6 or 7 which is a human or humanized IgG.

23. The anti-VEGF antibody of claim 1, 2, 3, 4, 5, 6 or 7, wherein the portion of the antibody is an antibody fragment.

24. The anti-VEGF antibody of claim 23, wherein the antibody fragment is a Fab.

25. The anti-VEGF antibody of claim 24, wherein the Fab comprises a light chain variable domain comprising the amino acid sequence beginning at aspartic acid at position 1 and ending at lysine at position 107 of SEQ ID NO: 3 and a heavy chain variable domain comprising the amino acid sequence beginning at glutamic acid at position 1 and ending at serine at position 120 of SEQ ID NO:l.

26. The anti-VEGF antibody of claim 25, wherein the Fab is joined to a Fc region.

27. The anti-VEGF antibody of claim 26, wherein the Fab joined to a Fc region comprises the amino acid shown in SEQ ID NO:5 or a portion thereof.

28. The anti-VEGF antibody of claim 23, wherein the antibody fragment is a F(ab')2.

29. The anti-VEGF antibody of claim 1, 2, 3, 4, 6 or 7 , wherein the variant is a scFv.

30. The anti-VEGF antibody of claim 29, wherein the scFv comprises the amino acid sequence beginning at aspartic acid at position 241 and ending at serine at position 483 of SEQ ID NO:9.

31. The anti-VEGF antibody of claim 30, wherein the scFv is joined to a Fc region.

32. The anti-VEGF antibody of claim 31, wherein the scFv joined to a Fc region comprises the amino acid shown in SEQ ID NO:9 or a portion thereof.

33. The anti-VEGF antibody of claim 23, wherein the antibody fragment is a Fv.

34. The anti-VEGF antibody of claim 1, 2, 3, 4, 6 or 7, wherein the variant of the antibody is a recombinant protein that recognizes and binds a VEGF comprising the antigen-binding region of the antibody of claim 1 , 2, 3, 4, 6 or 7.

35. The anti-VEGF antibody of claim 1, 2, 3, 4, 6 or 7 , wherein the recombinant protein comprises an alteration to the amino acid sequence of CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, or CDRL3.

36. The anti-VEGF antibody of claim 35, wherein the alteration is an insertion or deletion of an amino acid(s).

37. The anti-VEGF antibody of claim 36, wherein the insertion or deletion is less than or equal to 10 amino acids.

38. The anti-VEGF antibody of claim 35, wherein the alteration is an amino acid substitution.

39. The anti-VEGF antibody of claim 38, wherein the amino acid substitution affects 3 amino acids or less.

40. The anti-VEGF antibody of claim 38, wherein the amino acid substitution is a conservative amino acid substitution.

41. The anti-VEGF antibody of claim 35, wherein the alteration does not inhibit binding of the antibody to VEGF or a portion of thereof.

42. The anti-VEGF antibody of claim 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18 or 19, wherein the variant of the antibody is an antibody having an alteration to the amino acid sequence of FRH1, FRH2, FRH3, FRH4, FRL1, FRL2, FRL3, or FRL4.

43. The anti-VEGF antibody of claim 42, wherein the alteration is an insertion or deletion of an amino acid(s).

44. The anti-VEGF antibody of claim 42, wherein the alteration is an amino acid substitution.

45. The anti-VEGF antibody of claim 44, wherein the amino acid substitution is a conservative amino acid substitution.

46. The anti-VEGF antibody of claim 42, wherein the alteration does not inhibit binding of the antibody to VEGF or a portion thereof.

47. A Fab, Fab1, F(ab')2, scFv or Fv fragment of the antibody of claim 1, 2, 3, 4, 6 or 7.

48. The anti-VEGF antibody of claim 1, 2, 3, 4, 6 or 7, wherein the antibody or portion or variant thereof binds a human VEGF with a K(d) value of no more than about 50 nM.

49. The anti-VEGF antibody of claim 1, 2, 3, 4, 5, 6 or 7 , wherein the antibody or portion or variant thereof which has an EC50 value of no more than about 200 pM for inhibiting VEGF-induced proliferation of endothelial cells in vitro.

50. A bispecific antibody with a binding specificity for two different antigens, one of the antigens being that with which the antibody of claim 1, 2, 3, 4, 5, 6 or 7 binds or with which it competes.

51. A bispecific antibody of claim 50, wherein the antibody comprises an amino acid sequence comprising the amino acid sequence for CDRH1 of claim 1 or a portion thereof, CDRH2 of claim 1 or a portion thereof, CDRH3 of claim 1 or a portion thereof, CDRL1 of claim 2 or a portion thereof, CDRL2 of claim 2 or a portion thereof, or CDRL3 of claim 2 or a portion thereof, or a combination thereof.

52. A bispecific antibody of claim 51, wherein the antibody additionally comprises a Fc region.

53. The anti-VEGF antibody of claim 1, 2, 3, 4, 5, 6 or 7 which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a synthetic antibody, a single chain antibody or an antigen-binding fragment thereof.

54. The anti-VEGF antibody of claim 1, 2, 3, 4, 6 or 7 wherein the portion is an antigen binding fragment of the antibody of claim 1, 2 or 7 which is selected from the group consisting of Fab, F(ab')2, Fab', scFv, and F(V>.

55. The antibody of claim 1, 2, 3, 4, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18 or 19, wherein each CDR and/or each FR amino acid sequence as defined by abat method is substituted with a corresponding amino acid sequence of a CDR or FR as defined by IMGT method, wherein the substitutions are any of the following:

a) Kabat-defined CDRH1 comprising histidine at amino acid position 31 to histidine at amino acid position 35 (SEQ ID NO: 1) is replaced with an IMGT-defined CDRH1 comprising glycine at amino acid position 26 (SEQ ID NO: 1) to serine at amino acid position 33 (SEQ ID NO: 1);

b) Kabat-defined CDRH2 comprising tyrosine at amino acid position 50 to glycine at amino acid position 66 (SEQ ID NO: 1) is replaced with an IMGT-defined CDRH2 comprising isoleucine at amino acid position 51 (SEQ ID NO: 1) to threonine at amino acid position 58 (SEQ ID NO: 1);

c) Kabat-defined CDRH3 comprising histidine at amino acid position 99 to tyrosine at amino acid position 109 (SEQ ID NO: 1) is replaced with an IMGT-defined

CDRH3 comprising alanine at amino acid position 97 to tyrosine at amino acid position 109 (SEQ ID NO: 1);

d) abat-defined CDRL1 comprising arginine at amino acid position 24 to alanine at amino acid position 34 (SEQ ID NO: 3) is replaced with IMGT-defined CDRL1 comprising glutamine at amino acid position 27 to arginine at amino acid position 32 (SEQ ID NO: 3);

e) Kabat-defined CDRL2 comprising lysine at amino acid position 50 to alanine at amino acid position 56 (SEQ ID NO: 3) is replaced with IMGT-defined CDRL2 comprising lysine at amino acid position 50 to serine at amino acid position 52 (SEQ ID NO: 3);

f) Kabat-defined CDRL3 comprising glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 3) is replaced with IMGT- defined CDRL3 comprising glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 3);

g) Kabat-defined FRH1 comprising glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1) is replaced with IMGT- defined FRH1 comprising glutamic acid at amino acid position 1 to serine at amino acid position 25 (SEQ ID NO: 1);

h) Kabat-defined FRH2 comprising tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1) is replaced with IMGT-defined FRH2 comprising isoleucine at amino acid position 34 to tyrosine at amino acid position 50 (SEQ ID NO: 1);

i) Kabat-defined FRH3 comprising arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1) is replaced with IMGT-defined FRH3 comprising tyrosine at amino acid position 59 to cysteine at amino acid position 96 (SEQ ID NO: 1);

j) Kabat-defined FRH4 comprising tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1) is replaced with IMGT-defined FRH4 comprising tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1);

k) Kabat-defined FRL1 comprising aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3) is replaced with IMGT-defined FRL1 comprising aspartic acid at amino acid position 1 to serine at amino acid position 26 (SEQ ID NO: 3);

1) Kabat-defined FRL2 comprising tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3) is replaced with IMGT-defined FRL2 comprising valine at amino acid position 33 to tyrosine at amino acid position 49 (SEQ ID NO: 3);

m) Kabat-defined FRL3 comprising glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3) is replaced with IMGT-defined FRL3 comprising glutamic acid at amino acid position 53 to cysteine at amino acid position 88 (SEQ ID NO: 3); and

n) Kabat-defined FRL4 comprising phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3) is replaced with IMGT-defined FRL4 comprising phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3).

56. The antibody of claim 1, 2, 3, 4, 6, 7, 8, 9, 100, 11, 12, 13, 14, 15, 16, 17, 18 or 19 , wherein each CDR and/or each FR amino acid sequence as defined by Kabat method is substituted with a corresponding amino acid sequence of a CDR or FR as defined by IMGT method, wherein the substitutions are any of the following:

a) Kabat-defined CDRH1 comprising histidine at amino acid position 31 to histidine at amino acid position 35 (SEQ ID NO: 78) is replaced with an IMGT-defined CDRH1 comprising glycine at amino acid position 26 (SEQ ID NO: 78) to serine at amino acid position 33 (SEQ ID NO: 78);

b) Kabat-defined CDRH2 comprising tyrosine at amino acid position 50 to glycine at amino acid position 66 (SEQ ID NO: 78) is replaced with an IMGT-defined CDRH2 comprising isoleucine at amino acid position 51 (SEQ ID NO: 78) to threonine at amino acid position 58 (SEQ ID NO: 78);

c) Kabat-defined CDRH3 comprising histidine at amino acid position 99 to tyrosine at amino acid position 109 (SEQ ID NO: 78) is replaced with an IMGT-defined

CDRH3 comprising alanine at amino acid position 97 to tyrosine at amino acid position 109 (SEQ ID NO: 78);

d) Kabat-defined CDRL1 comprising arginine at amino acid position 24 to alanine at amino acid position 34 (SEQ ID NO: 76) is replaced with IMGT-defined CDRL1 comprising glutamine at amino acid position 27 to arginine at amino acid position 32 (SEQ ID NO: 76);

e) Kabat-defined CDRL2 comprising lysine at amino acid position 50 to alanine at amino acid position 56 (SEQ ID NO: 76) is replaced with IMGT-defined CDRL2 comprising lysine at amino acid position 50 to serine at amino acid position 52 (SEQ ID NO: 76);

f) Kabat-defined CDRL3 comprising glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 76) is replaced with IMGT- defined CDRL3 comprising glutamine at amino acid position 89 to threonine at amino acid position 97 (SEQ ID NO: 76);

g) Kabat-defined FRH1 comprising glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 78 ) is replaced with IMGT- defined FRH1 comprising glutamic acid at amino acid position 1 to serine at amino acid position 25 (SEQ ID NO: 78);

h) Kabat-defined FRH2 comprising tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 78) is replaced with IMGT-defined FRH2 comprising isoleucine at amino acid position 34 to tyrosine at amino acid position 50 (SEQ ID NO: 78);

i) Kabat-defined FRH3 comprising arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 78) is replaced with IMGT-defined FRH3 comprising tyrosine at amino acid position 59 to cysteine at amino acid position 96 (SEQ ID NO: 78);

j) Kabat-defined FRH4 comprising tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 78) is replaced with IMGT-defined FRH4 comprising tryptophan at amino acid position 1 10 to serine at amino acid position 120 (SEQ ID NO: 78);

k) Kabat-defined FRL1 comprising aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 76) is replaced with IMGT-defined FRL1 comprising aspartic acid at amino acid position 1 to serine at amino acid position 26 (SEQ ID NO: 76);

1) Kabat-defined FRL2 comprising tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 76) is replaced with IMGT-defined FRL2 comprising valine at amino acid position 33 to tyrosine at amino acid position 49 (SEQ ID NO: 76);

m) Kabat-defined FRL3 comprising glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 76) is replaced with IMGT-defined FRL3 comprising glutamic acid at amino acid position 53 to cysteine at amino acid position 88 (SEQ ID NO: 76); and

n) Kabat-defined FRL4 comprising phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 76) is replaced with IMGT-defined FRL4 comprising phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 76).

57. An immunoconjugate comprising an anti-VEGF antibody or portion or variant thereof of claim 1, 2, 3, 4, 6 or 7 joined to a therapeutic agent.

58. The immunoconjugate of claim 57, wherein the therapeutic agent is a cytotoxic agent selected from a group consisting of ricin, doxorubicin, daunorubicin, taxol, ethidium, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, dihydroxyanthraquinone, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, ricin, abrin, glucocorticoid and radioisotope.

59. The immunoconjugate of claim 56, wherein the portion of the antibody is selected from the group consisting of scFv, Fv, Fab, Fab', and F(ab')2 fragments.

60. A composition comprising the anti-VEGF antibody of claim 1, 2, 3, 4, 6 or 7 and a pharmaceutically acceptable carrier.

61. A composition comprising the immunoconjugate of claim 60 and a pharmaceutically acceptable carrier.

62. An isolated nucleic acid encoding the antibody of claim 1, 2, 3, 4, 6 or 7.

63. A vector comprising the nucleic acid of claim 62.

64. A host cell comprising the vector of claim 63.

65. A process of producing an anti-VEGF antibody comprising culturing the host cell of claim 64 so that the nucleic acid is expressed to produce the antibody.

66. The process of claim 65 further comprising recovering the anti-VEGF antibody from the host cell culture.

67. An isolated nucleic acid encoding an immunoglobulin heavy chain or a portion thereof, an immunoglobulin light chain or a portion thereof, or both, of a specific binding member that binds to human VEGF, said specific binding member comprising an antigen-binding portion of an antibody,

a) wherein the immunoglobulin heavy chain or a portion thereof, of the antigen- binding portion of the antibody comprises an immunoglobulin heavy chain CDRH1 comprising the nucleic acid sequence beginning at cytosine at position 91 and ending at cytosine at position 105 (SEQ ID NO: 1) or a portion thereof, an heavy chain CDRH2 comprising the nucleic acid sequence beginning at thymine at position 148 and ending at cytosine at position 198 (SEQ ID NO: 1) or a portion thereof, and an heavy chain CDRH3 comprising the nucleic acid sequence beginning at cytosine at position 295 and ending at cytosine at position 327 (SEQ ID NO: 1) or a portion thereof; and/or

b) wherein the immunoglobulin light chain or portion thereof, of the antigen-binding portion of the antibody comprises a light chain CDRL1 comprising the nucleic acid sequence beginning at cytosine at position 70 and ending at cytosine at

position 102 (SEQ ID NO: 3) or a portion thereof, a light chain CDRL2 comprising the nucleic acid sequence beginning at adenine at position 148 to cytosine at position 168 (SEQ ID NO: 3) or a portion thereof, and a light chain CDRL3 comprising the nucleic acid sequence beginning at cytosine at position 265 and ending at guanine at position 291 (SEQ ID NO: 3) or a portion thereof.

68. An isolated nucleic acid encoding the Fab antibody of claim 4 comprising a light chain nucleic acid sequence shown in Figure 16 and a heavy chain nucleic acid sequence shown in Figure 16.

69. The isolated nucleic acid of claim 67 , wherein any of the nucleic acid sequence as provided in (a) or (b) may be replaced with an equivalent nucleic acid sequence encoding for the same amino acid sequence.

70. The isolated nucleic acid according to claim 69, wherein the antibody further comprises a human or humanized constant region.

71. The isolated nucleic acid according to claim 70, wherein the human or humanized constant region is an IgGl constant region.

72. The isolated nucleic acid according to claim 70, wherein the human or humanized constant region is an IgG4 constant region.

73. The isolated nucleic acid according to claim 67, wherein the specific binding member is a single chain Fv molecule comprising the nucleic acid sequence beginning at guanine at position 721 to thymine at position 1449 as shown in SEQ ID NO: 9 or a portion thereof.

74. The isolated nucleic acid according to claim 67, wherein the specific binding member is a Fab comprising the nucleic acid sequences shown in SEQ ID NOS: 1 and 3 or a portion(s) thereof.

75. The isolated nucleic acid according to claim 67, wherein the specific binding member is a Fab comprising the nucleic acid sequence beginning at guanine at position 70 to thymine at position 711 as shown in SEQ ID NO: 16 and the nucleic acid sequence beginning at guanine at position 897 to adenine at position 1580 in SEQ ID NO: 16 or a portion(s) thereof.

76. The isolated nucleic acid according to claim 67, wherein the specific binding member is a Fab comprising the nucleic acid sequence beginning at adenine at position 1 to thymine at position 711 as shown in SEQ ID NO: 16 and the nucleic acid sequence beginning at adenine at position 828 to adenine at position 1580 in SEQ ID NO: 16 or a portion(s) thereof.

77. The isolated nucleic acid according to claim 67, wherein the specific binding member is a Fab comprising the nucleic acid sequences shown in SEQ ID NO: 16 or a portion thereof.

78. The isolated nucleic acid according to claim 67, wherein the immunoglobulin heavy chain or a portion thereof, of the antigen-binding portion of the antibody additionally comprises an immunoglobulin heavy chain framework region (FR) 1, FRH1, comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 90 (SEQ ID NO: 1 ) encoding an amino acid sequence glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1).

79. The isolated nucleic acid according to claim 67, wherein the immunoglobulin heavy chain or a portion thereof, of the antigen-binding portion of the antibody additionally comprises an immunoglobulin heavy chain framework region (FR) 2, FRH2, comprising the nucleic acid sequence beginning at thymine at position 106 and ending at adenine at position 147 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1).

80. The isolated nucleic acid according to claim 67, wherein the immunoglobulin heavy chain or a portion thereof, of the antigen-binding portion of the antibody additionally comprises an immunoglobulin heavy chain framework region 3, FRH3, comprising the nucleic acid

sequence beginning at cytosine at position 199 and ending at cytosine at position 294 (SEQ ID NO: 1) encoding an amino acid sequence arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1).

81. The isolated nucleic acid according to claim 67, wherein the immunoglobulin heavy chain or a portion thereof, of the antigen-binding portion of the antibody additionally comprises an immunoglobulin heavy chain framework region 4, FRH4, comprising the nucleic acid sequence beginning at thymine at position 328 and ending at guanine at position 360 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1).

82. The isolated nucleic acid according to claim 67, wherein the immunoglobulin heavy chain or a portion thereof, of the antigen-binding portion of the antibody additionally comprises a heavy chain framework region FRH1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 90 (SEQ ID NO: 1) encoding an amino acid sequence glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1), a heavy chain framework region FRH2 comprising the nucleic acid sequence beginning at thymine at position 106 and ending at adenine at position 147 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1 ), a heavy chain framework region FRH3 comprising the nucleic acid sequence beginning at cytosine at position 199 and ending at cytosine at position 294 (SEQ ID NO: 1) encoding an amino acid sequence arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1), and a heavy chain framework region FRH4 comprising the nucleic acid sequence beginning at thymine at position 328 and ending at guanine at position 360 (SEQ ID NO:

1) encoding an amino acid sequence tryptophan at amino acid position 1 10 to serine at amino acid position 120 (SEQ ID NO: 1), wherein the nucleic acid sequences encoding the heavy chain framework regions, FRH1 to FRH4, and the heavy chain complementarity determining regions, CDRH1 to CDRH3, are joined in the order FRH 1 -CDRH 1 -FRH2- CDRH2-FRH3-CDRH3-FRH4.

83. The isolated nucleic acid according to claim 82, wherein the heavy chain of the antibody comprises the nucleic acid sequence for a heavy chain variable region beginning at guanine at position 1 and ending at guanine at position 360 (SEQ ID NO: 1) encoding an amino acid sequence glutamic acid at amino acid position 1 to serine at amino acid position 120 (SEQ ID NO: 1).

84. The isolated nucleic acid according to claim 67, wherein the immunoglobulin light chain or portion thereof, of the antigen-binding portion of the antibody additionally comprises an immunoglobulin light chain framework region 1, FRL1, comprising the nucleic acid sequence beginning at guanine at position 1 and ending at cytosine at position 69 (SEQ

ID NO: 3) encoding an amino acid sequence aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3).

85. The isolated nucleic acid according to claim 67, wherein the immunoglobulin light chain or portion thereof, of the antigen-binding portion of the antibody additionally comprises an immunoglobulin light chain framework region 2, FRL2, comprising the nucleic acid sequence beginning at thymine at position 103 and ending at cytosine at position 147 (SEQ ID NO: 3) encoding an amino acid sequence tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3).

86. The isolated nucleic acid according to claim 67, wherein the immunoglobulin light chain or portion thereof, of the antigen-binding portion of the antibody additionally comprises an immunoglobulin light chain framework region 3, FRL3, comprising the nucleic acid sequence beginning at guanine at position 169 and ending at thymine at position 264 (SEQ ID NO: 3) encoding an amino acid sequence glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3).

87. The isolated nucleic acid according to claim 67, wherein the immunoglobulin light chain or portion thereof, of the antigen-binding portion of the antibody additionally comprises an immunoglobulin light chain framework region 4, FRL4, comprising the nucleic acid sequence beginning at thymine at position 292 and ending at adenine at position 321 (SEQ ID NO: 3) encoding an amino acid sequence phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3).

88. The isolated nucleic acid according to claim 67, wherein the immunoglobulin light chain or a portion thereof, of the antigen-binding portion of the antibody additionally comprises a light chain framework region FRL1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at cytosine at position 69 (SEQ ID NO: 3) encoding an amino acid sequence aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3), a light chain framework FRL2 comprising the nucleic acid sequence beginning at thymine at position 103 and ending at cytosine at position 147 (SEQ

ID NO: 3) encoding an amino acid sequence tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3), a light chain framework FRL3 comprising the nucleic acid sequence beginning at guanine at position 169 and ending at thymine at position 264 (SEQ ID NO: 3) encoding an amino acid sequence glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3), a light chain framework FRL4 comprising the nucleic acid sequence beginning at thymine at position 292 and ending at adenine at position 321 (SEQ ID NO: 3) encoding an amino acid sequence phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3), wherein the nucleic acid sequences encoding the light chain framework regions FRL1 to FRL4 and the light chain complementarity determining regions CDRL1 to CDRL3 are joined in the order FRL 1 -CDRL 1 -FRL2-CDRL2-FRL3-CDRL3-FRL4.

89. The isolated nucleic acid according to claim 88, wherein the light chain of the antibody comprises the nucleic acid sequence for a light chain variable region beginning at guanine at position 1 and ending at adenine at position 321 (SEQ ID NO: 3) encoding an amino acid sequence aspartic acid at amino acid position 1 to lysine at amino acid position 107 (SEQ ID NO: 3).

90. The isolated nucleic acid according to claim 67, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or 89, wherein the isolated nucleic acid has a silent mutation or mutations.

91. The isolated nucleic acid according to claim 67, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or 89, wherein the isolated nucleic acid has an inframe deletion or insertion, wherein the deletion or insertion occurs as a multiple of 3 bases or basepairs.

92. The isolated isolated nucleic acid according to claim 91, wherein the inframe deletion or insertion occurs at a complementarity determining region (CDR) and is less than or equal to 30 bases or basepairs in length.

93. The isolated isolated nucleic acid according to claim 91, wherein the inframe deletion or insertion occurs at a complementarity determining region (CDR) and is less than or equal to IS bases or basepairs in length.

94. The isolated isolated nucleic acid according to claim 91, wherein the inframe deletion or insertion occurs at a complementarity determining region (CDR) sequence and and is 6 bases or basepairs in length.

95. The isolated isolated nucleic acid according to claim 91, wherein the inframe deletion or insertion occurs at a complementarity determining region (CDR) sequence and and is 3 bases or basepairs in length.

96. The isolated isolated nucleic acid according to claim 67, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or 89, wherein the isolated nucleic acid has an inframe deletion or insertion at a framework sequence in a multiple of 3 bases.

97. The isolated nucleic acid according to claim 96, wherein the inframe deletion or insertion is less than or equal to 30 bases or basepairs in length.

98. The isolated nucleic acid according to claim 67, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or 891, wherein the isolated nucleic acid has a missense mutation or mutations.

99. The isolated nucleic acid according to claim 98, wherein the missense mutation(s) occurs at a CDR sequence altering up to 3 amino acids of the CDR sequence.

100. The isolated nucleic acid according to claim 67, wherein the specific binding member is an antibody, wherein the heavy chain of the antibody comprises the FF03046- 2 VH domain from glutamic acid at amino acid position 1 to serine at amino acid position 120 (SEQ ID NO: 1) and a CHI domain of a human IgGl constant region from alanine at amino acid position 121 to valine at amino acid position 218 (SEQ ID NO: 1), and wherein the light chain of the antibody comprises the FF03046-2 VL domain from aspartic acid at amino acid position 1 to lysine at amino acid position 107 (SEQ ID NO: 3) and a human kappa light chain constant region from arginine at amino acid position 108 to cysteine at amino acid position 214 (SEQ ID NO: 3).

101. The isolated nucleic acid according to claim 67, wherein the specific binding member is an antibody, wherein the heavy chain of the antibody comprises the FF03046- 2 VH domain from glutamic acid at amino acid position 1 to serine at amino acid position 120 (SEQ ID NO: 1), a CHI domain of a human IgGl constant region from alanine at amino acid position 121 to valine at amino acid position 218 (SEQ ID NO: 1), and a portion of a hinge region of a human IgGl constant region from glutamic acid at amino acid position 219 to threonine at amino acid position 228 (SEQ ID NO: 1), and wherein the light chain of the antibody comprises the FF03046-2 VL domain from aspartic acid at amino acid position 1 to lysine at amino acid position 107 (SEQ ID NO: 3) and a human kappa light chain constant region from arginine at amino acid position 108 to cysteine at amino acid position 214 (SEQ ID NO: 3).

102. The isolated nucleic acid according to claim 67, wherein the specific binding member is an antibody, wherein the heavy chain of the antibody comprises the FF03046- 2 VH domain from glutamic acid at amino acid position 261 to serine at amino acid position 380 (SEQ ID NO: 16) and a CHI domain of a human IgGl constant region from alanine at amino acid position 381 to valine at amino acid position 478 (SEQ ID NO: 16), and wherein the light chain of the antibody comprises the FF03046-2 VL domain from aspartic acid at amino acid position 24 to lysine at amino acid position 130 (SEQ ID NO: 16) and a human kappa light chain constant region from arginine at amino acid position 131 to cysteine at amino acid position 237 (SEQ ID NO: 16).

103. The isolated nucleic acid according to claim 67, wherein the specific binding member is an antibody, wherein the heavy chain of the antibody comprises the FF03046- 2 VH domain from glutamic acid at amino acid position 261 to serine at amino acid position 380 (SEQ ID NO: 16), a CHI domain of a human IgGl constant region from alanine at amino acid position 381 to valine at amino acid position 478 (SEQ ID NO: 16), and a portion of a hinge region of a human IgGl constant region from glutamic acid at amino acid position 479 threonine at amino acid position 488 (SEQ ID NO: 16), and wherein the light chain of the antibody comprises the FF03046-2 VL domain from aspartic acid at amino acid position 24 to lysine at amino acid position 130 (SEQ ID NO: 16) and a human kappa light chain constant region from arginine at amino acid position 131 to cysteine at amino acid position 237 (SEQ ID NO: 16).

104. The isolated nucleic acid of claim 102 or 103 as provided in SEQ ID NO: 16.

105. The isolated nucleic acid of claim 104, wherein the nucleic acid is expressed under the control of a constitutive promoter.

106. The isolated nucleic acid of claim 104, wherein the nucleic acid is expressed under the control of a regulatible promoter or an inducible promoter.

107. The isolated nucleic acid of claim 106, wherein the regulatible promoter or inducible promoter is a pho A promoter.

108. The isolated nucleic acid of claim 107, wherein the pho A promoter comprises a nucleic acid sequence as provided in SEQ ID NO: 15 or a portion thereof.

109. The isolated nucleic acid of claim 104, wherein termination of transcription of the nucleic acid is under the control of a transcriptional terminator.

110. The isolated nucleic acid of claim 109, wherein the transcriptional terminator is a ribosomal RNA gene terminator.

111. The isolated nucleic acid of claim 110, wherein the ribosomal RNA gene terminator comprises a nucleic acid sequence as provided in SEQ ID NO: 19 or a portion thereof.

112. The isolated nucleic acid of claim 104, wherein the nucleic acid is in a vector.

113. The isolated nucleic acid of claim 1 12, wherein the vector comprises a colE 1 origin of replication.

114. The isolated nucleic acid of claim 113, wherein the vector comprises a selectable marker or a screening marker.

115. The isolated nucleic acid of claim 114, wherein the selectable marker or screening marker is selected from the group consisting of a drug selectable marker, a fluorescent protein, a cell surface marker, an enzyme, a luminescent protein, a metabolic marker, a growth factor and a resistance factor.

116. The isolated nucleic acid of claim 112, wherein the vector is pBR322 deleted of tetracycline resistance gene as provided in SEQ ID NO: 20.

117. The isolated nucleic acid according to claim 67, wherein the antibody does not bind BSA or Fc.

118. The isolated nucleic acid of claim 67, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or 89, wherein the nucleic acid encoding for each CDR and each FR amino acid sequence as defined by Kabat method is substituted with a nucleic acid encoding a corresponding amino acid sequence of a CDR or FR as defined by IMGT method, wherein the nucleic acid substitutions are the following:

a) Kabat-defined CDRH 1 comprising the nucleic acid sequence beginning at cytosine at position 91 and ending at cytosine at position 105 (SEQ ID NO: 1) is replaced with IMGT-equivalent of CDRH1 comprising the nucleic acid sequence beginning at guanine at position 76 and ending at thymine at position 99 (SEQ ID NO: 1);

b) Kabat-defined CDRH2 comprising the nucleic acid sequence beginning at thymine at position 148 and ending at cytosine at position 198 (SEQ ID NO: 1) is replaced with IMGT-equivalent of CDRH2 comprising the nucleic acid sequence beginning at adenine at position 151 and ending at thymine at position 174 (SEQ ID NO: 1); c) Kabat-defined CDRH3 comprising the nucleic acid sequence beginning at cytosine at position 295 and ending at cytosine at position 327 (SEQ ID NO: 1) is replaced with IMGT-equivalent of CDRH3 comprising the nucleic acid sequence beginning at guanine at position 289 and ending at cytosine at position 327 (SEQ ID NO: 1); d) Kabat-defined CDRL 1 comprising the nucleic acid sequence beginning at cytosine at position 70 and ending at cytosine at position 102 (SEQ ID NO: 3) is replaced with IMGT-equivalent of CDRL1 comprising the nucleic acid sequence beginning at cytosine at position 79 and ending at cytosine at position 96 (SEQ ID NO: 3); e) Kabat-defined CDRL2 comprising the nucleic acid sequence beginning at adenine at position 148 to cytosine at position 168 (SEQ ID NO: 3) is replaced with IMGT- equivalent of CDRL2 comprising the nucleic acid sequence beginning at adenine at position 148 and ending at cytosine at position 156 (SEQ ID NO: 3);

f) Kabat-defined CDRL3 comprising the nucleic acid sequence beginning at cytosine at position 265 and ending at guanine at position 291 (SEQ ID NO: 3) is replaced with IMGT-equivalent of CDRL3 comprising the nucleic acid sequence beginning at cytosine at position 265 and ending at guanine at position 291 (SEQ ID NO: 3); g) Kabat-defined FRH1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 90 (SEQ ID NO: 1) encoding an amino acid sequence glutamic acid at amino acid position 1 to phenylalanine at amino acid position 30 (SEQ ID NO: 1) is replaced with IMGT-equivalent of FRH1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 75 (SEQ ID NO: 1) encoding an amino acid sequence glutamic acid at amino acid position 1 to serine at amino acid position 25 (SEQ ID NO: 1);

h) Kabat-defined FRH2 comprising the nucleic acid sequence beginning at thymine at position 106 and ending at adenine at position 147 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 36 to alanine at amino acid position 49 (SEQ ID NO: 1) is replaced with IMGT-equivalent of FRH2 comprising the nucleic acid sequence beginning at adenine at position 100 and ending at cytosine at position 150 (SEQ ID NO: 1) encoding an amino acid sequence isoleucine at amino acid position 34 to tyrosine at amino acid position 50 (SEQ ID NO: 1);

i) Kabat-defined FRH3 comprising the nucleic acid sequence beginning at cytosine at position 199 and ending at cytosine at position 294 (SEQ ID NO: 1) encoding an amino acid sequence arginine at amino acid position 67 to arginine at amino acid position 98 (SEQ ID NO: 1) is replaced with IMGT-equivalent of FRH3 comprising the nucleic acid sequence beginning at thymine at position 175 and ending at thymine at position 288 (SEQ ID NO: 1) encoding an amino acid sequence tyrosine at amino acid position 59 to cysteine at amino acid position 96 (SEQ ID NO: 1);

j) Kabat-defined FRH4 comprising the nucleic acid sequence beginning at thymine at position 328 and ending at guanine at position 360 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1) is replaced with IMGT-equivalent of FRH4 comprising the nucleic acid sequence beginning at thymine at position 328 and ending at guanine at position 360 (SEQ ID NO: 1) encoding an amino acid sequence tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 1);

k) Kabat-defined FRL1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at cytosine at position 69 (SEQ ID NO: 3) encoding an amino acid sequence aspartic acid at amino acid position 1 to cysteine at amino acid position 23 (SEQ ID NO: 3) is replaced with IMGT-equivalent of FRL1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 78 (SEQ ID NO: 3) encoding an amino acid sequence aspartic acid at amino acid position 1 to serine at amino acid position 26 (SEQ ID NO: 3);

1) Kabat-defined FRL2 comprising the nucleic acid sequence beginning at thymine at position 103 and ending at cytosine at position 147 (SEQ ID NO: 3) encoding an amino acid sequence tryptophan at amino acid position 35 to tyrosine at amino acid position 49 (SEQ ID NO: 3) is replaced with IMGT-equivalent of FRL2 comprising the nucleic acid sequence beginning at guanine at position 97 and ending at cytosine at position 147 (SEQ ID NO: 3) encoding an amino acid sequence valine at amino acid position 33 to tyrosine at amino acid position 49 (SEQ ID NO: 3);

m) Kabat-defined FRL3 comprising the nucleic acid sequence beginning at guanine at position 169 and ending at thymine at position 264 (SEQ ID NO: 3) encoding an amino acid sequence glycine at amino acid position 57 to cysteine at amino acid position 88 (SEQ ID NO: 3) is replaced with IMGT-equivalent of FRL3 comprising the nucleic acid sequence beginning at guanine at position 157 and ending at thymine at position 264 (SEQ ID NO: 3) encoding an amino acid sequence glutamic acid at amino acid position 53 to cysteine at amino acid position 88 (SEQ ID NO: 3); and

n) Kabat-defined FRL4 comprising the nucleic acid sequence beginning at thymine at position 292 and ending at adenine at position 321 (SEQ ID NO: 3) encoding an amino acid sequence phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3) is replaced with IMGT-equivalent of FRL4 comprising the nucleic acid sequence beginning at thymine at position 292 and ending at adenine at position 321 (SEQ ID NO: 3) encoding an amino acid sequence phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 3).

119. The isolated nucleic acid of claim 4 or 68, wherein the nucleic acid encoding for each CDR and each FR amino acid sequence is defined by IMGT method are the following:

a) IMGT-defined CDRH 1 comprising the nucleic acid sequence beginning at guanine at position 76 and ending at thymine at position 99 (SEQ ID NO: 79); b) IMGT-defined CDRH2 comprising the nucleic acid sequence beginning at adenine at position 151 and ending at thymine at position 174 (SEQ ID NO: 79);

c) IMGT-defined CDRH3 comprising the nucleic acid sequence beginning at guanine at position 289 and ending at cytosine at position 327 (SEQ ID NO: 79);

d) IMGT-defined CDRL1 comprising the nucleic acid sequence beginning at cytosine at position 79 and ending at cytosine at position 96 (SEQ ID IMGT- defined CDRL2 comprising the nucleic acid sequence beginning at adenine at position 148 and ending at cytosine at position 156 (SEQ ID NO: 77);

e) IMGT-defined CDRL3 comprising the nucleic acid sequence beginning at cytosine at position 265 and ending at guanine at position 291 (SEQ ID NO: 77); f) IMGT-defined FRH1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 75 (SEQ ID NO: 79) encoding an amino acid sequence glutamic acid at amino acid position 1 to serine at amino acid position 25 (SEQ ID NO: 79);

g) IMGT-defined FRH2 comprising the nucleic acid sequence beginning at adenine at position 100 and ending at cytosine at position 150 (SEQ ID NO: 79) encoding an amino acid sequence isoleucine at amino acid position 34 to tyrosine at amino acid position 50 (SEQ ID NO: 79);

h) IMGT-defined FRH3 comprising the nucleic acid sequence beginning at thymine at position 175 and ending at thymine at position 288 (SEQ ID NO: 79) encoding an amino acid sequence tyrosine at amino acid position 59 to cysteine at amino acid position 96 (SEQ ID NO: 79);

i) IMGT-dewfined FRH4 comprising the nucleic acid sequence beginning at thymine at position 328 and ending at guanine at position 360 (SEQ ID NO: 79) encoding an amino acid sequence tryptophan at amino acid position 110 to serine at amino acid position 120 (SEQ ID NO: 79);

j) IMGT-defined FRL1 comprising the nucleic acid sequence beginning at guanine at position 1 and ending at thymine at position 78 (SEQ ID NO: 77) encoding an amino acid sequence aspartic acid at amino acid position 1 to serine at amino acid position 26 (SEQ ID NO: 77);

k) IMGT-defined FRL2 comprising the nucleic acid sequence beginning at guanine at position 97 and ending at cytosine at position 147 (SEQ ID NO: 77) encoding an amino acid sequence valine at amino acid position 33 to tyrosine at amino acid position 49 (SEQ ID NO: 77);

1) IMGT-defined FRL3 comprising the nucleic acid sequence beginning at guanine at position 157 and ending at thymine at position 264 (SEQ ID NO:77) encoding an amino acid sequence glutamic acid at amino acid position 53 to cysteine at amino acid position 88 (SEQ ID NO: 77); and

m) IMGT-defined FRL4 comprising the nucleic acid sequence beginning at thymine at position 292 and ending at adenine at position 321 (SEQ ID NO: 77) encoding an amino acid sequence phenylalanine at amino acid position 98 to lysine at amino acid position 107 (SEQ ID NO: 77).

120. A host cell transformed with the nucleic acid according to claim 67 or 68.

121. A method of producing a specific binding member that recognizes and binds a VEGF, comprising: culturing a host cell according to claim 120 under conditions for production of said specific binding member, and isolating and/or purifying said specific binding member, wherein the host cell comprises a nucleotide sequence encoding the heavy chain or portion thereof, and a nucleotide sequence encoding the light chain or portion thereof, of the antigen-binding portion of the antibody.

122. The method according to claim 121, wherein the specific binding member that recognizes and binds a VEGF is a scFv antibody molecule or a Fab antibody molecule.

123. The method according to claim 121, wherein the specific binding member that recognizes and binds a VEGF is a full length antibody.

124. A method for inhibiting VEGF-induced angiogenesis in a mammal comprising administering a therapeutically effective amount of the anti-VEGF antibody of claim 1 , 2, 3, 4, 6 or 7 to the mammal.

125. The method of claim 124, wherein the mammal is selected from the group of: a murine, feline, canine, porcine, caprine, ovine, bovine, leporine, equine, simian, and a human subject.

126. The method of claim 124, wherein the mammal has a disease selected from retinal disorder, wet age-related macular degeneration, diabetic maculopathy, proliferative diabetic retinopathy, macular edema in retinal vein occulusion (RVO), iris neovascularization, choroidal neovascularization caused by pathological myopia, retinopathy of prematurity (ROP), retinopathy of maturity, neovascular glaucoma, and cancer.

127. The method of claim 126, wherein the retinal disorder is associated with poor vision at night (night blindness), trouble adjusting from brightly lit to dim areas, sudden or unexplained loss of vision, loss of peripheral vision, loss of vision in a particular visual field, a rapid, involuntary oscillatory motion of the eyeball (nystagmus), abnormal sensitivity to or intolerance of light (photophobia) or a combination thereof.

128. A method for inhibiting macular degeneration in a mammal comprising administering a therapeutically effective amount of the humanized anti-VEGF antibody of claim 1, 2, 3, 4, 6 or 7 to the mammal.

129. The method of claim 128, wherein macular degeneration is wet macular degeneration.

130. The method of claim 129, wherein wet macular degeneration is age-related.

131. The method of claim 128, wherein macular degeneration is age-related.

132. The method of claim 128, wherein macular degeneration is associated with neovascularization or angiogenesis.

133. The method of claim 128, wherein administration is intravitreal administration.

134. The method of claim 133, wherein administration inhibits, prevents or reverses neovascularization or angiogenesis.

135. A method for inhibiting a cell proliferative disorder in a mammal comprising administering a therapeutically effective amount of the anti-VEGF antibody of claim 1, 2, 3, 4, 6 or 7 to the mammal.

136. The method of claim 128 or 135, wherein the mammal is selected from the group of: a murine, feline, canine, porcine, caprine, leporine, ovine, bovine, equine, simian, and a human subject.

137. The method of claim 135, wherein the cell proliferative disorder is selected from the group consisting of wet age related macular degeneration, diabetic maculopathy, proliferative diabetic retinopathy, macular edema in retinal vein occlusion (RVO), iris neovascularization, choroidal neovascularisation caused by pathological myopia, retinopathy of maturity, neovascular glaucoma, diabetic retinopathy, retinal neovascularization, pars plana vitrectomy (PPV), diabetic macular edema (DME) and cancer.

138. An isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Tables 5a and 5c.

139. An isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising a heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Tables 5b and 5d.

140. An isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising any of the light chain of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 comprising amino acid sequence as provided in Table 5a or 5c.

141. An isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising any of the heavy chain of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 comprising amino acid sequence as provided in Table 5b or 5d.

142. An isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising

a. a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Table 5a, and

b. a corresponding heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Table 5b.

143. An isolated human anti-VEGF antibody or portion or variant thereof that specifically recognizes and binds a VEGF comprising

a. a light chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Table 5c, and

b. a corresponding heavy chain variable domain comprising the following hypervariable region or complementarity determining region (CDR) amino acid sequences of variant 201, 202, 203, 204, 205, 206, 207, 208, 209, 212, 213, 214, 215, 216, 217, 218, 219, or 220 as shown in Table 5d.