# Patent

## Title
VEGF-specific human antibody

## Abstract

The present invention relates to a vascular endothelial growth factor (VEGF)-specific human antibody, and more particularly to a human antibody including a complementarity determining region (CDR) and a framework region (FR) derived from a human antibody specifically bound to VEGF. The VEGF-specific human antibody of the present invention may be used in diagnosis of diseases caused by the VEGF-overexpression, classification of the diseases, visualization, treatment, and prognostic evaluation.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. §119 of Korean Patent application number KR 10-2008-0118124, filed on Nov. 26, 2008, which is herein incorporated by reference in its entirety.
TECHNICAL FIELD
The present disclosure relates to a vascular endothelial growth factor (VEGF)-specific human antibody.
BACKGROUND ART
Vascular endothelial growth factor (VEGF) is known to play a critical role in vasculogenesis and angiogenesis of the developmental process (Soker S et al., J Cell Biochem. 85:357-368, 2002). As it has been reported that VEGFR1 is overexpressed not only in vascular endothelial cells, but also in colonic and pancreatic cancer cells, and is directly involved in tumor progression and metastasis, VEGFR1 is considered to play an important role in angiogenesis, tumor growth and metastasis, inflammation, etc (Wey J S et al., Cancer 104:427-438, 2005; Fan F et al., Oncogene 24:2647-2653, 2005). VEGF is one of the most important factors in tumor angiogenesis, is expressed in most tumor tissues, such as renal cell cancer (Tomisawa M et al., Eur J Cancer 35:133-137, 1999), lung cancer (Volm M et al., Int J Cancer 74: 64-68, 1997), breast cancer (Yoshiji H et al., Cancer Res. 56:2013-2016, 1996), ovarian cancer (Sowter H M et al., Lab Invest. 77:607-614, 1997), etc., and is secreted not only in tumor cells, but also in tumor stromal cells. Although mouse anti-human VEGF monoclonal antibodies did not have much effect on the ex vivo growth of tumor cells in an attempt to use VEGF antagonists to inhibit tumor growth, the antibodies showed significant inhibiting effects for tumor angiogenesis and tumor growth in vivo (Kim K J et al., Nature 362: 841-844, 1993; Borgstrom P et al., Cancer Res. 56:4032-4039).
It is widely known that VEGF is strongly associated with not only tumors, but also other diseases, and various efforts have been made to develop therapeutics for these diseases.
Representative examples of these diseases include rheumatoid arthritis (RA) which is a disease associated with angiogenesis, diabetic retinopathy, ischemic retinopathy, psoriasis, etc., and it has been revealed that VEGF functions as an important factor to theses diseases. In the case of RA, it was confirmed that the amount of serum VEGF from RA patients increased compared to that from patients in a control group (Ikeda M et al., J pathol. 191:426-33, 2000). The amount of serum VEGF from diabetic patients also increased and the increased blood sugar level caused toxic effects on the endothelium to induce a hyperglycemic pseudo-hypoxic state which induced VEGF production. This showed a correlation between endothelial damage in diabetes and dysfunction (Lim H S et al., Diabetes Care 27:2918-24; 2004). Excessive secretion of VEGF in the retina causes ocular neovascularization and hematoma, resulting in visual impairment/blindness. In an effort to prevent visual loss associated with proliferative diabetic retinopathy (PDR) and diabetic macular edema and avoid side effects associated with destructive treatments such as laser treatment, humanized monoclonal anti-VEGF antibody fragments which are selectively bound to all the subtypes of VEGF are used. The compound known as rhuFab V2, produced by Genetech Co., Ltd. is now under clinical research and known to show some effectiveness in prevention of PDR or diabetic macular edema (Heier J S, Program and abstracts of the American Academy of Opthalmology 2002 Annual Meeting; October 20-23, Orlando, Fla.).
Various types of 40 or more angiogenesis inhibitors are currently under clinical development for various kinds of tumors. VEGF and VEGF receptors are the most representative targets, and include agents which inhibit activity, signal transduction, and production. VEGF inhibitors include antibodies, aqueous VEGF receptors (VEGF traps), etc. Because bevacizumab (AvastinTM, Genetech), which is a humanized anti-VEGF monoclonal antibody as an angiogenesis inhibitor for tumor treatment, showed life-prolonging effects on patients with metastatic colorectal cancer, the drug was approved by the FDA in February, 2004. Therefore, the development of these anti-VEGF human monoclonal antibodies has advantages in that the antibodies are a promising candidate for treatment of angiogenesis and various diseases associated with it and may be used in clinical and preclinical settings due to their minimal side effects, and thus the development of various therapeutic agents using these antibodies warrants due attention.
Thus, the present inventors have selected 14 kinds of human antibodies specifically bound to VEGF, confirmed that the human antibodies have binding and neutralizing capacities similar to those of AvastinTM and exhibit cross reactivity with the mouse VEGF, proposed that the human antibodies of the present invention may be effectively used in treatment of VEGF-overexpressed diseases, and have made the present invention.
DISCLOSURE
Technical Problem
One object of the present invention is to provide a VEGF-specific human antibody.
Another object of the present invention is to provide a polynucleotide encoding a heavy chain of the human antibody or a fragment thereof, and an expression vector including the polynucleotide and a constant region of human heavy chain.
Still another object of the present invention is to provide a polynucleotide encoding a light chain of the human antibody or a fragment thereof, and an expression vector including the polynucleotide and a constant region of human light chain.
Even another object of the present invention is to provide a transformant prepared by introducing an expression vector including a polynucleotide encoding the heavy chain of the human antibody or an immunologically active fragment thereof into a host cell.
Yet another object of the present invention is to provide a transformant prepared by introducing an expression vector including a polynucleotide encoding the light chain of the human antibody or an immunologically active fragment thereof into a host cell.
Further another object of the present invention is to provide a transformant prepared by introducing an expression vector including a polynucleotide encoding the heavy chain of the human antibody or a fragment thereof and an expression vector including a polynucleotide encoding the light chain or a fragment thereof simultaneously into a host cell.
Still further another object of the present invention is to provide a method for preparing a VEGF-specific human antibody by incubating the transformant.
The present invention also provides a composition including the human antibody.
The present invention also provides a pharmaceutical composition including the human antibody.
Another object of the present invention is to provide a method for treating diseases caused by VEGF-overexpression, the method including administering a pharmaceutically effective amount of the human antibody to a subject.
Still another object of the present invention is to provide a composition including the human antibody and a radioactive isotope.
Even another object of the present invention is to provide an immunodetection method for detecting an ex vivo VEGF-overexpressed cancer, including contacting a composition including the radioactive isotope with a cancer cell.
Yet another object of the present invention is to provide a method for imaging an in vivo VEGF-overexpressed cancer, including administering a composition including the radioactive isotope to a subject.
Further another object of the present invention is to provide a method for treating an in vivo VEGF-overexpressed cancer by using a composition including the radioactive isotope.
Still further another object of the present invention is to provide a method for prognostic evaluation of a VEGF-overexpressed cancer treatment using a composition including the radioactive isotope.
Even further another object of the present invention is to provide a method for measuring side effects of the human antibody, including administering the human antibody to an animal experiment model.
Technical Solution
To achieve the objects, the present invention provides a VEGF-specific human antibody including a heavy chain including a heavy chain variable region (VH) including a heavy chain complementarity determining region (hereinafter, HCDR) 1 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 5 to 17, HCDR 2 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 18 to 30, and HCDR 3 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 31 to 43, or a fragment thereof; and
a light chain including a light chain variable region (VL) including a light chain complementarity determining region (hereinafter, LCDR) 1 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 57 to 69 and SEQ ID Nos. 130 to 142, LCDR 2 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 70 to 82 and SEQ ID Nos. 143 to 152, and LCDR 3 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 83 to 93 and SEQ ID Nos. 153 to 164, or a fragment thereof.
The present invention also provides a polynucleotide encoding a heavy chain of the human antibody or an immunologically active fragment thereof, and an expression vector including the polynucleotide.
The present invention also provides a polynucleotide encoding a light chain of the human antibody or an immunologically active fragment thereof, and an expression vector including the polynucleotide.
The present invention also provides a transformant prepared by introducing an expression vector including a polynucleotide encoding a heavy chain of the human antibody or an immunologically active fragment thereof into a host cell.
The present invention also provides a transformant prepared by introducing an expression vector including a polynucleotide encoding a light chain of the human antibody or an immunologically active fragment thereof into a host cell.
The present invention also provides a transformant prepared by introducing an expression vector including a polynucleotide encoding a heavy chain of the human antibody or an immunologically active fragment thereof and an expression vector including a polynucleotide encoding a light chain of the human antibody or an immunologically active fragment thereof simultaneously into a host cell.
The present invention also provides a method for preparing a VEGF-specific human antibody by incubating the transformant.
The present invention also provides a composition including the human antibody.
The present invention also provides a pharmaceutical composition including the human antibody.
The present invention also provides a method for treating a disease caused by VEGF-overexpression, including administering a pharmaceutically effective amount of the human antibody to a subject with the disease.
The present invention also provides a composition including the human antibody, a light or heavy chain of the human antibody or an immunologically active fragment thereof, and a radioactive isotope.
The present invention also provides an immunodetection method for detecting an ex vivo VEGF-overexpressed cancer, including contacting a composition including the radioactive isotope with a cancer cell.
The present invention also provides a method for imaging an in vivo VEGF-overexpressed cancer, the method including:
1) administering a diagnostically effective amount of a composition including the radioactive isotope to a subject; and
2) obtaining a detection image for the subject.
The present invention also provides a method for treating an in vivo VEGF-overexpressed cancer, the method including:
1) intravenously administering a composition including the radioactive isotope to a subject;
2) detecting the composition of Step 1) to identify tumor cells; and
3) eliminating the tumor cells identified in Step 2) by surgical operation.
The present invention also provides a method for prognostic evaluation of a cancer patient, the method including:
1) intravenously administering a composition including the radioactive isotope to a patient whose tumor has been eliminated;
2) detecting the composition of Step 1) to identify tumor cells; and
3) judging that all tumor cells have been eliminated when tumor cells are not detected in step 2).
Furthermore, the present invention provides a method for measuring side effects of the human antibody, including administering the human antibody to an animal experiment model with a disease caused by VEGF-overexpression.
Advantageous Effect
The VEGF-specific human antibody of the present invention may be used in diagnosis of diseases caused by the VEGF-overexpression, classification of the diseases, visualization, treatment, and prognostic evaluation.



DESCRIPTION OF DRAWINGS
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a photo illustrating results of VEGF protein expression in a pYK602-VEGF vector, confirmed by Western blot.
FIG. 2 is a photo illustrating results of VEGF protein expression in pYK602-VEGF, pYK602-His-VEGF, and pYK603-VEGF vectors, compared by Western blot.
FIG. 3 is a photo illustrating results of VEGF expression in a pYK603-VEGF vector, confirmed by Western blot.
FIG. 4 is a photo illustrating results of a purified VEGF, confirmed by SDS-PAGE.
FIG. 5 is a graph illustrating results of phage screening in 1st to 3rd pannings.
FIG. 6 is a photo illustrating results of diversity of human VEGF monoclonal phage antibodies to VEGF, confirmed by fingerprinting.
FIG. 7 is a list of sequences illustrating analysis results of a polypeptide used in a heavy chain CDR of human VEGF monoclonal phage antibodies to VEGF.
FIG. 8 is a list of sequences illustrating analysis results of a polypeptide used in a light chain CDR of human VEGF monoclonal phage antibodies to VEGF.
FIG. 9 is a group of graphs illustrating results comparing binding specificities of human VEGF monoclonal antibodies:
a: C5, E9, F6, G12, A4, C11, and F2; and b: H7, G9, C9, B12, F9, D12, and C12.
FIG. 10 is a group of photos illustrating results, confirming that neutralizing capacities inhibited the tube formation of human VEGF monoclonal antibodies in HUVEC cells.
FIG. 11 is a group of graphs illustrating results of the measurement of cross-reactivity of human VEGF monoclonal antibodies with hVEGF and mVEGF.
FIG. 12 is a group of drawings illustrating cleavage maps of pNATAB H and pNATAB L vectors:
a: pNATAB H vector, b: pNATAB L vector
FIG. 13 is a group of drawings illustrating cleavage maps of pYK602, pYK602-His, and pYK603 vectors:
a: pYK602 vector; b: pYK602-His vector; and c: pYK603 vector.
FIG. 14 is a group of drawings illustrating results of phage antibody search from a LC shuffling library in 1st to 3rd pannings.
FIG. 15 is a group of drawings illustrating results of a diversity of an LC shuffling monoclonal phage antibody confirmed through a fingerprinting process.
FIG. 16 is a group of drawings illustrating analysis results of a polypeptide used in a light chain CDR of an LC shuffling monoclonal phage antibody.



BEST MODE
Features and advantages of the present invention will be more clearly understood by the following detailed description of the present preferred embodiments by reference to the accompanying drawings. It is first noted that terms or words used herein should be construed as meanings or concepts corresponding with the technical sprit of the present invention, based on the principle that the inventor can appropriately define the concepts of the terms to best describe his own invention. Also, it should be understood that detailed descriptions of well-known functions and structures related to the present invention will be omitted so as not to unnecessarily obscure the important point of the present invention.
Hereinafter, the terms of the present invention will be described.
“Variable region” means a region of an antibody molecule which specifically binds to an antigen and demonstrates modifications in sequence, which is exemplified by CDR1, CDR2, and CDR3. Between the CDRs, there is a framework region (FR) which supports the CDR loop.
“Complementarity determining region” is a loop-shaped site involved in antigen recognition, and specificity of an antibody against antigen depends on modification in that site.
“Panning” refers to a process of selecting only a phage expressing a peptide which binds to a target molecule (antibody, enzyme, cell-surface receptor, etc.) on the coat of the phage from a phage library displaying the peptide on the coat.
Hereinafter, the present invention will be described in detail.
The present invention provides a VEGF-specific human antibody, including: a heavy chain including a heavy chain variable region (VH) including a heavy chain complementarity determining region (hereinafter, HCDR) 1 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 5 to 17, HCDR 2 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 18 to 30, and HCDR 3 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 31 to 43, or a fragment thereof; and
a light chain including a light chain variable region (VL) including a light chain complementarity determining region (hereinafter, LCDR) 1 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 57 to 69 and SEQ ID Nos. 130 to 142, LCDR 2 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 70 to 82 and SEQ ID Nos. 143 to 152, and LCDR 3 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 83 to 93 and SEQ ID Nos. 153 to 164, or a fragment thereof.
Preferably, the heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID Nos. 44 to 56, and the light chain variable region has an amino acid sequence selected from the group consisting of SEQ ID Nos. 94 to 106 and SEQ ID Nos. 165 to 178.
The antibody includes not only a whole antibody, but also a functional fragment of the antibody molecule. The whole antibody has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to heavy chain by disulfide bond. The functional fragment of an antibody molecule indicates a fragment retaining a antigen-binding function, and examples of the antibody fragment include (i) Fab fragment consisting of light chain variable region (VL), heavy chain variable region (VH), light chain constant region (CL), and heavy chain 1st constant region (CH1); (ii) Fd fragment consisting of VH and CH1 domains; (iii) Fv fragment consisting of VL and VH domains of a monoclonal antibody; (iv) dAb fragment consisting of VH domain (Ward E S et al., Nature 341:544-546 (1989)); (v) separated CDR region; (vi) F(ab′)2 fragment including two linked Fab fragments, as a divalent fragment; (vii) single chain Fv molecule (ScFv) in which VH and VL domains are linked by a peptide linker to form an antigen binding site; (viii) bi-specific single chain Fv dimmer (PCT/US92/09965), and (ix) multivalent or multi-specific diabody fragment (WO94/13804) prepared by gene fusion.
In the present invention, a human antibody against VEGF was obtained as ScFV by using phage display technology and screened as a mono phage clone. As a result, 14 kinds of VEGF-specific monoclonal phages were obtained.
In a specific example of the present invention, VEGF obtained through recombinant technology (see FIGS. 1 to 4) was used in preparation of monoclonal antibodies. The VEGF was reacted with a library phage constructed from human naive scFV library cells, followed by panning and screening of mono clones strongly binding to the VEGF antigen (see Tables 1 & 2 and FIG. 5). The selected mono clones were identified by fingerprinting (see FIG. 6), followed by sequencing to identify CDR regions of VH and VL of the antibody (see Table 5 and FIGS. 7 and 8). The Ig BLAST program of NCBI (//www.ncbi.nlm.nih.gov/igblast/) was used for identification of similarity between the antibody and a germ line antibody group (see Table 6). As a result, 14 kinds of VEGF-specific phage antibodies were obtained. The selected monoclonal antibodies exhibited binding capacities in the order of G12> D12> E9> F6> H7> C5> B12> G9> F9> C11> F2> A4> C9> C12 (see FIGS. 9a and 9b), and it was observed that they significantly inhibited a capillary-like tube formation of HUVEC cells, induced by VEGF (see FIG. 10). E9, F6, and G12 monoclonal antibodies showing binding capacities similar to that of AvastinTM all exhibited strong affinities for mouse VEGF similar to those for human VEGF, leading to a cross reaction (see Table 8 and FIG. 11). Because AvastinTM is not reacted with mouse VEGF at all, it has been difficult to perform animal experimental studies on side effects by AvastinTM However, the VEGF neutralizing human antibody of the present invention is cross reacted with the mouse, indicating that it is highly different from the conventional anti-cancer drug AvastinTM in terms of epitope. Because the VEGF neutralizing human antibody of the present invention exhibits a high cross reactivity with mouse, it may be used in animal experimental studies.
A library phage with a light chain shuffling was prepared for F6 and G12 monoclonal antibodies exhibiting neutralizing capacities similar to those of the AvastinTM. The prepared library phage was reacted with VEGF for panning, followed by screening of monoclones strongly binding to VEGF antigen (see Tables 10 & 11 and FIG. 14). The selected monoclones were confirmed by a fingerprinting process (see FIG. 15), followed by sequencing to confirm CDR regions of VH and VL of the antibody. Because CDR regions of VH of the antibody selected by an LC shuffling were identical to each other, CDR regions of VL of the antibody were prepared (see Table 12 and FIG. 16). Homology between the antibody and germ line antibody group was investigated (see Table 13). As a result, 15 VEGF specific LC shuffling monoclonal phage antibodies were obtained and their VEGF binding capacities were confirmed (see FIG. 14). A confirmation result of human VEGF binding capacities of the LC shuffling monoclonal phage antibodies showed that the antibodies had high affinities similar to those of F6 and G12, except for 2C11, 2G03, 2C05, and 2F10.
The present invention also provides a polynucleotide encoding a heavy chain of the human antibody or an immunologically active fragment thereof, and an expression vector including the polynucleotide.
The present invention also provides a polynucleotide encoding a light chain of the human antibody or an immunologically active fragment thereof, and an expression vector including the polynucleotide.
In a specific embodiment of the present invention, VEGF obtained by recombinant technology was used to screen mono clones strongly binding to VEGF antigens (see Tables 1 and 2 and FIG. 5). The selected monoclones were identified by fingerprinting (see FIG. 6), followed by sequencing to identify CDR regions of VH and VL of the antibody (see Table 5 and FIGS. 7 and 8). The identification of similarity between the antibody and a germ line antibody group was performed (see FIG. 6). As a result, 14 kinds of VEGF-specific phage antibodies were obtained. Binding capacities to VEGF of the selected monoclonal antibodies were measured (see FIGS. 9a & 9b), and it was observed that the antibodies significantly inhibited a capillary-like tube formation of HUVEC cells, induced by VEGF (see FIG. 10). E9, F6, and G12 monoclonal antibodies showing binding capacities similar to that of AvastinTM all exhibited strong affinities for mouse VEGF similar to those for human VEGF, leading to a cross reaction (see Table 8 and FIG. 11). Because AvastinTM is not reacted with mouse VEGF at all, it has been difficult to perform animal experimental studies on side effects by AvastinTM However, the VEGF neutralizing human antibody of the present invention is cross reacted with the mouse, indicating that it is highly different from the conventional anti-cancer drug AvastinTM in terms of epitope. Because the VEGF neutralizing human antibody of the present invention exhibits a high cross reactivity with mouse VEGF, it may be used in animal experimental studies.
In the polynucleotide encoding light and heavy chains of the human antibody of the present invention or a fragment thereof, due to degeneracy of the codon or in consideration of a preferred codon in an organism where light and heavy chains of the human antibody or a fragment thereof are to be expressed, various modifications may be made in a coding region within a scope that the amino acid sequences of light and heavy chains or a fragment thereof are not changed, and various changes or modifications may be made even in portions other than the coding region within a scope that the gene expression is not affected. It will be appreciated by those skilled in the art that these modified genes are also included within the scope of the present invention. That is, one or more nucleotides may be modified by substitution, deletion, insertion, or a combination thereof as long as the polynucleotide of the present invention encodes a protein with an equivalent activity thereof, and they are also included in the present invention. The sequence of the polynucleotide may be a single or double chain, and a DNA or RNA (mRNA) molecule.
In preparation of the expression vector, an expression control sequence such as a promoter, a terminator, an enhancer, etc., a sequence for membrane targeting or secretion, etc. may be appropriately selected according to a kind of host cell in which light and heavy chains of the human antibody or a fragment thereof are to be produced, and may be variously combined according to its purpose.
The expression vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage, and a viral vector. A suitable expression vector may include expression regulatory elements such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer and a signal sequence or leader sequence for membrane targeting or secretion, and may be variously prepared according to its purpose. A promoter of the expression vector may be constitutive or inductive. Examples of the signal sequence for use may include, but is not limited to, a PhoA signal sequence and an OmpA signal sequence for genus Escherichia hosts; an α-amylase signal sequence and a subtilicin signal sequence for genus Bacillus hosts; an MFα signal sequence and an SUC2 signal sequence for yeast hosts; and an insulin signal sequence, an α-interferon signal sequence, and an antibody molecule signal sequence for animal cell hosts. In addition, the expression vector may include a selection marker for selecting host cells containing the vector, and a replication origin when it is a replicable expression vector.
The present invention also provides a transformant prepared by introducing an expression vector including a polynucleotide encoding a heavy chain of the human antibody or an immunologically active fragment thereof into a host cell.
The present invention also provides a transformant prepared by introducing an expression vector including a polynucleotide encoding a light chain of the human antibody or an immunologically active fragment thereof into a host cell.
The present invention also provides a transformant prepared by introducing an expression vector including a polynucleotide encoding a heavy chain of the human antibody or a fragment thereof and an expression vector including a polynucleotide encoding a light chain of the human antibody or a fragment thereof simultaneously into a host cell.
In a specific example of the present invention, genes encoding light and heavy chains of a monoclonal phage were obtained and linked to a vector, respectively, and then a whole human IgG antibody expressed by introducing the expression vectors simultaneously into a host cell was identified. The human antibody was obtained to identify a binding capacity (see FIGS. 9a & 9b), a neutralizing capacity to VEGF (see FIG. 10), and a cross reactivity with mouse and human VEGF (see Table 8 and FIG. 11).
The expression vector according to the present invention may be transformed into a suitable host cell, for example, E. coli or yeast cell, and the transformed host cell may be incubated to produce light and heavy chains of the human antibody of the present invention or a fragment thereof in mass quantities. Incubation methods and media conditions suitable for a kind of host cell may be easily chosen from those known to those skilled in the art. The host cell may be a prokaryote such as E. coli or Bacillus subtilis. In addition, it may be a eukaryotic cell derived from a yeast such as Saccharomyces cerevisiae, an insect cell, a vegetable cell, and an animal cell. More preferably, the animal cell may be an autologous or allogeneic animal cell. A transformant prepared through introduction into an autologous or allogeneic animal cell may be administered to a subject for use in cellular therapy for cancer. As for a method for introducing an expression vector into the host cell, it is possible to use any method if it is known to those skilled in the art.
The present invention also provides a method for preparing VEGF-specific human antibodies, the method including:
1) incubating the transformant; and
2) purifying the human antibody from the medium.
As for the culture medium, it is desirable to select and use a culture medium suitable for the transformant among those known to those skilled in the art. As for the method for purifying human antibodies, it is possible to use any method known to those skilled in the art.
In a specific example of the present invention, genes encoding light and heavy chains of a monoclonal phage were obtained and linked to a vector, respectively, and then a whole human IgG antibody expressed by introducing the expression vectors simultaneously into a host cell was identified. The human antibody was obtained to identify a binding capacity (see FIGS. 9a & 9b), a neutralizing capacity to VEGF (see FIG. 10), and a cross reactivity with mouse and human VEGF (see Table 8 and FIG. 11).
The present invention also provides a composition including the human antibody.
The present invention also provides a pharmaceutical composition including the human antibody.
The disease caused by VEGF-overexpression includes one associated with cancer or angiogenesis. The cancer is preferably one selected from the group consisting of, but not limited to, colorectal cancer, renal cell cancer, lung cancer, breast cancer, and ovarian cancer, and includes all the VEGF-overexpressed cancers. The diseases associated with angiogenesis also include rheumatoid arthritis (RA), diabetic retinopathy, ischemic retinopathy, psoriasis, proliferative diabetic retinopathy (PDR), diabetic macular edema, etc.
In a specific example of the present invention, it was observed that VEGF monoclonal antibodies exhibiting binding capacities to VEGF (see FIGS. 9a and 9b) significantly inhibited a capillary-like tube formation of HUVEC cells, induced by VEGF in a human umbilical vein endothelial cell line (see FIG. 10). E9, F6, and G12 monoclonal antibodies showing binding capacities similar to that of AvastinTM all exhibited strong affinities for mouse VEGF similar to those for human VEGF, leading to a cross reaction (see Table 8 and FIG. 11). Because AvastinTM is not reacted with mouse VEGF at all, it has been difficult to perform animal experimental studies on side effects by AvastinTM. However, the VEGF neutralizing human antibody of the present invention is cross reacted with the mouse, indicating that it is highly different from the conventional anti-cancer drug AvastinTM in terms of epitope. Because the VEGF neutralizing human antibody of the present invention exhibits a high cross reactivity with mouse VEGF, it may be used in animal experimental studies. Thus, the monoclonal antibody of the present invention may be useful as a composition for prevention and treatment of VEGF-overexpressed diseases.
The pharmaceutical composition of the present invention may selectively contain the VEGF-specific human antibody or the transformant, and may additionally contain one or more effective ingredients exhibiting functions identical or similar to those of the ingredient. For administration, the pharmaceutical composition of the present invention may be formulated by including one or more pharmaceutically acceptable carriers in addition to the effective ingredients described above. For example, the pharmaceutically acceptable carrier includes saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and at least one combination thereof, and other general additives such as antioxidants, buffer solution, bacteriostatic agents, etc. may be added if necessary. In addition, it may be formulated in the form of an injectable formulation such as aqueous solution, suspension, emulsion, etc. by additionally adding diluent, dispersing agent, surfactant, binder and lubricant, and antibodies and other ligands specific to a target cell may be used in combination with the carrier to be specifically reacted with the target cell. Furthermore, the composition may be preferably formulated according to each disease or ingredient using a suitable method in the art or a method which is taught in Remington's Pharmaceutical Science, Mack Publishing Company, Easton Pa.
The pharmaceutical composition of the present invention may be parenterally administered, and the parenteral administration is effected by subcutaneous injection, intravenous injection, intramuscular injection, or intrapleural injection. For parenteral administration, the pharmaceutical composition of the present invention may be mixed with a stabilizer or buffer to prepare a solution or suspension, which may then be provided as ampoules or vials each containing a unit dosage form.
The pharmaceutical composition of the present invention may be prepared in various forms according to the route of administration. For example, the pharmaceutical composition of the present invention may be formulated to a sterilized aqueous solution or dispersion for injection, or may be prepared in a freeze-dried form through a freeze-drying technique. The freeze-dried pharmaceutical composition may be stored typically at about 4° C. and may be reconstituted with a stabilizer that may contain an adjuvant such as saline solution and/or HEPE.
In a method of the present invention, factors affecting the amount of the pharmaceutical composition to be administered include, but are not limited to, administration mode, administration frequency, specific disease under treatment, severity of disease, history of disease, whether the subject is under treatment in combination with other therapeutics, the subject's age, height, weight, health, and physical conditions. As the patient's weight under treatment increases, the pharmaceutical composition of the present invention may preferably be administered in increasing amounts.
In addition, the pharmaceutical composition of the present invention may be administered in combination with chemotherapy.
The present invention also provides a method for treating a disease caused by VEGF-overexpression, the method including administering a pharmaceutically active amount of the human antibody to a subject with the disease caused by VEGF-overexpression.
The disease caused by VEGF-overexpression includes one associated with cancer or angiogenesis. The cancer is preferably one selected from the group consisting of, but not limited to colorectal cancer, renal cell cancer, lung cancer, breast cancer, and ovarian cancer, and includes all the VEGF-overexpressed cancers. The diseases associated with angiogenesis also include rheumatoid arthritis, diabetic retinopathy, ischemic retinopathy, psoriasis, proliferative diabetic retinopathy (PDR), diabetic macular edema, etc.
In a specific example of the present invention, it was observed that VEGF monoclonal antibodies showing binding capacities to VEGF (see FIGS. 9a & 9b) significantly inhibited a capillary-like tube formation of HUVEC cells, induced by VEGF in a human umbilical vein endothelial cell line (see FIG. 10). E9, F6, and G12 monoclonal antibodies showing binding capacities similar to that of AvastinTM all exhibited strong affinities for mouse VEGF similar to those for human VEGF, leading to a cross reaction (see Table 8 and FIG. 11). Because AvastinTM is not reacted with mouse VEGF at all, it has been difficult to perform animal experimental studies on side effects by AvastinTM. However, the VEGF neutralizing human antibody of the present invention is cross reacted with the mouse, indicating that it is highly different from the conventional anti-cancer drug AvastinTM in terms of epitope. Because the VEGF neutralizing human antibody of the present invention exhibits a high cross reactivity with mouse VEGF, it may be used in animal experimental studies. Thus, the monoclonal antibody of the present invention may be useful as a composition for prevention and treatment of VEGF-overexpressed diseases.
The subject applicable in the present invention is a vertebrate, preferably a mammal, more preferably an experimental animal such as mouse, rabbit, guinea pig, hamster, dog, and cat, and most preferably a primate such as chimpanzee and gorilla.
The method for administering the human antibody of the present invention may be conducted by parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, or local administration) according to the purpose of use, and preferably by intravenous administration. In administration for solid cancer, local administration may be often preferable for rapid and facilitated access of the antibody. The dose may vary depending on weight, age, sex, and health condition of a patient, diet, administration time, administration method, excretion rate, and severity of disease. The single dose is in the range of 5 to 500 mg/m2, which may be administered daily or weekly. The effective amount may be controlled at the discretion of a doctor treating the patient.
The human antibody of the present invention may be used alone or in combination with surgery, hormone therapy, chemical therapy, and a biological response controller for treatment of a patient.
The present invention also provides a composition including the human antibody, light and heavy chains of the human antibody, or an immunologically active fragment thereof, and a radioactive isotope.
The composition may be used for radioimmuno treatment and detection of a disease caused by VEGF-overexpression. The disease caused by VEGF-overexpression includes one associated with cancer or angiogenesis. The cancer is preferably one selected from the group consisting of, but not limited to, colorectal cancer, renal cell cancer, lung cancer, breast cancer, and ovarian cancer, and includes all the VEGF-overexpressed cancers. The diseases associated with angiogenesis also include rheumatoid arthritis, diabetic retinopathy, ischemic retinopathy, psoriasis, proliferative diabetic retinopathy (PDR), diabetic macular edema, etc.
In a specific example of the present invention, it was confirmed that the monoclonal VEGF antibody showed strong binding capacity to VEGF (see FIGS. 9a and 9b). Thus, the monoclonal antibody of the present invention may be useful as a composition for radioimmuno treatment of diseases caused by VEGF-overexpression and including a radioactive isotope.
Examples of preferred radioactive isotopes include 3H, 11C, 14C, 18F, 64Cu, 76Br, 86Y, 99mTc, 111In, 123I, 177Lu, and a mixture or combination thereof. The radioactive isotope is characterized by the fact that it is bound to a human antibody and included in a carrier to which the human antibody is bound.
The present invention also provides an immunodetection method for detecting an ex vivo VEGF-overexpressed cancer, the method including: contacting a composition including the radioactive isotope with cancer cells.
The VEGF-overexpressed cancer is preferably one selected from the group consisting of, but not limited to, colorectal cancer, renal cell cancer, lung cancer, breast cancer, and ovarian cancer, and includes all the VEGF-overexpressed cancers.
In a specific example of the present invention, it was confirmed that the monoclonal VEGF antibody showed strong binding capacity to VEGF (see FIGS. 9a and 9b). Thus, the monoclonal antibody of the present invention may be useful as a composition for radioimmuno treatment of VEGF-overexpressed diseases and including a radioactive isotope.
The composition including the radioactive isotope may be bound to a solid substrate in order to facilitate the subsequent steps such as washing or separation of complexes. The solid substrate includes, for example, synthetic resin, nitrocellulose, glass substrate, metal substrate, glass fiber, microsphere, microbead, etc. The synthetic resin includes polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, nylon, etc.
In addition, cancer cell may be appropriately diluted before it is contacted with a composition including the radioactive isotope.
The present invention also provides a method for imaging a VEGF-overexpressed cancer, the method including 1) administering a diagnostically effective amount of a compound including the radioactive isotope to a subject; and
2) obtaining a detection image for the subject.
The VEGF-overexpressed cancer is preferably one selected from the group consisting of, but not limited to, colorectal cancer, renal cell cancer, lung cancer, breast cancer, and ovarian cancer, and includes all the VEGF-overexpressed cancers.
In a specific example of the present invention, it was confirmed that the monoclonal VEGF antibody showed strong binding capacity to VEGF (see FIGS. 9a and 9b). Thus, the monoclonal antibody of the present invention may be useful in a method for imaging a VEGF-overexpressed cancer.
The detection image is characterized by the fact that it is obtained by near-infrared light imaging, PET, MRI, or ultrasonic imaging.
The present invention also provides a method for treating an in vivo VEGF-overexpressed cancer, the method including:
1) intravenously administering a composition including the radioactive isotope to a subject;
2) detecting the composition of Step 1) to identify tumor cells; and
3) eliminating the tumor cells identified in Step 2) by surgical operation.
The VEGF overexpressed cancer is preferably one selected from the group consisting of, but not limited to, colorectal cancer, renal cell cancer, lung cancer, breast cancer, and ovarian cancer, and includes all the VEGF-overexpressed cancers.
In a specific example of the present invention, it was observed that VEGF monoclonal antibodies showing binding capacities to VEGF (see FIGS. 9a and 9b) significantly inhibited a capillary-like tube formation of HUVEC cells, induced by VEGF in a human umbilical vein endothelial cell line (see FIG. 10). E9, F6, and G12 monoclonal antibodies showing binding capacities similar to that of AvastinTM all exhibited strong affinities for mouse VEGF similar to those for human VEGF, leading to a cross reaction (see Table 8 and FIG. 11). Because AvastinTM is not reacted with mouse VEGF at all, it has been difficult to perform animal experimental studies on side effects by AvastinTM. However, the VEGF neutralizing human antibody of the present invention is cross reacted with the mouse, indicating that it is highly different from the conventional anti-cancer drug AvastinTM in terms of epitope. Because the VEGF neutralizing human antibody of the present invention exhibits a high cross reactivity with mouse VEGF, it may be used in animal experimental studies. Thus, the monoclonal antibody of the present invention may be useful as a composition for prevention and treatment of VEGF-overexpressed diseases. Thus, the monoclonal antibodies of the present invention may be useful in prevention and treatment of VEGF-overexpressed cancers.
The present invention also provides a method for prognostic evaluation of a cancer patient, the method including:
1) intravenously administering a composition including the radioactive isotope to a patient whose tumor has been eliminated;
2) detecting the composition of Step 1) to identify tumor cells; and
3) judging that all tumor cells have been eliminated when tumor cells are not detected in step 2).
In addition, the present invention provides a method for measuring side effects of the human antibody, the method including administering the human antibody to an animal experiment model.
The animal experiment model may be preferably an animal with a disease caused by VEGF-overexpression.
Mode For Invention
Hereinafter, the present invention will be described in more detail with reference to examples.
However, the following examples are provided for illustrative purposes only, and the scope of the present invention should not be limited thereto in any manner.
Example 1
Preparation of VEGF Antigen Protein
<1-1> VEGF Gene Cloning
<1-1-1> Cloning Using YK602
A plasmid (UG-0036-D12) containing human VEGF gene was provided from KUGI (Korean UniGene Information) of the Center for Functional Analysis of Human Genome in Korea Research Institute of Bioscience and Biotechnology. The plasmid was used as a template DNA. In order to express only the domain of the VEGF, a forward primer (SEQ ID No. 1: 5′-GCTCTAGAGTGATGAACTTTCTGCTGTCTT-3′) and a reverse primer (SEQ ID No. 2: 5′-CGGAATTCCCGCCTCGGCTTGTCACA-3′) were used to amplify the gene under the following conditions. The gene was treated with XbaI and EcoRI, followed by subcloning into a pcDNA3.1/myc-His (−) vector (V80020: Invitrogen, USA) using a ligase. PCR conditions are as follows: when a total reaction reagent was 50 μl, 500 ng of the template was introduced and a reaction at 94° C. for 5 minutes, 25 cycles of reactions at 95° C. for 30 seconds, at 55° C. for 30 seconds, and at 72° C. for one and a half minutes, and at a reaction at 72° C. for 10 minutes were performed to get a PCR product.
The pcDNA3.1-VEGF was used as a template DNA, and a forward primer (SEQ ID No. 3: 5′-CAGGGGGCCGTGGGGGCCGCAGAAGGAGGAGGGCAG-3′) and a reverse primer (SEQ ID No. 4: 5′-TAGCGGCCGACGCGGCCAACCGCCTCGGCTTGTCACA-3′) were used to amplify the gene under the following conditions. The gene was treated with SfiI, and a ligase was used to subclone the gene into a pYK602 vector (FIG. 13a). PCR conditions are as follows: when a total reaction reagent was 50 μl, 500 ng of the template was introduced and a reaction at 95° C. for 5 minutes, 25 cycles of reactions at 95° C. for 30 seconds, at 58° C. for 30 seconds, and at 72° C. for 1 minute, and at a reaction at 72° C. for 10 minutes were performed to get a PCR product. Furthermore, the base sequence of the subcloned pYK602-VEGF vector was identified.
<1-1-2> Subcloning Using pYK602-His and pYK603
pcDNA3.1-VEGF was used as a template by the same manner as in Example 1-1-1 to subclone the VEGF into a pYK602-His vector (FIG. 13b) and a pYK603 vector (FIG. 13c). In addition, the base sequences of the subcloned pYK602-His-VEGF and pyK603-VEGF vectors were identified.
<1-2> Expression and Purification of VEGF Protein
<1-2-1> Identification of VEGF Expression Using pYK602
First, 5×106 293E cells were plated in ten 150 mm dishes. The next day, 10 μg of the subcloned pYK602-VEGF vector was treated with PEI (23966: Polysciences, Inc, USA) for transformation. The subsequent day, the dishes were changed with a growth medium (serum-free DMEM) and then a supernatant was obtained every other day. The obtained supernatant was electrophoresized in a 10% SDS-PAGE gel and identified by Western blot.
Specifically, two sheets of 10% SDS-PAGE gel loaded with 20 μl of a supernatant VEGF of the 1st to 7th subcultures were electrophoresized at 100 V for 2 hours and then transferred at 85 V for 2 hours to NC membrane (HATF00010: Millipore, USA). Subsequently, the membrane was blocked overnight at 4° C. in 4% skim milk in TEST. Subsequently, 0.8 mg/Ml of commercially available anti-human Fc-HRP (product No. 31413: Thermo Sci, USA) was diluted at 1:4000 in 4% skim milk in TEST, followed by reaction at room temperature for 1 hour. The mixture was washed five times in a cycle of once every 10 minutes with TEST, followed by development (12145: Intron, USA) to compare the expression levels of protein.
As a result, as shown in FIG. 1, VEGF was expressed until the 7th subculture, and the expression level of VEGF was not generally high while the expression levels of the 2nd and 3rd subcultures were high.
In addition, Protein A column (17-1279-03: GE Healthcare, USA) was used to purify the supernatant at the rate of 1.5 Ml/min. Protein was obtained at a concentration of 0.25 mg/Ml and dialysis of the protein was performed with PBS to confirm that a precipitate was produced.
Thus, the protein was resubcloned into pYK602-His and pYK603 vectors which produced relatively low amounts of precipitation.
<1-2-2> Identification of VEGF Expression Using pYK602-His and pYK603
Transduction was performed on the subcloned pYK602-His-VEGF and pYK603-VEGF vectors by the same manner as in Example 1-2-1, followed by several subcultures to obtain a supernatant.
The supernatants of the 1st and 2nd subcultures of pYK602-VEGF, pYK602-His-VEGF, and pYK603-VEGF vectors were electrophoresized on a 10% SDS-PAGE gel and identified by Western blot. Precipitate productions were also confirmed.
As a result, as shown in FIG. 2, the pYK602-VEGF and pYK602-His-VEGF vectors have similar expression levels with relatively low precipitate productions, while the PYK603-VEGF vector has a relatively lower expression level than those of the two.
Thus, the pYK602-His-VEGF vector was ultimately used to express and purify VEGF.
<1-2-3> Identification of pYK602-His-VEGF Vector Expression
5×106 293E cells were plated in ten 150 mm dishes and transduction of a pYK602-His-VEGF vector was performed, followed by subculture to obtain 300 Ml of supernatants of the 1st to 7th subcultures, respectively. The supernatant was electrophoresized on a 10% SDS-PAGE gel, and then identified by Western blot.
As a result, as shown in FIG. 3, expression levels were highest in supernatants of the 2nd and 3rd subcultures.
<1-2-4> Expression and Purification of VEGF Protein
Each of supernatants of the 2nd and 3rd subcultures was concentrated into 50 Ml, exchanged with 300 Ml of Ni-NTA binding buffer (Qiagen, USA), and then concentrated again into 50 Ml. Ni-NTA beads (1024473, Qiagen, USA) were added into the concentrate, followed by binding at 4° C. for 2 hours to separate and elute the mixture.
The elution was introduced into a membrane (10K, 132574: SPECTRAPOR, USA). Buffer exchange was performed at 4° C. in 4 L of PBS solution for 4 hours or more, followed by dialysis in 4 L of pre-cooled PBS solution overnight to change the buffer solution. After the overnight dialysis, the solution was transferred to an e-tube. The concentration of the protein was measured by the Bradford method and identification was performed on a 10% SDS-PAGE gel (FIG. 4).
Example 2
Construction of Library Phage
2.7×1010 human naive scFv library cells having diversity were incubated in a medium (3 L) containing 2×YTCM [17 g of Tryptone (CONDA, 1612.00), 10 g of yeast extract (CONDA, 1702.00), 5 g of NaCl (Sigma, S7653-5 kg), 34 μg/Ml of chloramphenicol (Sigma, C0857)], 2% glucose (Sigma, G5400), and 5 mM MgCl2 (Sigma, M2393) at 37° C. for 2-3 hours (OD600=0.5 ̃0.7). Then, the cells were infected with helper phage, followed by incubation in a medium containing 2×YTCMK [2×YT CM, 70 μg/Ml of Kanamycin (Sigma, K1876), 1 mM IPTG (ELPISBIO, IPTG025)] at 30° C. for 16 hours. The incubated cells was centrifuged (4500 rpm, 15 min, 4° C.) to obtain a supernatant. The supernatant was treated with PEG (Fluka, 81253) and NaCl (Sigma, S7653) until the two reagents became 4% and 3%, respectively. The reactant was centrifuged again (8000 rpm, 20 min, 4° C.). The pellet was dissolved in PBS, which proceeded to centrifugation again (12000 rpm, 10 min, 4° C.). As a result, the supernatant containing library phage was obtained, which was transferred to a new tube and stored at 4° C.
Example 3
Preparation of Monoclonal Antibody
<3-1> Panning Process
An immunosorb tube (Nunc 470319) was coated with 50 μg of VEGF-antigen obtained in Example 1 using 4 Ml of a coating buffer [1.59 g of Na2CO3 (Sigma, S7795), 2.93 g of NaHCO3 (Sigma, S8875), 0.2 g of NaN3 (Sigma, S2002)] at 4° C. for 16 hours with rotator. Then, the antigen was dissolved in PBS at room temperature for 2 hours, followed by blocking in the immunotube using skim milk [(BD,232100)-4% in 1×PBS]. 2 Ml of library phage constructed in Example 2 was added into the immunotube, followed by reaction at room temperature for 2 hours. The immunotube was washed five times with PEST (0.05%) and twice with PBS. After washing, antigen specific scFV-phage was eluted using 100 mM TEA (Sigma T-0886). E. coli (XL1-blue, stratagene, 200249) was transfected with the eluted phage, followed by amplification. The 2nd and 3rd pannings was performed on the phage amplified at the first panning by the same manner as described above except that washing times with PEST were increased (2nd: 13 times, 3rd: 23 times).
As a result, as shown in Table 1, it was confirmed that colony titer against the antigen was increased at least 1000 times from the 2nd panning.












TABLE 1









Initial phage
Binding phage



Target antigen
Panning
number
number









VEGF
1st
4.2 × 1013
6.0 × 105




2nd
2.6 × 1013
1.3 × 108




3rd
1.4 × 1013
1.9 × 108










<3-2> Screening of Phage Antibody by Phage ELISA
<3-2-1> Identification of Panning Results
Cell stocks obtained from the 1st-3rd pannings and stored as frozen were dissolved in a medium containing 5 Ml of 2×YTCM, 2% glucose, and 5 mM MgCl2 to make OD600 as 0.1. Then, the cells were incubated at 37° C. for 2-3 hours (OD600=0.5 ̃0.7), which were infected with M1 helper phage. Then, the cells were incubated in a medium containing 2×YTCMK, 5 mM MgCl2 and 1 mM IPTG at 30° C. for 16 hours. At this point, a single phage antibody (#39), which was specifically binding to WW45 antigen associated with a developmental process unrelated to VEGF and constructed by a method in Example of the present invention, was used as a control group. The incubated cells were centrifuged (4500 rpm, 15 min, 4° C.), and the supernatant was transferred to a new tube (1st ̃3rd panning poly scFv-phage). A 96-well immuno-plate (NUNC 439454) was coated with VEGF antigen (100 ng/well) using a coating buffer at 4° C. for 16 hours, followed by blocking with skim milk dissolved in PBS (4%). Each well of the 96-well immuno-plate was washed with 0.2 Ml of PBS-tween20 (0.05%). 100 μl of the 1st-3rd panning poly ScFV-phage was added into each well, followed by reaction at room temperature for 2 hours. Again, each well was washed four times with 0.2 Ml of PBS-tween20 (0.05%). The secondary antibody anti-M13-HRP (Amersham 27-9421-01) was diluted at 1:2000, followed by reaction at room temperature for 1 hour. The reactant was washed with 0.2 Ml of PBS-tween20 (0.05%). An OPD tablet (Sigma 8787-TAB) was added into a PC buffer [5.1 g of C6H8O7H2O (Sigma, C0706), 7.3 g of Na2HPO4 (Sigma, S7907)] to make a substrate solution, which was added into each well by 100 μl/well, followed by color development for 10 minutes. The optical density was measured at 490 nm by using a spectrophotometer (MolecularDevice, USA).
As a result, as shown in FIG. 5, it was confirmed that binding capacities to VEGF antigen were increased from the 2nd polyclonal ScFV-phage pool to reach a saturated state, and values for the tagged His were relatively low.
<3-2-2> Selection of Monoclonal Antibodies
Colonies obtained from a polyclonal antibody group (the 3rd panning) having strong binding capacity were incubated in a 96-deep well plate (Bioneer, 90030) containing 1 Ml of a medium supplemented with 2×YTCM, 2% glucose and 5 mM MgCl2 at 37° C. for 16 hours. 100-200 μl of the solution was incubated in 1 Ml of a medium supplemented with 2×YTCM, 5 mM MgCl2, and 1 mM IPTG, which was loaded in a 96-deep well plate at 37° C. for 2-3 hours, followed by inoculation at an initial OD600 value of 0.1. The cells were infected with M1 helper phage (MOI=1:20) and the infected cells were cultured in a medium supplemented with 2×YTCMK, 5 mM MgCl2, and 1 mM IPTG at 30° C. for 16 hours. The cultured cells were centrifuged (4500 rpm, 15 min, 4° C.) and a supernatant was obtained, to which 4% PEG 6000 and 3% NaCl were added. Upon completion of dissolving, reaction was induced in ice for 1 hour. The reactant was centrifuged (8000 rpm, 20 min, 4° C.) and pellet was dissolved in PBS. Centrifugation (12000 rpm, 10 min, 4° C.) was performed again and a supernatant was obtained, from which the 3rd panning monoclonal ScFv phage was obtained. The phage was transferred to a new tube and stored at 4° C.
A 96-well immuno-plate was coated with VEGF antigen (100 ng/well) at 4° C. for 16 hours, followed by blocking with skim milk dissolved in PBS (4%). Each well of the 96-well immuno-plate was washed with 0.2 Ml of PBS-tween20 (0.05%). 100 μl of the 3rd panning monoclonal scFV-phage was added to each well, followed by reaction at room temperature for 2 hours. Each well was washed four times with 0.2 Ml of PBS-tween20 (0.05%). The secondary antibody anti-M13-HRP was diluted at 1:2000, followed by reaction at room temperature for 1 hour. The plate was washed with 0.2 Ml of PBS-tween20 (0.05%), followed by color development. The optical density was measured at 490 nm.
As a result, 50 monoclonal phages having antigen VEGF binding capacity values of 1.5 or more (highlighted in Table 2) were selected.
[Table 2]





















VEGF ≧ 1.5
VEGF
anti-myc
His

VEGF
anti-myc
His

VEGF
anti-myc
His







A1
0.452
0.624
0.080
A5
1.991
1.142
0.076
A9 
0.954
0.121
0.058


B1
1.561
0.692
0.055
B5
1.451
1.054
0.067
B9 
1.696
0.987
0.364


C1
0.237
0.148
0.047
C5
1.927
1.134
0.091
C9 
1.640
1.171
0.074


D1
1.372
0.893
0.068
D5
0.628
1.117
0.082
D9 
2.091
0.771
0.065


E1
1.594
0.484
0.049
E5
0.142
0.045
0.059
E9 
2.162
0.705
0.066


F1
1.494
0.962
0.049
F5
1.759
0.983
0.113
F9 
2.077
1.525
0.096


G1
1.909
1.088
0.065
G5
0.607
0.336
0.137
G9 
1.852
1.091
0.089


H1
0.832
0.598
0.520
H5
1.312
1.160
0.069
H9 
1.550
0.889
0.156


A2
1.663
0.313
0.058
A6
1.035
1.202
0.083
A10
1.811
1.110
0.061


B2
1.504
0.945
0.062
B6
1.148
1.143
0.061
B10
0.553
0.335
0.062


C2
1.153
0.953
0.051
C6
0.291
0.118
0.074
C10
1.676
1.005
0.063


D2
1.117
0.832
0.056
D6
1.544
0.807
0.075
D10
1.711
0.849
0.055


E2
1.825
0.566
0.068
E6
1.454
1.215
0.083
E10
1.661
1.401
0.084


F2
1.517
0.778
0.064
F6
2.003
0.637
0.063
F10
1.711
0.862
0.071


G2
0.724
0.167
0.065
G6
1.763
0.975
0.050
G10
1.721
0.661
0.060


H2
1.213
0.928
0.049
H6
1.710
0.778
0.063
H10
1.719
1.152
0.072


A3
0.581
0.134
0.056
A7
1.910
1.124
0.056
A11
1.265
1.312
0.068


B3
1.509
0.915
0.066
B7
0.180
0.278
0.059 
B11
0.440
0.099
0.062


C3
1.399
1.070
0.072
C7
0.436
0.224
0.054
C11
1.719
1.484
0.069


D3
1.607
0.644
0.062
D7
1.999
0.684
0.063
D11
0.047
0.032
0.049


E3
1.602
1.040
0.078
E7
2.000
0.750
0.066
E11
0.429
0.138
0.052


F3
1.332
0.911
0.062
F7
0.205
1.457
0.343
F11
1.393
1.142
0.058


G3
1.877
0.950
0.071
G7
1.417
1.198
0.096
G11
2.045
1.207
0.100


H3
1.765
0.830
0.058
H7
1.780
1.420
0.185
H11
0.534
0.483
0.072


A4
1.699
0.904
0.055
A8
0.992
1.008
0.066
A12
0.049
0.164
0.063


B4
0.045
0.051
0.054
B8
1.472
1.201
0.092
B12
1.686
1.288
0.073


C4
1.419
0.969
0.061
C8
1.688
0.862
0.061
C12
1.688
1.390
0.084


D4
0.725
0.910
0.066
D8
1.233
0.962
0.087
D12
1.849
0.833
0.054


E4
1.307
0.959
0.054
E8
1.650
1.314
0.065
E12
2.139
1.012
0.064


F4
1.153
0.915
0.061
F8
1.452
1.039
0.072
F12
2.109
0.868
0.048


G4
1.270
0.844
0.060
G8
1.433
1.264
0.089
G12
2.142
1.752
0.104


H4
1.469
0.623
0.063
H8
1.486
0.620
0.064
H12
2.165
1.319
0.107









<3-3> Identification of Monoclonal Phages and Examination Thereof
<3-3-1> Verification by Fingerprinting
1 μl of the ten monoclonal cells firstly selected, 0.2 μl of Taq DNA polymerase (Gendocs, Korea) (5 U/ul), 50 p/μl of each forward primer (pelB5, SEQ. ID. No. 125: 5′-CTAGATAACGAGGGCAAATCATG-3′) and reverse primer (cla3, SEQ. ID. No. 126: 5′-CGTCACCAATGAAACCATC-3′), 3 μl of 10× buffer, 0.6 μl of 10 mM dNTP mix, and 24.8 μl of distilled water were mixed to perform a colony PCR (iCycler iQ, BIO-RAD). PCR conditions are as shown in Table 3.












TABLE 3







Temperature

Time
Cycle





















95° C.
5
min




95° C.
30
sec
30



56° C.
30
sec



72° C.
1
min



72° C.
10
min



 4° C.










The colony PCR product was identified on a 1% agarose gel (Seakem LE, CAMERES 50004). 0.2 μl of BstNI (Roche11288075001, 10 U/μl) was added to perform a reaction at 37° C. for 2-3 hours. Reaction conditions are as shown in Table 4. The fragmented product was identified on an 8% DNA polyacrylamide gel.











TABLE 4









10X Buffer
3
μl



colony PCR product
10
μl



BstNI (10 U/μl)
0.2
μl



Distilled water
16.8
μl










The colony PCR product was identified on a 1% agarose gel (Seakem LE, CAMERES 50004). 0.2 μl of BstNI (Roche11288075001, 10 U/μl) was taken to perform a reaction at 37° C. for 2-3 hours. Reaction conditions are as shown in Table 5. The fragmented product was identified on an 8% DNA polyacrylamide gel.
As a result, as shown in Table 6, fragments of monoclonal phage antibodies digested by BstNI were proved to have diversity.
<3-3-2> Verification by Base Sequence Analysis
50 kinds of the monoclonal phages were incubated in a medium (5 ml) supplemented with 2×YTCM, 2% glucose, and 5 mM MgCl2 at 37° C. for 16 hours. A DNA purification kit (Nuclogen 5112) was used for the incubated monoclones to obtain a DNA, and then sequencing was performed by using a pelB5 primer of SEQ ID No. 125 (Solgent, Korea).
As a result, as shown in Table 5 and FIGS. 7 & 8, CDR regions of VH and VL of the selected antibody were identified.
Similarity between the antibody and germ line antibody group was investigated by Ig BLAST program of NCBI (//www.ncbi.nlm.nih.gov/igblast/). As a result, 14 kinds of VEGF specific phage antibodies were obtained, and the result was summarized and presented in Table 6. Specifically, the heavy chain exhibited 89.9% to 96% homology with human germ cell family sequence, while the light chain exhibited 89.2% to 97% homology. In addition, polypeptides used in CDR3 of heavy and light chains of each human antibody were analyzed, and it was confirmed that their sequences were different.











TABLE 5









Clone
Heavy Chain
Light Chain














Group
name
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3


















1
A4
DYAMH
FINEDGGNIYYGDS
EPSGSLTFDY
RASQTISSYLN
AASRLQS
QQSYSTPYT





SEQ
VKG
SEQ ID
SEQ ID
SEQ ID
SEQ ID




ID
SEQ ID No. 18
No. 31
No. 57
No. 70
No. 83




No. 5


 


2
B12
SYAIS
GIIPIFGTANYAQK
DRSGYTAMDY
RASQGISSYLA
AASTLQS
QQGHTTPYT




SEQ
FQG
SEQ ID
SEQ ID
SEQ ID
SEQ ID




ID
SEQ ID No. 19
No. 32
No. 58
No. 71
No. 84




No. 6


 


3
C11
SDAIS
GVIPIFATTTYAQG
GQMDRGGGLDP
RASQGIGNYLN
AASSLQR
QQSYTTPYS




SEQ
FQG
SEQ ID
SEQ ID
SEQ ID
SEQ ID




ID
SEQ ID No. 20
No. 33
No. 59
No. 72
No. 85




No. 7


 


4
C12
SYGMN
SISSSSSSIHYADS
LGPYDAFDF
PGGTSNIDSKY
RNDQRPS
QSYDTSLSA




SEQ
VKG
SEQ ID
VH
SEQ ID
PYV




ID
SEQ ID No. 21
No. 34
SEQ ID
No. 73
SEQ ID




No. 8


No. 60

No. 86


 


5
C5
SYSMH
GISYDGSSKQFGDS
DGVPGHSYGIG
RASQGISSWLA
AASILQT
QQANSFPYT




SEQ
VKG
MDV
SEQ ID
SEQ ID
SEQ ID




ID
SEQ ID No. 22
SEQ ID
No. 61
No. 74
No. 87




No. 9

No. 35


 


6
C9
SYAMH
VISYDGSNKYYADS
DVDSWSQGWFPH
RASQTISTFVN
SASSLQS
QQNYSTPLT




SEQ
VKG
SEQ ID
SEQ ID
SEQ ID
SEQ ID




ID
SEQ ID No. 23
No. 36
No. 62
No. 75
No. 88




No. 10


 


7
D12
EYAMH
LISGDDYNTFYADS
DAGPAGGGGLDH
RTSQTITNFLN
GASSLQS
QQSHGTPYT




SEQ
VKG
SEQ ID
SEQ ID
SEQ ID
SEQ ID




ID
SEQ ID No. 24
No. 37
No. 63
No. 76
No. 89




No. 11


 


8
E9
TSGVA
LIYWDNDKRYSPSL
GDGWLFDF
TGSNSNIGAGH
GNTNRAS
QSYDNSLSG




VG
KN
SEQ ID
DVH
SEQ ID
YV




SEQ
SEQ ID No. 25
No. 38
SEQ ID
No. 77
SEQ ID




ID


No. 64

No. 90




No. 12


 


9
F2
SYAMS
YISSSGHDIYYADP
DKLATPGAFDI
RASQSISNWLA
EASSLES
QQSHGTPYT




SEQ
VKG
SEQ ID
SEQ ID
SEQ ID
SEQ ID




ID
SEQ ID No. 26
No. 39
No. 65
No. 78
No. 89




No. 13


 


10
F6
TSGVA
LIYWDNDKRYSPSL
GDGWLFDF
TGSNSNIGAGH
GNTNRAS
QSYDNSLSG




VG
KN
SEQ ID
DVH
SEQ ID
YV




SEQ
SEQ ID No. 25
No. 38
SEQ ID
No. 77
SEQ ID




ID


No. 64

No. 90




No. 12


 


11
F9
TTAIT
WITPFNGNTFYAQK
SQAAELGTGAF
SGSYSNIGTNY
KNTQRPS
SAWDDSLSA




SEQ
FQD
DI
VY
SEQ ID
VL




ID
SEQ ID No. 27
SEQ ID
SEQ ID
No. 79
SEQ ID




No. 14

No. 40
No. 66

No. 91


 


12
G12
NYAIS
RIIPIYGTPTYAQK
ERSFWNWFAP
TGSSSNIGAGY
GNNNRPS
QSYDSRLGVV




SEQ
FRD
SEQ ID
DVH
SEQ ID
SEQ ID




ID
SEQ ID No. 28
No. 41
SEQ ID
No. 80
No. 92




No. 15


No. 67


 


13
G9
TYALH
VISHDGTTDYYRDS
DGSGYFFDY
TGSSSDVGGYN
DVTKRPS
SSYSSSTFYV




SEQ
VKG
SEQ ID
YVS
SEQ ID
SEQ ID




ID
SEQ ID No. 29
No. 42
SEQ ID
No. 81
No. 93




No. 16


No. 68


 


14
H7
KYGMH
FIWFDGSNKFYADS
DRDYYGSGPLDY
RASQRIATYLH
AASSLQS
QQSYSTPYT




SEQ
VKG
SEQ ID
SEQ ID
SEQ ID
SEQ ID




ID
SEQ ID No. 30
No. 43
No. 69
No. 82
No. 83




No. 17









[Table 6]






















Clone name
VH
Identities
VL
Identities
VH(CDR-a.a seq)
Vk(CDR-a.a seq)
VEGF
anti-myc
His
Ratio
Group


























A4
VH3-43
93.2(272/292)
O12
97.2(276/284)
EPSGSLTFDY
QQSYSTPYT
1.699
0.904
0.055
1.880
1


 B12
VH1-69
99.3(294/296)
O12
93.3(265/284)
DRSGYTAMDY
QQGHTTPYT
1.686
1.288
0.073
1.309
2


 C11
VH1-69
90.8(268/295)
O12
93.3(265/284)
GQMDRGGGLDP
QQSYTTPYS
1.719
1.484
0.069
1.159
3


 C12
VH3-21
95.6(280/293)
V1-17
87.4(250/286)
LGPYDAFDF
QSYDTSLSAPYV
1.688
1.390
0.084
1.214
4


C5
VH3-30
94.3(279/296)
L5   
97.5(277/284)
DGVPGHSYGIGMDV
QQANSFPYT
1.927
1.134
0.091
1.700
5


C9
VH3-30
96.6(286/296)
O2 
93.4(267/286)
DVDSWSQGWFPH
QQNYSTPLT
1.640
1.171
0.074
1.400
6


 D12
VH3-43
91.8(269/293)
O12
94.0(267/284)
DAGPAGGGGLDH
QQSHGTPYT
1.849
0.833
0.054
2.221
7


D9
VH2-5 
94.3(280/297)
V1-13
93.2(272/292)
GDGWLFDF
QSYDNSLSGYV
2.091
0.771
0.065
2.712
8


E2
VH2-5 
94.3(280/297)
V1-13
93.2(272/292)
GDGWLFDF
QSYDNSLSGYV
1.825
0.566
0.068
3.226
8


E7
VH2-5 
94.3(280/297)
V1-13
93.2(272/292)
GDGWLFDF
QSYDNSLSGYV
2.000
0.750
0.066
2.668
8


E9
VH2-5 
94.3(280/297)
V1-13
93.2(272/292)
GDGWLFDF
QSYDNSLSGYV
2.162
0.705
0.066
3.069
8


F2
VH3-11
89.9(266/296)
L12a
94.1(254/270)
DKLATPGAFDI
QQSHGTPYT
1.517
0.778
0.064
1.949
9


F6
VH2-5 
94.3(280/297)
V1-13
93.2(272/292)
GDGWLFDF
QSYDNSLSGYV
2.003
0.637
0.063
3.143
10


F9
VH1-45
90.5(268/296)
V1-17
89.2(255/286)
SQAAELGTGAFDI
SAWDDSLSAVL
2.077
1.525
0.096
1.362
11


 G12
VH1-69
94.3(279/296)
V1-13
96.5(279/289)
ERSFWNWFAP
QSYDSRLGVV
2.142
1.752
0.104
1.222
12


G9
VH3-30
91.6(271/296)
V1-4 
94.3(264/280)
DGSGYFFDY
SSYSSSTFYV
1.852
1.091
0.089
1.698
13


H7
VH3-33
94.5(276/292)
O12
97.5(278/285)
DRDYYGSGPLDY
QQSYSTPYT
1.780
1.420
0.185
1.253
14









Example 4
Analysis of Characteristics of Human Antibody Against VEGF
<4-1> Measurement of Binding Capacity
In order to measure binding capacities to VEGF of 14 kinds of monoclonal phage antibodies selected in Example 3, each binding capacity was measured by the manner as in Example 3-2.
As a result, as shown in FIGS. 9a & 9b, binding capacities of 14 kinds of the monoclonal phage antibodies were in the order of G12> D12> E9> F6> H7> C5> B12> G9> F9> C11> F2> A4> C9> C12.
<4-2> Analysis of Whole IgG Conversion
In order to convert monoclonal phage antibodies against VEGF from phage to whole IgG vector, 1 μl of heavy chain monoclonal DNA, 10 pmole/μl of each heavy chain forward primer and heavy chain reverse primer in Table 7, 5 μl of 10× buffer, 1 μl of 10 mM dNTP mix, 0.5 μl of pfu DNA polymerase (Solgent, 2.5 U/μl), and distilled water (iCycler iQ, BIO-RAD) were mixed to perform a colony PCR (iCycler iQ, BIO-RAD). In addition, colony PCR was performed on light chain by the same manner by using light chain forward primer and reverse primer in Table 7.










TABLE 7









Heavy Chain










Clone
Reverse
Light Chain











name
Forward primer(Sfi I)
primer(NheI)
Forward primer(Sfi I)
Reverse primer(Bgl II)



















A4
NATVH7-1
TTGGTGGCCACAGC
NATJH-ALL
GAGGAGG
NATVK
TTGGTGGCCACAGC
NATJK-R7
GAGGAGAGA




SEQ ID
GGCCGATGTCCACT
SEQ ID
CTAGCTGA
1-1
GGCCGATGTCCACT
SEQ ID No.
TCTTTTGATA



No. 107
CGCAGATGCAGCTG
No. 114
GGAGACG
SEQ ID
CGGACATCCAGATG
119
TCCACCTTGGT




GTGGAGTC

GTGA
No. 115
ACCCAGTC


C5
NATVH3-1
TTGGTGGCCACAGC




NATJK-R1
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT




SEQ ID No.
TCTTTTTGAT



No. 108
CGCAGGTGCAGCTG




120
CTCTACCTTG




GTGGAGTC





GT


C11
NATVH1-2
TTGGTGGCCACAGC




NATJK-R2
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT




SEQ ID No.
TCTTTTTGAT



No. 109
CGCAGATGCAGCTG




121
CTCCACTTTG




GTGCAGTC





GT


C12
NATVH7-1
TTGGTGGCCACAGC


NATVL10
TTGGTGGCCACAGC
NATJL1-R
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT


SEQ ID
GGCCGATGTCCACT
SEQ ID No.
TCTTTAGGAC



No. 107
CGCAGATGCAGCTG


No. 116
CGCAGCTCGTGCTG
122
GGTGACCTT




GTGGAGTC



ACTCAGCC

GGTCCC


E9
NATVH2-1
TTGGTGGCCACAGC


NATVL2
TTGGTGGCCACAGC



SEQ ID
GGCCGATGTCCACT


SEQ ID
GGCCGATGTCCACT



No. 110
CGCAGGTCACCTTG


No. 117
CGCAGCCTGTGCTG




AAGGAGTC



ACTCAGCC


F2
NATVH3-2
TTGGTGGCCACAGC


NATVK1-1
TTGGTGGCCACAGC
NATJK-R5
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT


SEQ ID
GGCCGATGTCCACT
SEQ ID No.
TCTTTTTGAT



No. 111
CGCAGGTGCAGCTG


No. 115
CGGACATCCAGATG
123
TTCCAGCTTG




GTGGAGTC



ACCCAGTC

GT


F6
NATVH2-1
TTGGTGGCCACAGC


NATVL2
TTGGTGGCCACAGC
NATJL1-R
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT


SEQ ID
GGCCGATGTCCACT
SEQ ID No.
TCTTTAGGAC



No. 110
CGCAGGTCACCTTG


No. 117
CGCAGCCTGTGCTG
122
GGTGACCTT




AAGGAGTC



ACTCAGCC

GGTCCC


G9
NATVH3-2
TTGGTGGCCACAGC


NATVL9
TTGGTGGCCACAGC



SEQ ID
GGCCGATGTCCACT


SEQ ID
GGCCGATGTCCACT



No. 111
CGCAGGTGCAGCTG


No. 118
CGAATTTTATGCTGA




GTGGAGTC



CTCAGCC


G12
NATVH1-1
TTGGTGGCCACAGC


NATVL2
TTGGTGGCCACAGC
NATJL2-R
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT


SEQ ID
GGCCGATGTCCACT
SEQ ID No.
TCTTTAGGAC



No. 112
CGCAGGTGCAGCTG


No. 117
CGCAGCCTGTGCTG
124
GGTCAGCTT




GTGCAGTC



ACTCAGCC

GGTCCC


 


H7
NATVH7-2
TTGGTGGCCACAGC
NATJH-ALL
GAGGAGG
NATVK1-1
TTGGTGGCCACAGC
NATJK-R1
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT
SEQ ID
CTAGCTGA
SEQ ID
GGCCGATGTCCACT
SEQ ID No.
TCTTTTTGAT



No. 113
CGCAGATGCAGCTG
No. 114
GGAGACG
No. 115
CGGACATCCAGATG
120
CTCTACCTTG




GTAAAGTC

GTGA

ACCCAGTC

GT


D12
NATVH7-1
TTGGTGGCCACAGC




NATJK-R5
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT




SEQ ID No.
TCTTTTTGAT



No. 107
CGCAGATGCAGCTG




123
TTCCAGCTTG




GTGGAGTC





GT


C9
NATVH7-3
TTGGTGGCCACAGC


NATVK1-1
TTGGTGGCCACAGC
NATJK-R3
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT


SEQ ID
GGCCGATGTCCACT
SEQ ID No.
TCTTTTGATC



No. 128
CGCAGGTGCAGCTG


No. 115
CGGACATCCAGATG
129
TCCAGTCGT




GTAAAGTC



ACCCAGTC

GT


F9




NATVL10
TTGGTGGCCACAGC
NATJL2-
GAGGAGAGA







SEQ ID
GGCCGATGTCCACT
RSEQ ID
TCTTAGGAC







No. 116
CGCAGCTCGTGCTG
No. 124
GGTCAGCTT








ACTCAGCC

GGTCCC


B12
NATVH1-1
TTGGTGGCCACAGC


NATVK1-1
TTGGTGGCCACAGC
NATJK-R5
GAGGAGAGA



SEQ ID
GGCCGATGTCCACT


SEQ ID
GGCCGATGTCCACT
SEQ ID No.
TCTTTTGATT



No. 112
CGCAGGTGCAGCTG


No. 115
CGGACATCCAGATG
123
TCCAGCTTG




GTGCAGTC



ACCCAGTC

GT









The heavy chain gene obtained through PCR was purified with DNA-gel extraction kit (Qiagen). 1 μl of pNATAB H vector (FIG. 12a) (10 ng), 15 μl of heavy chain (100-200 ng), 2 μl of 10× buffer, 1 μl of Ligase (1 U/μl), and distilled water were mixed with the gene and the mixture was left still at room temperature for 1-2 hours for linkage to the vector. The vector was left still in ice for 30 minutes along with a cell for transformation (XL1-blue), followed by heat shock at 42° C. for 90 sec for transfection. It was again left still in ice for 5 minutes and 1 Ml of LB medium was injected, followed by incubation at 37° C. for 1 hour. The mixture was smeared in LB Amp liquid medium, followed by incubation at 37° C. for 16 hours. Single colony was inoculated into 5 Ml of LB Amp liquid medium, followed by incubation at 37° C. for 16 hours. A DNA-prep kit (Nuclogen) was used for the medium to extract a DNA.
In addition, pNATAB L vector (FIG. 12b) was used by the same manner to extract a DNA of the light chain.
Sequencing of the obtained DNA was performed by using a CMV-proF primer (SEQ ID No. 127: AAA TGG GCG GTA GGC GTG) (Solgent).
As a result, it was confirmed that the sequences of heavy and light chains of the 14 clone phages against VEGF converted into whole IgG were identical to those of the phage antibodies.
<4-3> Verification of Whole IgG
40 μg of PEI and 10 μg of each antibody heavy chain DNA and light chain DNA in the whole form were added into 293E cells (Invitrogen) for co-transfection to obtain a supernatant, which was identified by Western blot. Normal human IgG (Jacson Lab) was used as a control group.
As a result, it was confirmed that it was successfully converted into whole IgG form compared to a control group.
<4-4> Identification of Neutralizing Capacities of Anti-VEGF Antibodies Through Analysis of Inhibition of Tube Formation
A monolayer of HUVEC cell (C2517A: Lonza), a human umbilical vein endothelial cell of about 80% confluence, was exchanged with EBM-2 (CC3156, Cambrex) supplemented with 1% FBS and a starvation state was made in a 37° C./5% CO2 incubator for 4 hours. Subsequently, the medium was treated with trypsin to collect and suspend the cells in a starvation medium at 3×105/Ml. 100 μl of cell suspension, 150 μl of the purified human VEGF Ab solution of the present invention (VEGF Ab is diluted with a starvation medium and treated at a concentration of 4 μg/Ml), and a normal human IgG as a negative control group were treated at a concentration of 4 μg/Ml, or AvastinTM (Genetech) as a positive control group was treated at a concentration of 4 μg/Ml, followed by pre-incubation at 37° C. for 1 hour. During the pre-incubation, 200 μl of Matrigel (354230: BD Bio-Science, USA) cooled with ice was introduced into a 24-well plate, and then homogenization was performed in a 37° C./5% CO2 incubator for 30 minutes. Recombinant human VEGF was added into a mixture of the cells for which reaction had been completed and Ab at a concentration of 20 ng/Ml and placed onto the Matrigel, followed by incubation in a 37° C./5% CO2 incubator. In order to observe a tube formation after incubation for 16 hours, 500 μl of 3.7% paraformaldehyde was introduced into the incubator, followed by immobilization at room temperature for 30 minutes. Next, the mixture was stained with 500 μl of 0.01% crystal violet/100 mM NaBorate and dried to observe a tube formation under a magnification of about 100×.
As a result, as shown in FIG. 10, it was observed that the purified VEGF Ab exhibited an obvious inhibition of tube formation, compared to a positive control group.
It was observed that the three antibodies except for C5 among E9, F6, G12, and C5 all inhibited a capillary-like tube formation of HUVEC cells, induced by VEGF significantly, and thus neutralizing capacities of human antibodies against VEGF of the present invention were identified.
<4-5> Identification of Cross Reactivity of Anti-VEGF Antibodies in a Mouse
In addition, ELISA was performed to see if each antibody could have cross-reactivity not only with human VEGF, but also with mouse VEGF. Two 96-well immuno-plates were coated with (50 ng/well) of each recombinant human VEGF (293-VE, R&D systems, USA) and recombinant mouse VEGF (493-MV, R&D systems, USA) using a coating buffer at 4° C. for 16 hours, followed by blocking with skim milk dissolved in PBS (4%). Each well of the 96-well immuno-plate was washed with 0.2 Ml of PBS-tween20 (0.05%). E9, F6, and G12 monoclonal antibodies were diluted gradually from 333 nM by 1/3, and each of 100 μl of the diluted monoclonal antibodies was added into each well of the two antigen coated plates with AvastinTM as a control group at room temperature for 2 hours. Each well was washed four times with 0.2 Ml of PBS-tween20 (0.05%). The secondary antibody anti-human Fc-HRP was diluted at 1:3000, followed by reaction at room temperature for 30 minutes. The reactant was washed with 0.2 Ml of PBS-tween20 (0.05%). An OPD tablet was added into a PC buffer to make a substrate solution, which was added into each well by 100 μl/well, followed by color development for 5 minutes. The optical density was measured at 490 nm by using a spectrophotometer.
Graphpad prism ver.4 software (CA 92037: Graphpad Software Inc., USA) was used to analyze the ELISA results.
As a result, E9, F6, and G12 monoclonal antibodies, showing neutralizing capacities similar to that of AvastinTM in FIG. 10, all exhibited strong affinities for mouse VEGF similar to those for human VEGF, leading to a cross reaction (Table 8 and FIG. 11). The F6 antibody showing the strongest neutralizing capacity consistently exhibited an about 2-fold lower KD value than that of AvastinTM. Thus, the probabilities are certainly high that the VEGF neutralizing human antibody of the present invention is different from the conventional anti-cancer agent AvastinTM in terms of epitope.









TABLE 8









Ab











Ag
E9 (R2)
F6 (R2)
G12 (R2)
Avastin (R2)





mVEGF
6.5 × 10−11 (0.99)
3.4 × 10−11 (0.98)
2.6 × 10−10 (0.99)
N.D


hVEGF
5.1 × 10−11 (0.99)
2.8 × 10−11 (0.97)
2.4 × 10−11 (0.99)
5.9 × 10−11 (0.87)


















TABLE 9









nM




















Ag
Ab
50
25
12.5
6.25
3.13
1.56
0.78
0.39
0.20
0.10
0.049
0.024























mVEGF
E9
2.4569
2.2777
2.3099
2.0685
2.3331
2.2543
2.2338
2.0163
1.7289
1.4132
0.9682
0.6036



F6
2.3555
2.3232
2.3049
2.3041
2.1922
2.1811
2.1885
2.1425
2.1341
1.7251
1.3023
0.8829



G12
2.4617
2.302
2.2623
2.166
2.1757
2.0375
1.7438
1.3811
0.9585
0.721
0.3959
2.159



Avastin
0.6949
0.1074
0.1041
0.0424
0.0489
0.0455
0.0438
0.0569
0.0433
0.147
0.0687
0.0467















Not bolgG















(50 nM)


VEGF
E9
2.5106
2.4445
2.4182
2.3279
2.4466
2.3016
2.1899
2.0938
1.9197
1.7033
1.1523
0.6822



F6
2.5407
2.3745
2.3649
2.3101
2.2942
2.2765
2.2617
2.1785
2.1829
1.8819
1.5032
0.9789



G12
2.5305
2.4915
2.2875
2.251
2.0766
1.9873
1.8751
1.4892
0.9924
0.7689
0.4748
0.226



Avastin
2.5166
2.4828
2.4441
2.4332
2.4087
2.3666
2.1938
2.187
2.0882
1.7951
1.3556
0.052















Not bolgG















(50 nM)









Example 5
Performance of LC Shuffling of VEGF F6 and G12 Monoclonal Antibodies
<5-1> Construction of LC Shuffling Library
Heavy chain portions from scFv-phages of the monoclonal F6 and G12 obtained in Example 3 were treated with SfiI at about 50° C., to which SfiI was added every 2 hours, and this process was repeated three times for gel elution. pYG100, a library plasmid, was also treated with SfiI at about 50° C. for about 4 hours, treated with SfiI once again, and restricted overnight for gel elution. Concentrations of heavy chain fragments of the monoclonal F6 and G12 digested and the concentration of vector were measured, followed by ligating with 10 units of ligase at a molar ratio of vector: heavy chain fragments=1:5 prepared for a total solution at 1 μg/100 μl at room temperature for about 2 to about 4 hours. A ligated product was concentrated with ethanol, followed by electroporation of 20 μl of the ligated product in distilled water with XL1-BLUE cells at 1×109 cells/100 μl for transduction. Incubation of the product was performed at about 37° C. overnight, followed by storage in 500 μl of 15% glycerol 2XYT at about −70° C. A titer measurement resulted in a diversity of 2.1×106.
<5-2> Construction of LC Shuffling Library Phage
A library phage was constructed for human naïve VEGF F6 & G12 LC shuffling scFv library cells having a diversity of 2.1×106 prepared in Example 5-1 by the same manner as described above in Example 2.
<5-3> Construction of LC Shuffling Monoclonal Antibodies
A phage panning was conducted using 50 μg of the purified VEGF antigen obtained in Example 1 and 2 Ml of the library phage constructed in Example 5-2 by the same manner as in Example 3, a panning result was confirmed, a monoclonal antibody selected, and a monoclonal phage was classified and examined.
As a result of the panning, it was confirmed that a high titer was shown from the 1st panning as indicated in Table 10 and colony titer of a phage against the antigen at the 2nd panning was increased at least 5 times.












TABLE 10









Initial phage
Binding phage



Target antigen
Panning
number
number









VEGF
1st
2.0 × 1013
2.2 × 105




2nd
1.2 × 1014
4.6 × 109










As shown in FIG. 14, the panning result confirmed by ELISA also showed that a VEGF antigen binding capacity was increased from LC shuffled polyclonal scFv-phage pools, a binding capacity to the 1st panning reached a saturated state, and a value for the tagged H is was relatively low.
61 monoclonal phages with a VEGF antigen binding capacity of at least 2.0 (highlighted in Table 11) were also selected by a monoclonal antibody selection. Then, F6 and G12 in the LC shuffling were used as a positive control group.
[Table 11]





















VEGF ≧ 2
VEGF
anti-myc
His

VEGF
anti-myc
His

VEGF
anti-myc
His


























F6(P)
2.143
0.468
0.068
2A5
0.089
0.350
0.049
2A9 
2.333
0.544
0.040


2B1
2.058
0.485
0.049
2B5
2.099
0.364
0.042
2B9 
0.133
1.131
0.059


2C1
0.103
0.273
0.069
2C5
2.035
0.515
0.050
2C9 
2.408
0.800
0.064


2D1
2.100
0.530
0.046
2D5
2.325
1.387
0.044
2D9 
2.336
0.536
0.075


2E1
2.195
1.599
0.062
2E5
2.124
0.270
0.044
2E9 
2.424
1.845
0.077


2F1
2.104
0.518
0.046
2F5
1.711
0.292
0.044
2F9 
2.478
1.946
0.075


2G1
2.282
1.525
0.052
2G5
1.900
0.290
0.047
2G9 
2.172
0.463
0.076


2H1
2.137
1.504
0.061
2H5
1.997
0.509
0.055
2H9 
2.497
0.857
0.076


G12(P)
2.205
1.569
0.072
2A6
0.089
0.088
0.058
2A10
2.390
0.524
0.071


2B2
0.181
0.996
0.081
2B6
0.080
0.285
0.089
2B10
2.220
0.605
0.062


2C2
0.130
0.992
0.044
2C6
1.250
0.508
0.053
2C10
2.158
0.593
0.088


2D2
2.020
0.402
0.046
2D6
2.154
0.366
0.048
2D10
2.047
3506.000
0.073


2E2
2.289
1.745
0.042
2E6
0.232
0.138
0.042
2E10
2.294
0.579
0.077


2F2
1.899
0.189
0.047
2F6
0.087
0.225
0.043
2F10
2.195
1.396
0.109


2G2
2.186
0.763
0.054
2G6
2.253
1.472
0.107
2G10
2.428
0.698
0.077


2H2
2.142
1.547
0.062
2H6
1.986
0.861
0.054
2H10
0.153
0.733
0.088


2A3
0.098
0.292
0.053
2A7
0.109
0.062
0.050
2A11
2.321
0.629
0.045


2B3
0.092
0.131
0.099
2B7
0.063
0.056
0.045
2B11
2.351
1.790
0.047


2C3
1.933
0.573
0.052
2C7
2.436
0.330
0.047
2C11
2.042
0.152
0.041


2D3
0.089
0.264
0.051
2D7
1.082
0.070
0.031
2D11
2.306
0.590
0.056


2E3
2.119
0.191
0.045
2E7
2.267
0.132
0.055
2E11
2.533
0.557
0.052


2F3
0.143
0.094
0.050
2F7
2.521
1.706
0.050
2F11
0.674
0.052
0.047


2G3
2.093
0.648
0.051
2G7
2.512
0.421
0.047
2G11
2.285
0.072
0.061


2H3
0.213
0.690
0.059
2H7
2.546
0.331
0.070
2H11





2A4
0.092
0.156
0.046
2A8
1.718
0.072
0.028
2A12
0.791
1.957
0.037


2B4
0.087
0.083
0.059
2B8
2.515
0.454
0.017
2B12
0.132
0.280
0.066


2C4
2.165
0.399
0.091
2C8
2.586
1.690
0.066
2C12
2.256
0.950
0.052


2D4
0.650
1.662
0.046
2D8
2.451
0.358
0.056
2D12
2.371
1.814
0.046


2E4
2.029
0.283
0.046
2E8
2.542
1.690
0.024
2E12
2.382
1.774
0.050


2F4
2.174
0.471
0.044
2F8
2.507
1.826
0.046
2F12
2.286
0.667
0.052


2G4
2.119
0.657
0.046
2G8
2.535
1.747
0.031
2G12
2.265
0.785
0.059


2H4
1.973
1.146
0.059
2H8
2.423
0.190
0.086
2H12









A verification result by a fingerprinting process also confirmed that fragments of monoclonal phage antibodies digested by BstNI had diversity as shown in FIG. 15.
Furthermore, a verification result by nucleotide sequencing analysis confirmed CDR regions of VH and VL of the selected antibody as described in Table 12. Homology between the selected antibody and germ line antibody group was investigated. AS a result, 12 VEGF specific phage antibodies with the same HC except for LC different from F6 were obtained, and 3 VEGF specific phage antibodies with the same HC except for LC different from G12 were obtained (Table 13). In particular, about 94.4% homology was shown in 90.1% of light chain. A polypeptide in CDR1, 2, and 3 of light chain of each human antibody was analyzed and it was confirmed that polypeptides with each different sequence were different each other. In FIG. 16, because CDR regions of VH of the antibody selected by LC shuffling were identical to each other, CDR regions of VL were compared.










TABLE 12









Heavy Chain
Light Chain













Clone
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3





F6(P)
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
TGSNSNIGAGHDVH:
GNTNRAS:
QSYDNSLSGYV:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 64
SEQ ID No. 77
SEQ ID No. 90


2A09
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
TGSSSNIGAG--YDVH:
GNSNRPS:
QSYDNSLS---AY:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 67
SEQ ID No. 143
SEQ ID No. 153


2B05
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
TGGDSNIGAG--YDVN:
GDTFPPS:
QSYDSSLS---GY:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID NO. 130
SEQ ID No. 144
SEQ ID No. 154


2C11
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
RSSQSLVRSDGTTYLS:
KISNRFS:
MQATFFPFT----:



SEQ ID No. 12
SEQ IDNo. 25
SEQ ID No. 38
SEQ ID No. 131
SEQ ID No. 145
SEQ ID No. 155


2D10
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
TGSSSNLGAP--NDVH:
GSTNRPS:
QSYDNSLS---AY:



SEQ ID No. 12
SEQ IDNo. 25
SEQ ID No. 38
SEQ ID No. 132
SEQ ID No. 146
SEQ ID No. 153


2E11
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
TGSSSNIGAG--YDVH:
GNNNRPS:
QSNDPSLGGL--H:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 67
SEQ ID No. 80
SEQ ID No. 156


2F01
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
TGSSSNIGAP--NDVH:
GNTNRP3:
QSYDNGLSAS--Y:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 133
SEQ ID No. 147
SEQ ID No. 157


2G03
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
RSSQSLVRSDGTTYLS:
KISNRFS:
MQATFFPFT----:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 134
SEQ ID No. 144
SEQ ID No. 155


2G04
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
TGSRSNFGAG--HDVH:
GNNNRPS:
QSFDNTLNG---W:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 135
SEQ ID No. 80
SEQ ID No. 158


2G12
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
TGSSSNIGAG--SDVH:
GNNNRPS:
QSYDSSLSG---Y:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 136
SEQ ID No. 80
SEQ ID No. 154


2H07
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
TGSSTNIGAG--YDVH:
GNSNRPS:
QSYDSSLSG-SLY:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 137
SEQ ID No. 143
SEQ ID No. 159


2H08
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
SESSSNIGAG--FDYH:
GNTDRPS:
QSYDSSLR---AY:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 138
SEQ ID No. 148
SEQ ID No. 160


2H09
TSGVAVG:
LIYWDNDKRYSPSLKN:
GDGWLFDF:
RASQGIVS-----WLA:
AASELQS:
QQLNNFLFA----:



SEQ ID No. 12
SEQ ID No. 25
SEQ ID No. 38
SEQ ID No. 139
SEQ ID No. 149
SEQ ID No. 161


G12(P)
NYAIS:
RIIPIYGTPTYAQKFRD:
ERSFWNWFAP:
TGSSSNIGAGYDVH:
GNNNRPS:
QSYDSRLGVV:



SEQ ID No. 15
SEQ ID No. 28
SEQ ID No. 41
SEQ ID No. 67
SEQ ID No. 80
SEQ ID No. 92


2C05
NYAIS:
RIIPIYGTPTYAQKFRD:
ERSFWNWFAP:
GSSAGAVISGHYPF:
DISNRHF:
FVAYG--AIW:



SEQ ID No. 15
SEQ ID No. 28
SEQ ID No. 41
SEQ ID No. 140
SEQ ID No. 150
SEQ ID No. 162


2F09
NYAIS:
RIIPIYGTPTYAQKFRD:
ERSFWNWFAP:
TGSSSNLGAGYDVH:
GDVNRPS:
QSYDTSLVGS:



SEQ ID No. 15
SEQ ID No. 28
SEQ ID No. 41
SEQ ID No. 141
SEQ ID No. 151
SEQ ID No. 163


1F10
NYAIS:
RIIPIYGTPTYAQKFRD:
ERSFWNWFAP:
RASQP---ISNWLA:
ATSILQS:
QQHRD--YPL:



SEQ ID No. 15
SEQ ID No. 28
SEQ ID No. 41
SEQ ID No. 152
SEQ ID No. 142
SEQ ID No. 164

























TABLE 13





Clone Name
Vl.
identities
CDR1-aaseq
CDR2-aaseq
CDR3-aaseq
VEGF
a-myc
Ratio
Group
























F06(P)
V1-13
272/292(93.2%)
TGSNSNIGAG--HDVH
GNTNRAS
QSYDNSLS---GY
2.1429
0.4678
4.5808
P


A09
V1-13
282/298(64.6%)
TGSSSNIGAG--YDVH
GNSNRPS
QSYDNSLS---AY
2.3329
0.5437
4.2908
1


B05
V1-13
266/292(91.1%)
TGGDSNIGAG--YDVN
GDTFRPS
QSYDSSLS---GY
2.0985
0.3641
5.7635
2


C11
A29
280/299(93.6%)
RSSQSLVRSDGTTYLS
KISNRFS
MQATFFPFT----
2.0415
0.1523
13.4045
3


D10
V1-13
268/292(91.8%)
TGSSSNIGAP--NDVH
GSTNRPS
QSYDNSLS---AY
2.0469
0.3506
5.8383
4


E11
V1-13
269/291(92.4%)
TGSSSNIGAG--YDVH
GNNNRPS
QSNDPSIGGL--H
2.5328
0.5573
4.5448
5


F01
V1-13
273/289(94.5%)
TGSSSNIGAP--NDVH
GNTNRPS
QSYDNGLSAS--Y
2.1038
0.5177
4.0637
6


G03
A23
282/299(94.3%)
RSSQSLVRSDGTTYLS
KISNRFS
MQATFFPFT----
2.0927
0.6483
3.2880
7


G04
V1-13
267/290(92.1%)
TGSRSNPGAG--HDVH
GNNNRPS
QSFDNTLNG---W
2.1193
0.6572
3.2247
8


G12
V1-13
283/292(96.9%)
TGSSSNIGAG--SDVH
GNNNRPS
QSYDSSLSG---Y
2.2646
0.7852
2.8841
9


H07
V1-13
277/293(94.5%)
TGSSTNIGAG--YDVH
GNSNRPS
QSYDSSLSG-SLY
2.546
0.3305
7.7035
10


H08
V1-13
275/293(92.3%)
SESSSNIGAG-FDVH
GNTDRPS
QSYDSSLR---AY
2.4231
0.1815
12.7868
11


H09
L8
258/283(91.2%)
RASQGIVS-----WLA
AASELQS
QQLNNFLFA----
2.4965
0.8568
2.9137
12


G12(P)
V1-13
279/289(96.5%)
TGSSSNIGAGYDVH
GNNNRPS
QSYDS-RLGV
2.2051
1.5689
1.4055
P


C05
V3-3
270/287(94.4%)
GSSAGAVTSGHYPF
DTSNRHF
FVAYG--AIN
2.0353
0.5153
3.9497
13


F09
V1-13
281/299(94.0%)
TGSSSNLGAGYDVH
GDVNRPS
QSYDTSLVGS
2.4779
1.9456
1.2736
14


F10
L15
256/284(90.1%)
RSQP---ISNWLA
ATSILQS
QQBRD--YPL
2.1954
1.3957
1.5730
15









Example 6
Characterization of LC Shuffling Human Antibodies
<6-1> Measurement of Binding Capacity
A binding capacity was measured for an LC shuffling monoclonal antibody selected in Example 5-3 by the same manner as described in Example 4-1. As a result, binding capacities of the 15 LC shuffling monoclonal phage antibodies against the antigen were confirmed as indicated in Table 14.









TABLE 14









VEGF-His













(Dilution)
3125
625
125
25
5
1
















F6(P)
0.4502
1.0161
1.7492
2.3787
2.6334
2.6027


G12(P)
1.2041
1.8817
2.3696
2.5961
2.7582
2.5957


2A9
0.5874
1.1848
1.8134
2.352
2.6411
2.5364


2B5
0.1951
0.4776
1.1497
1.9639
2.428
2.6934


2C5
0.071
0.1258
0.3388
0.9994
2.1226
2.6528


2C11
0.0592
0.0863
0.2195
0.7511
1.7178
2.4353


2D10
0.0955
0.1876
0.5045
1.1569
2.0359
2.4843


2E11
0.36
0.7239
1.4011
2.0133
2.4173
2.3286


2F1
0.2977
0.5735
1.173
1.9021
2.3977
2.3666


2F9
0.941
1.4798
2.194
2.4754
2.5794
2.4680


2F10
0.0798
0.1158
0.324
0.8996
1.9159
2.4501


2G3
0.0888
0.1682
0.4737
1.1641
2.1681
2.5639


2G4
0.5673
1.1624
1.8694
2.4342
2.6262
2.6711


2G12
0.5464
0.9802
1.9239
2.4643
2.7957
2.7999


2H7
0.4001
0.6833
1.6313
2.228
2.7175
2.7383


2H8
0.1404
0.2725
0.9064
1.617
2.4888
2.8512


2H9
0.1476
0.3282
0.9177
1.9178
2.6841
3.0981









<6-2> Conversion Analysis of Whole IgG
In order to convert LC shuffling monoclonal antibodies selected in Example 5-3 into a whole IgG vector in the phage, a colony PCR was performed by the same manner as described in Example 4-2 by using a primer pair in Table 15. As a result, the heavy chain was ligated to a pNATAB H vector (FIG. 12a) and the light chain was ligated to a pNATAB L vector (FIG. 12b). A nucleotide sequencing analysis was performed by extracting DNA from the vector. As a result, it was confirmed that sequences of the heavy and light chains of 15 clone phages for an LC shuffling monoclonal phage antibody converted into a whole IgG were identical to those of phage antibodies.










TABLE 15









Heavy chain
Light chain













Forwardprimer
Reverseprimer
Forwardprimer
Reverseprimer



Clones
(Sfi I)
(Nhe I)
(Sfi I)
(Bgl II)



















F06(P)
NATVH2-
TTGGTGG
NATJH-
GAG
NATVL10:
TTGGTGGCCACA
NATJL1-R:
GAGGAGAG




3:
CCACAGC
ALL:
GAG
SEQ
GCGGCCGATGTC
SEQ ID No.
ATCTTAGGA



SEQ
GGCCGAT
SEQ
GCT
ID No.
CACTCGCAGCTC
186
CGGTGACCT



ID
GTCCACT
ID
AGC
116
GTGCTGACTCAG

TGGTCCC



No.
CGCAGGT
No.
TGA

CC


2A09
179
CACCTTG
114
GGA
NATVL13:
TTGGTGGCCACA




AGGGAG

GAC
SEQ
GCGGCCGATGTC




TC

GGT
ID No.
CACTCGCAGTTCG






GA
180
TGCTGACTCAGCC


2B05




NATVL10:
TTGGTGGCCACA







SEQ
GCGGCCGATGTC







ID No.
CACTCGCAGCTC







116
GTGCTGACTCAG








CC


2C11




NATVK3:
TTGGTGGCCACA
NATJK-R2:
GAGGAGAG







SEQ ID
GCGGCCGATGTC
SEQ ID No.
ATCTTTTGAT







No. 181
CACTCGGATATTG
187
CTCCACTTT








TGATGACCCAGA

GGT








CTCC


2D10




NATVL10:
TTGGTGGCCACA
NATJL1-R:
GAGGAGAG


2E11




SEQ
GCGGCCGATGTC
SEQ ID No.
ATCTTAGGA


2F01




ID No.
CACTCGCAGCTC
188
CGGTGACCT







116
GTGCTGACTCAG

TGGTCCC








CC


2G03




NATVK3
TTGGTGGCCACA
NATJK-R7:
GAGGAGAG








GCGGCCGATGTC
SEQ ID No.
ATCTTTTGAT








CACTCGGATATTG
119
ATCCACCTT








TGATGACCCAGA

GGT








CTCC


2G04




NATVL10:
TTGGTGGCCACA
NATJL2-R:
GAGGAGAG







SEQ
GCGGCCGATGTC
SEQ ID No.
ATCTTAGGA







ID No.
CACTCGCAGCTC
124
CGGTCAGCT







116
GTGCTGACTCAG

TGGTCCC


2G12





CC
NATJL1-R
GAGGAGAG


2H07






SEQ ID No.
ATCTTAGGA


2H08




NATVL13:
TTGGTGGCCACA
189
CGGTGACCT







SEQ
GCGGCCGATGTC

TGGTCCC







ID No.
CACTCGCAGTTCG







182
TGCTGACTCAGCC


2H09




NATVK1-
TTGGTGGCCACA
NATJK-R2:
GAGGAGAG







1:
GCGGCCGATGTC
SEQ ID No.
ATCTTTTGAT







SEQ ID
CACTCGGACATC
190
CTCCACTTT







No. 183
CAGATGACCCAG

GGT








TC


 


G12(P)
NATVH1-
TTGGTGG
NATJH-

NATVL10:
TTGGTGGCCACA
NATJL2-R:
GAGGAGAG



1:
CCACAGC
ALL:

SEQ
GCGGCCGATGTC
SEQ ID No.
ATCTTAGGA



SEQ
GGCCGAT
SEQ

ID No.
CACTCGCAGCTC
124
CGGTCAGCT



ID
GTCCACT
ID

116
GTGCTGACTCAG

TGGTCCC



No.
CGCAGGT
No.


CC


2C05
112
GCAGCTG
114

NATVL12:
TTGGTGGCCACA
NATJL5-R:
GAGGAGAG




GTGCAGTC


SEQ
GCGGCCGATGTC
SEQ ID No.
ATCTTAGGA







ID No.
CACTCGCAGGCT
191
CGGTCAGCT







184
GTGGTGACTCAG

CGGT








GA


2F09




NATVL13:
TTGGTGGCCACA
NATJL2-R:
GAGGAGAG







SEQ
GCGGCCGATGTC
SEQ ID No.
ATCTTAGGA







ID No.
CACTCGCAGTTCG
124
CGGTCAGCT







185
TGCTGACTCAGCC

TGGTCCC


1F10




NATVK1-
TTGGTGGCCACA
NATJK-R7:
GAGGAGAG







1:
GCGGCCGATGTC
SEQ ID No.
ATCTTTTGAT







SEQ ID
CACTCGGACATC
119
ATCCACCTT







No. 115
CAGATGACCCAG

GGT








TC









<6-3> Confirmation of Binding Capacities of Anti-VEGF Antibodies
In order to confirm a VEGF binding capacity by comparing LC shuffling monoclonal phage antibodies with F6 and G12, respectively, ELISA was performed by the same manner as described in Example 4-5.
As a result, degrees of binding of only 2C11, 2G03, 2C05, and 2F10 were too low while others exhibited high affinities similar to that of a positive control group, as indicated in Table 16.




















TABLE 16





(nM)
50
25
12.5
6.25
3.125
1.5625
0.78125
0.39063
0.19531
0.09766
0.04883
0.02441
Kd value(M)(R2)




























IgG
0.0952
0.1015
0.1032
0.1227
0.0951
0.0993
0.0941
0.1011
0.0911
0.092
0.091
0.0874
—


F06(P)
2.5809
2.4608
2.6248
2.5662
2.5017
2.5287
2.4768
2.2867
1.8512
1.3136
0.864
0.4938
8.8 * 10−11 M















(0.99)


2A09
2.529
2.5265
2.6023
2.6243
2.4743
2.5441
2.4944
2.234
1.8063
1.1968
0.8211
0.4838
9.8 * 10−11 M















(0.99)


2B05
2.479
2.5411
2.499
2.4371
2.4739
2.4681
2.5128
2.1458
1.6658
1.1279
0.6944
0.4271
1.1 * 10−10 M















(0.98)


2C11
0.0881
0.0942
0.0919
0.0912
0.0982
0.1021
0.0989
0.101
0.0928
0.0895
0.0879
0.0865
—


2D10
2.1757
2.0057
1.4823
1.0899
0.7038
0.3882
0.2463
0.1615
0.1252
0.1033
0.0956
0.0892
8.4 * 10−9 M















(0.98)


2E11
2.1736
1.7494
1.3507
0.8021
0.5537
0.3098
0.1866
0.1332
0.1077
0.0986
0.0929
0.0907
1.3 * 10−8M















(0.94)


2F01
2.6062
2.5367
2.2299
2.2665
1.9197
1.6427
1.2476
0.7042
0.4147
0.2151
0.1299
0.1146
9.4 * 10−10M















(0.99)


2G03
0.0886
0.0926
0.092
0.939
0.0927
0.096
0.0985
0.0929
0.0986
0.1
0.086
0.0927
—


2G04
2.5279
2.5187
2.4115
2.4195
2.5001
2.4575
2.424
2.4811
2.3615
1.6605
1.2495
0.7616
3.3 * 10−11 M















(1)


2G12
2.5233
2.546
2.3541
2.6124
2.4604
2.4384
2.4091
2.0182
1.6568
1.1688
0.6642
0.4813
1.1 * 10−10M















(0.99)


2H07
2.3654
2.4546
2.548
2.4666
2.3474
1.8805
1.5607
1.152
0.5821
0.3146
0.1824
0.1408
5.3 * 10−10M















(0.99)


2H08
2.5012
2.5022
2.6496
2.5016
2.5143
2.1478
2.1006
1.7333
1.0913
0.6665
0.3348
0.2256
2.5 * 10−10M















(0.99)


2H09
1.1042
2.0759
2.0025
1.7631
1.3284
0.8864
0.5223
0.3525
0.2049
0.1341
0.1055
0.0873
2.4 * 10−9M















(1)


G12(P)
2.7337
2.4535
2.5613
2.4466
2.3736
2.0751
1.8909
1.2653
0.7613
0.4329
0.2541
0.1633
4.2 * 10−10M















(0.99)


2C05
0.1073
0.1434
0.1019
0.1036
0.0974
0.1023
0.1175
0.1004
0.0951
0.0985
0.0935
0.0937
—


2F09
2.6076
2.3879
2.4921
2.4349
2.2943
2.0733
2.1207
1.6154
1.0599
0.6351
0.3728
0.231
2.5 * 10−10M















(0.99)


2F10
0.807
0.4553
0.2579
0.1709
0.149
0.1266
0.1185
0.1107
0.1049
0.1058
0.1009
0.093
7.8 * 10−8M















(0.96)









Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

## Claims

1. A VEGF-specific human antibody comprising:
a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 having the amino acid sequence of SEQ ID NO: 12, a HCDR 2 having the amino acid sequence of SEQ ID NO: 25, and a HCDR 3 having the amino acid sequence of SEQ ID NO: 38; and
a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 having the amino acid sequence of SEQ ID NO: 64, a LCDR 2 having the amino acid sequence of SEQ ID NO: 77, and a LCDR 3 having the amino acid sequence of SEQ ID NO: 90.

2. The human antibody as set forth in claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51.

3. The human antibody as set forth in claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 101.

4. A composition comprising the human antibody of claim 1.

5. A pharmaceutical composition comprising the human antibody of claim 1.

6. A method for preparing a VEGF-specific human antibody, the method comprising:
1) constructing a first polynucleotide encoding a heavy chain of the human antibody of claim 1;
2) constructing a second polynucleotide encoding a light chain of the human antibody of claim 1;
3) constructing a first expression vector comprising the polynucleotide of the first polynucleotide;
4) constructing a second expression vector comprising the polynucleotide of the second polynucleotide;
5) preparing a transformant by introducing the first expression vector and the second expression vector simultaneously into a host cell;
6) incubating the transformant of step 5); and
7) purifying the human antibody of claim 1 from the medium.

7. A pharmaceutical composition for preventing or treating a disease caused by VEGF-overexpression, comprising a pharmaceutically acceptable carrier and a VEGF-specific human antibody, wherein the VEGF-specific human antibody comprises:
a heavy chain variable region (VH) comprising a HCDR1 having the amino acid sequence of SEQ ID NO: 12, a HCDR2 having the amino acid sequence of SEQ ID NO: 25, and a HCDR3 having the amino acid sequence of SEQ ID NO: 38; and
a light chain variable region (VL) comprising a LCDR1 having the amino acid sequence of SEQ ID NO: 64, a LCDR2 having the amino acid sequence of SEQ ID NO: 77, and a LCDR3 having the amino acid sequence of SEQ ID NO: 90.

8. The pharmaceutical composition as set forth in claim 7, wherein the disease caused by VEGF-overexpression is a disease associated with cancer or angiogenesis.

9. The pharmaceutical composition as set forth in claim 8, wherein the cancer is selected from the group consisting of colorectal cancer, renal cell cancer, lung cancer, breast cancer, and ovarian cancer.

10. The pharmaceutical composition as set forth in claim 8, wherein the disease associated with angiogenesis is selected from the group consisting of rheumatoid arthritis, diabetic retinopathy, ischemic retinopathy, psoriasis, proliferative diabetic retinopathy, and diabetic macular edema.

11. A method for treating a disease caused by VEGF-overexpression, the method comprising administering to a subject with the disease a pharmaceutically effective amount of a VEGF-specific human antibody comprising:
a heavy chain variable region (VH) comprising a HCDR1 having the amino acid sequence of SEQ ID NO: 12, a HCDR2 having the amino acid sequence of SEQ ID NO: 25, and a HCDR3 having the amino acid sequence of SEQ ID NO: 38; and
a light chain variable region (VL) comprising a LCDR1 having the amino acid sequence of SEQ ID NO: 64, a LCDR2 having the amino acid sequence of SEQ ID NO: 77, and a LCDR3 having the amino acid sequence of SEQ ID NO: 90.

12. The method as set forth in claim 11, wherein the VEGF-specific human antibody is administered in combination with chemotherapy.

13. The method as set forth in claim 11, wherein the disease caused by VEGF-overexpression is a disease associated with cancer or angiogenesis.

14. The method as set forth in claim 13, wherein the cancer is selected from the group consisting of colorectal cancer, pancreatic cancer, renal cell cancer, lung cancer, breast cancer, and ovarian cancer.

15. The method as set forth in claim 13, wherein the disease associated with angiogenesis is selected from the group consisting of rheumatoid arthritis, diabetic retinopathy, ischemic retinopathy, psoriasis, proliferative diabetic retinopathy, and diabetic macular edema.