Abstract:
The present invention provides a fully human anti-VEGF monoclonal antibody, the preparation method and use thereof. The fully human anti-VEGF monoclonal antibody is obtained by using antibody phage display technology, which has higher antibody affinity and stronger capacity for inhibiting tumor cell proliferation in comparison with humanized antibody bevacizumab, and can be used to prepare anti-tumor medicines.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a National Stage of International Application No. PCT/CN2010/000513, filed on Apr. 16, 2010, which claims priority to Chinese Patent Application No. 201010125263.7, filed Feb. 25, 2010. The contents of the prior applications are incorporated herein by reference in their entirety. 
     FIELD OF THE INVENTION 
     The present invention relates to the field of biotechnology. In particular, the present invention relates to a fully human monoclonal antibody, the preparation method and use thereof. 
     BACKGROUND OF THE INVENTION 
     Tumor, particularly malignant tumor is a disease which causes serious harm to the health of human bodies and is the second leading cause of death among all diseases in the world today. The incidence tendency of malignant tumor is rising obviously in recent years. However, the treatment effect is limited, with high metastasis rate in advanced stage and poor prognosis. Although, conventional treatments currently used in clinical such as radiotherapy, chemotherapy and surgical treatment can ease the pain to a large extent and prolong the life span, there are still many limitations of these therapies and it&#39;s difficult to improve the treatment effect further. 
     There are two distinct phases of tumor growth, namely from the avascular phase of slow growth to the vascular phase of rapid proliferation. Angiogenesis enables the tumors to obtain adequate nutrition and therefore complete the angiogenic switch. If there is no angiogenesis, the primary tumor will not grow to exceed 1-2 mm and tumor metastasis will not be realized. Vascular endothelial growth factor (VEGF) is a kind of growth factor which can promote endothelial cell differentiation and proliferation, promote new blood vessel formation and increase vascular permeability. It binds to vascular endothelial growth factor receptor on the surface of cells, and functions by activating tyrosine kinase signal transduction pathways. In tumor tissues, the tumor cells, invasive tumor macrophages and mast cells can secrete high levels of VEGF, stimulate the tumor vascular endothelial cells in the form of paracrine secretion, promote the proliferation and migration of endothelial cells, induce angiogenesis, promote the sustainable growth of tumor and increase vascular permeability, cause fibrin calm of the surrounding tissues, promote the invasion of monocytes, fibroblast cells and endothelial cells, facilitate the formation of tumor stroma and the entries of tumor cells into new vessels and promote tumor metastasis. Thus, the inhibition of tumor angiogenesis is considered to be one of the most promising treatments of tumors. 
     Bevacizumab (Avastin) is a humanized anti-human VEGF antibody (Cancer Res 1997; 57: 4593-9). It was approved as a first-line drug for the treatment of metastatic colorectal cancer by the U.S. Food and Drug Administration in 2004. However, bevacizumab is a humanized antibody that retains murine CDR regions and a few murine residues of FR regions. It is still not a fully humanized antibody and still has a problem of low affinity. 
     SUMMARY OF THE INVENTION 
     The present invention constructs a large-capacity human natural phage antibody library and obtains a fully human anti-VEGF antibody 11A7 by selecting therefrom. 
     More particularly, the present invention provides a fully human anti-VEGF antibody, having an amino acid sequence of heavy chain variable region as shown in SEQ ID NO: 6, and an amino acid sequence of light chain variable region as shown in SEQ ID NO: 8. 
     The above fully human anti-VEGF antibody according to the present invention has an amino acid sequence of heavy chain as shown in SEQ ID NO: 10, and an amino acid sequence of light chain as shown in SEQ ID NO: 12. 
     The present invention also provides an isolated nucleotide encoding the above fully human anti-VEGF antibody. 
     The above nucleotide according to the present invention has a nucleotide sequence encoding heavy chain variable region of the fully human anti-VEGF antibody as shown in SEQ ID NO: 5, and a nucleotide sequence encoding light chain variable region of the fully human anti-VEGF antibody as shown in SEQ ID NO: 7. 
     The above nucleotide according to the present invention has a nucleotide sequence encoding heavy chain of the fully human anti-VEGF antibody as shown in SEQ ID NO: 9, and a nucleotide sequence encoding light chain of the fully human anti-VEGF antibody as shown in SEQ ID NO: 11. 
     The present invention also provides an expression vector containing the above nucleotide, which is pcDNA3.1/ZEO (+) or pcDNA3.1 (+). 
     The present invention also provides a host cell transfected with the above expression vector, which is CHO-K1 cell. 
     The present invention further provides a method for preparing the above fully human antibody, comprising selecting human phage antibody library to obtain a fully human anti-VEGF single-chain antibody with high affinity; constructing an eukaryotic expression vector of the complete molecular of the fully human anti-VEGF antibody; expressing the complete molecular of fully human anti-VEGF antibody in CHO cells; and purifying the complete molecular of the fully human anti-VEGF antibody. 
     The present invention also provides a use of the above fully human anti-VEGF antibody in preparing medicines for treatment of tumors, wherein said tumor is colorectal cancer. 
     The obtained antibody are used to perform a series of experiments in the present invention and the experiment results show that compared to humanized antibody bevacizumab and human anti-VEGF antibody 6A6 (which is prepared according to the method disclosed in Chinese Patent Application No. 02111093.X entitled “Humanized Anti-vascular Endothelial Growth Factor Monoclonal Antibody, Preparation Method and Pharmaceutical Composition Thereof” filed on Mar. 20, 2002, the antibody obtained according to the present invention has higher antibody affinity and stronger inhibition effect on tumor cell proliferation; and the results of antitumor experiment in vivo show that the antibody of the present invention can inhibit tumor growth significantly. 
    
    
     
       DESCRIPTION OF DRAWINGS 
         FIG. 1  shows the results of cell proliferation assay. 
         FIG. 2  shows the curve of tumor growth. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The following examples and experiment examples are used to further illustrate the present invention only and should not be construed to limit the present invention. 
     (1) Cloning of Genes Encoding Human Antibody Light and Heavy Chain Constant Region 
     Healthy human lymphoma cells were isolated with lymphocyte separation medium (Dingguo Biotechnology Development Company, China) and total RNA was extracted using Trizol reagent (Invitrogen). The genes encoding antibody heavy and light chain constant region were amplified by RT-PCR reaction, with the primers designed according to the sequences reported in the reference (Cell, 1980, 22: 197-207) and reference (Nucleic Acids Research, 1982, 10: 4071-4079), respectively. The PCR products were purified by agarose gel electrophoresis and recovered and cloned into pGEM-T vectors (Promega). Correct clones were obtained by sequencing verification. SEQ ID NO: 1 and SEQ ID NO: 2 showed the nucleotide sequence and amino acid sequence of the heavy chain constant region (C H ), respectively. SEQ ID NO: 3 and SEQ ID NO: 4 showed the nucleotide sequence and amino acid sequence of the light chain constant region (C L ), respectively. In this example, the correct clones were designated as pGEM-T/C H  and pGEM-T/C L . 
     (2) Preparation of cDNA 
     20 ml of peripheral blood was collected from each of 50 healthy people and mononuclearcells were isolated with lymphocyte separation medium (Tianjin blood research Institute of Medical Science). Total cellular RNA was extracted from the isolated human peripheral blood lymphocytes using Trizol reagent (Invitrogen). cDNA was reverse transcribed using cDNA reverse transcription kit (Shanghai Biocolor Biotechnolgy Ltd.). The above procedures were performed according to the manufacturer&#39;s instructions. 
     (3) Design of Primers 
     V H Back, V H For, V L Back and V L For, the primers for cloning genes of human antibody heavy chain variable region (V H ) and light chain variable region (V L ), were designed and synthesized according to the reference (Immunotechnology, 1998, 3:271-278). Sequences of V H Back, V H For, V L Back and V L For were shown in Immunotechnology, 1998, 3:271-278. Wherein, V H Back primer was added with an Sfi I site-containing sequence: atg gcc cag ccg gcc atg gcc (SEQ ID NO: 13) at the 5′ end; V H For primer was added with a sequence: gcc aga acc acc gcc gcc gga gcc acc acc gcc ESEQ ID NO:14) at the 5′ end; V L Back primer was added with a sequence: tcc ggc ggc ggt ggt tct ggc gga ggc gga tct (SEQ ID NO:15) at the 5′ end; and V L For primer was added with a Not I site-containing sequence: atg cgg ccg c (SEQ ID NO:16) at the 5′ end. 
     (4) Construction and Selection of Phage Antibody Library 
     Phage single-chain antibody library was constructed with the cDNA of (2) and the primers of (3) using recombinant Phage antibody system kit (Amersham Biosciences) and then selected with a specific antigen. The methods of constructing and selecting the antibody library were performed according to the instructions of recombinant Phage antibody system kit. The specific antigen “Recombinant Human VEGF165 Protein” was purchased from R&amp;D. 
     A anti-human VEGF single-chain antibody 11A7ScFv was obtained after several times of selection, and its gene sequence was obtained by sequencing. SEQ ID NO: 5 and SEQ ID NO: 6 show the nucleotide sequence and amino acid sequence of the heavy chain variable region (VH) of 11A7ScFv, respectively. SEQ ID NO: 7 and SEQ ID NO: 8 show the nucleotide sequence and amino acid sequence of the light chain variable region (V L ) of 11A7ScFv, respectively. 
     (5) Expression of Fully Human Antibody in Eukaryotic Cells 
     3E12ScFv genes and pGEM-T/C H  vectors were used as template to synthesize fully human antibody heavy chain genes by overlapping PCR. The reaction conditions were: 95° C. for 15 min; 94° C. for 50 sec, 58° C. for 50 sec, 72° C. for 50 sec, for 30 cycles; 72° C. for 10 min Besides, the fully human antibody heavy chain genes were allowed to contain HindIII restriction enzyme sites and a signal peptide gene sequence at the 5′ end and contain translation stop codons TAA and EcoRI restriction enzyme sites at the 3′ end. The sequence encoding the signal peptide was: (ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCAT AATATCCAGAGGA)(SEQ ID NO:17). Finally, PCR amplification products were separated by agarose gel electrophoresis and the band of interest was recovered and cloned into pGEM-T vectors (Promega) to select and sequence positive clones. Clones with the correct sequence were selected and digested with Hind III and EcoRI, and the fully human antibody heavy chain fragments 3E12V H C H  were purified and recovered by agarose gel electrophoresis and ligated into the HindIII and EcoRI-digested plasmids pcDNA3.1(+) (Invitrogen) to construct fully human heavy chain eukaryotic expression vectors pcDNA3.1(+) (3E12V H C H ). 
     3E12ScFv genes and pGEM-T/C L  vectors were used as template to synthesize fully human antibody light chain genes by overlapping PCR. The reaction conditions were: 95° C. for 15 min; 94° C. for 50 sec, 58° C. for 50 sec, 72° C. for 50 sec, for 30 cycles; 72° C. for 10 min. The obtained PCR products contained HindIII restriction enzyme sites and a signal peptide gene sequence at the 5′ end and contained translation stop codons TAA and EcoRI restriction enzyme sites at the 3′ end. The sequence encoding the signal peptide was: (ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCAT AATATCCAGAGGA)(SEQ ID NO:17). Clones with the correct sequences were selected and digested with Hind III and EcoRI, and the fully human antibody light chain fragments 3E12V L C L  were purified and recovered by agarose gel electrophoresis and ligated into the HindIII and EcoRI-digested plasmids pcDNA3.1/ZEO(+) (Invitrogen) to construct fully human light chain eukaryotic expression vectors pcDNA3.1/ZEO(+) (3E12V L C L ). 
     3×10 5  CHO-K1 cells (ATCC CRL-9618) were inoculated into 3.5cm tissue culture dishes, and transfected when the cells were cultured to 90-95% confluence: 10 μg of plasmids (4 μg of plasmids pcDNA3.1(+) (11A7V H C H ), 6 μg of plasmids pcDNA3.1/ZEO(+) (11A7V L C L )) and 20 μl of Lipofectamine2000 Reagent (Invitrogen) were taken to perform transfection according to the instructions of Lipofectamine2000 Reagent kit. After transfection for 24 hours, the cells were transferred to DMEM medium containing 600 μg/ml G418 (Invitrogen) and 250 μg/ml Zeocin (Invitrogen) to select resistant clones. Cell culture supernatants were taken to select high-expressing clones by ELISA: ELISA plates were coated with goat anti-human IgG (Fc) overnight at 4° C. and blocked with 2% BSA-PBS at 37° C. for 2h; the culture supernatants of resistant clones to be tested or standard sample (Human myeloma IgG1, κ) (Sigma) were added and warm incubated at 37° C. for 2 h; HRP-goat anti-human IgG (κ) (Southern Biotechnology Associates) was added and warm incubated at 37° C. for 1 h for combining reaction, and chromogenic reagent TMB was added and reacted at 37° C. for 5 min, finally H 2 SO 4 was used to stop the reaction and A 450  value was measured. The high-expressing clones obtained by selection were enlarged cultured in serum-free medium, and fully human antibodies 11A7 were isolated and purified by Protein A affinity column (GE). The purified antibodies were dialyzed against PBS and finally quantified by UV absorbance. SEQ ID NO: 9 and SEQ ID NO: 10 show the nucleotide sequence and amino acid sequence of the heavy chain of fully human antibody 11A7, respectively. SEQ ID NO: 11 and SEQ ID NO: 12 show the nucleotide sequence and amino acid sequence of the light chain of fully human antibody 11A7, respectively. 
     EXPERIMENT EXAMPLES 
     Human anti-VEGF antibody 6A6 was prepared according to the method disclosed in Chinese Patent Application No. 02111093.X entitled “Humanized Anti-vascular Endothelial Growth Factor Monoclonal Antibody, Preparation Method and Pharmaceutical Composition Thereof” filed on Mar. 20, 2002. 
     Experiment Example 1 
     Affinity Detection of VEGF Antibody 
     Affinity constant of VEGF antibody was detected using Biacore T100 system (Biacore AB, Uppsala, Sweden). VEGF165 (R&amp;D) was covalently linked to CMS biological sensor chips (Biacore) by amino-coupling. Fully human antibody 11A7, Bevacizumab, human antibody 6A6 (prepared according to the method disclosed in Chinese Patent Application No. 02111093.X entitled “Humanized Anti-vascular Endothelial Growth Factor Monoclonal Antibody, Preparation Method and Pharmaceutical Composition Thereof” filed on Mar. 20, 2002) and negative control antibody (Rituximab, commercial available) were formulated with PBS/0.05% TWEEN-20 (ICI Americas) (an eradicator) into solutions with different concentrations (2-fold dilution)) and passed through the chips at a flow rate of 50 μl/min. After each examination, they were washed with 5 μl of 50 mM hydrochloric acid aqueous solution at a flow rate of 3 μl/min so as to wash away the residual antibodies from the immobilized ligands. The binding curves were subjected to nonlinear regression analysis using BIAevalution software (T100 evalution version 2.0, Biacore). The results are shown in table 1. The KD value of fully human antibody 11A7 was significantly lower than that of Bevacizumab and fully human antibody 6A6, demonstrating that the affinity of fully human antibody 11A7 to VEGF was higher than that of Bevacizumab and fully human antibody 6A6. The results of affinity experiment are shown in Table 1. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Results of affinity experiment 
               
             
          
           
               
                 Antibody 
                 K on  (M −1 S −1 /10 4 ) 
                 K off  (10 4 S −1 ) 
                 K D  (nM) 
               
               
                   
               
               
                 11A7 
                 8.24 
                 0.71 
                 0.86 
               
               
                 Bevacizumab 
                 5.12 
                 1.03 
                 2.01 
               
               
                 6A6 
                 4.19 
                 0.95 
                 2.27 
               
               
                 Rituximab 
                 ND 
                 ND 
                 ND 
               
               
                   
               
             
          
         
       
     
     Experiment Example 2 
     Experiment of Inhibition of HUVEC Cells Proliferation by VEGF Antibody 
     Experiment steps: The well-growing HUVEC cells (Cascade Biologics) were adjusted to a cell density of 2.5×10 4 /ml, inoculated into 96 cell culture plates with 200 μl/well and cultured in a 5% CO 2  incubator at 37° C. for 24 h and then cultured in serum-free medium for another 72 h, added with VEGF antibody of different concentrations and incubated at 37° C. for 1 h, with anti-CD20 antibody Rituximab as negative control and three parallel wells for each concentration; added with VEGF 165 (R&amp;D) until reaching a final concentration of 25 ng/ml and cultured for another 24 h; then added with 10 μl of [ 3 H]-TdR(18.5 kBq/well) and incubated in a incubator at 37° C. for 7 h. The cells were collected onto a glass fiber filtration film with cell collector and counted with [ 3 H] liquid scintillation counter. The results are shown as  FIG. 1 . 
     The results showed that the negative control antibody (Rituximab) cannot effectively inhibit VEGF-induced HUVEC proliferation, however, all of fully human antibodyl1A7, Bevacizumab and human antibody 6A6 can effectively inhibit VEGF-induced HUVEC proliferation. The activity of inhibiting VEGF-induced HUVEC proliferation of fully human antibody 11A7 was significantly stronger than that of Bevacizumab and human antibody 6A6 (P&lt;0.05, t-test, the concentration range of the antibodies was 0.4-3.2 nM). 
     Experiment Example 3 
     In vivo Tumor Growth Inhibition Eexperiment of VEGF Antibody 
     Experiment steps: In order to detect the activity of inhibiting tumor growth of the VEGF antibodies in vivo, first LM3 cells (Human hepatoma cells, from Liver Cancer Institute, Medical College of the Fudan University, Shanghai, China) were inoculated into the right axillary lateral subcutaneous of female mice with severe immune deficient (from the Animal Experiment Center of the Second Military Medical University, China). Each mouse was administrated with 25 mg/kg of VEGF antibody and unrelated control protein Rituximab on the same day and then subjected to subcutaneous injection every other day for 4 weeks. After six weeks, the length and width of the tumors were measured to calculate the volume of the tumors every 3 days. The results are shown in  FIG. 2 . 
     The results showed that the negative control antibody (Rituximab) cannot inhibit tumor growth effectively, however, all of fully human antibody 11A7, Bevacizumab and human antibody 6A6 can inhibit tumor growth effectively. And from the 18 th  day, the activity of inhibiting tumor growth of fully human antibody 11A7 was stronger than that of Bevacizumab and human antibody 6A6, with significant difference (P&lt;0.05, Mann-Whitney test, when observation time is longer than 35 days).