Abstract:
The invention provides a fully human anti-HER2 monoclonal antibody, which has 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 invention also discloses the nucleotide sequence encoding the antibody, the expression vector and the host cell comprising the nucleotide sequence, and the use of the antibody for manufacturing the medicament for the treatment of tumor.

Description:
FIELD OF THE INVENTION 
       [0001]    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 
       [0002]    Breast cancer is one of the most common malignant tumors in women. More than one million new cases of breast cancer occur worldwide annually, and nearly 400 thousand people died from breast cancer every year. In recent years, the incidence of breast cancer showed a clear upward trend in the world. Despite in high and low endemic areas, the incidence of breast cancer increases by 5-20%. The growth trend of the incidence of breast cancer in Asian women has been significantly higher than that in the United States and Europe. In China, breast cancer has become the primary malignant tumor in women in some cities. The common treatments for breast cancer include surgery, chemicotherapy and endocrine therapy and so on. Although these conventional treatments may prolong survival in patients to a large extent, their side effects are serious and their therapeutic effect is hard to be further improved. Targeted cancer therapy is a new treatment for cancer that has arisen in recent years, of which the representative is antitumor monoclonal antibody. 
         [0003]    HER2 (human epidermal growth factor receptor 2) is a transmembrane protein with tyrosine kinase activity, having a molecular weight of about 185 KD. Anti-HER2 humanized monoclonal antibody may specifically bind to HER2, and has antitumor mechanisms as follows: specifically binding to the extracellular domain of HER2 receptor to block the constitutive activation of HER2 homodimers and interfere the heterodimer formation of HER2 with other ErbB family members; mediating the endocytosis and the degradation in lysosomes of HER2 receptor; activating PTEN (phosphatase and tensin homology) and blocking PI3K 
         [0004]    (Phosphatidylinositol 3-kinase) signal channel; inhibiting tumor cell proliferation by regulation of cell cycle; promoting tumor cell apoptosis; inhibiting tumor angiogenesis; ADCC (antibody-dependent cell-mediated cytotoxicity) effect; inhibiting DNA repair; increasing the cytotoxicity of chemotherapeutic agents; reversing the resistance of tumor cells to the killing effects of host cell factors, and etc. (Pergram M, Ngo D, Application and potential limitations of animal models utilized in the development of trastuzumab (Herceptin®): A case study. Adv Drug Deliv Rev. 2006; 58:723-34). 
         [0005]    Anti-HER2 humanized monoclonal antibody (Trastuzumab, trade name: Herceptin) has been used in clinical trials to treat patients with HER2 overexpressing metastatic breast cancer as single drug, who had received but failed one or more chemotherapy regimens for their metastases. This drug may be used in combination with paclitaxel or anthracyclines (doxorubicin or epirubicin) plus cyclophosphamide in clinical trials as first-line drugs to treat HER2 overexpressing metastatic breast cancer (Merlin JL, Barberi-Heyob M, Bachmann N, In vitro comparative evaluation of trastuzumab (Herceptin) combined with paclitaxel (Taxol) or docetaxel (Taxotere) in HER2-expressing human breast cancer cell lines. Ann Oncol. 2002; 13:1743-8). But Trastuzumab is a humanized antibody that maintains the murine CDR regions and a small amount of murine FR residues, which still has not been fully humanized and the affinity is not high. 
       SUMMARY OF THE INVENTION 
       [0006]    The present invention constructs a very large human natural phage antibody library and obtains a fully human anti-HER2 antibody 3E12 by selecting therefrom. 
         [0007]    More particularly, the present invention provides a fully human anti-HER2 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. 
         [0008]    The above fully human anti-HER2 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. 
         [0009]    The present invention also provides an isolated nucleotide encoding the above fully human anti-HER2 antibody. 
         [0010]    The above nucleotide according to the present invention has a nucleotide sequence encoding heavy chain variable region of the fully human anti-HER2 antibody as shown in SEQ ID NO: 5, and a nucleotide sequence encoding light chain variable region of the fully human anti-HER2 antibody as shown in SEQ ID NO: 7. 
         [0011]    The above nucleotide according to the present invention has a nucleotide sequence encoding heavy chain of the fully human anti-HER2 antibody as shown in SEQ ID NO: 9, and a nucleotide sequence encoding light chain of the fully human anti-HER2 antibody as shown in SEQ ID NO: 11. 
         [0012]    The present invention also provides an expression vector containing the above nucleotide, which is pcDNA3.1/ZEO(+) or pcDNA3.1 (+). 
         [0013]    The present invention also provides a host cell transfected with the above expression vector, which is a CHO-K1 cell. 
         [0014]    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-HER2 single-chain antibody with high affinity; constructing an eukaryotic expression vector of the complete molecular of the fully human anti-HER2 antibody; expressing the complete molecular of fully human anti-HER2 antibody in CHO cells; and purifying the complete molecular of the fully human anti-HER2 antibody. 
         [0015]    The present invention also provides a use of the above fully human antibody in preparing medicines for treatment of tumor. The tumor is a Her2-overexpressing tumor, and more particularly is breast cancer. 
         [0016]    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 Trastuzumab (rhumAb 4D5), and humanized antibody hGH0/1 disclosed in Chinese Patent Application No.01132225.X entitled “Humanized Anti-HER2 Monoclonal Antibody, Preparation Method and Pharmaceutical Composition Thereof” filed on Nov. 16, 2001, 3E12 has higher antibody affinity and stronger inhibition effect on the cell proliferation of Her2-overexpressing breast cancer cells, and apoptosis-inducing activity; the results of in vivo antitumor experiment show that the antibody of the present invention can significantly inhibit tumor growth. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0017]      FIG. 1  shows the results of the apoptosis experiment of anti-HER2 antibody, wherein  FIG. 1-1 : SK-BR3 cell;  FIG. 1-2 : BT-474 cell;  FIG. 1-3 : MCF-7 cell); 
           [0018]      FIG. 2  shows the results of the growth inhibition experiment of anti-HER2 antibody, wherein  FIG. 2-1 : SK-BR3 cell;  FIG. 2-2 : BT-474 cell;  FIG. 2-3 : MCF-7 cell); 
           [0019]      FIG. 3  shows the results of the in vivo antitumor experiment of anti-HER2 antibody. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0020]    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. 
       Example: Preparation of Antibody 
     (1) Cloning of Genes Encoding Human Antibody Light and Heavy Chain Constant Region 
       [0021]    Healthy human peripheral blood lymphocytes 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 regions 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 . 
         [0000]    (2) Preparation of cDNA 
         [0022]    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 
       [0023]    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 at the 5′ end; V H For primer was added with a sequence: gcc aga acc acc gcc gcc gga gcc acc acc gcc at the 5′ end; V L Back primer was added with a sequence: tcc ggc ggc ggt ggt tct ggc gga ggc gga tct at the 5′ end; and V L For primer was added with a Not I site-containing sequence: atg cgg ccg c at the 5′ end. 
       (4) Construction and Selection of Phage Antibody Library 
       [0024]    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 “human HER2 extracellular protein” used for selection was prepared according to the method disclosed in the reference (Proc Natl Acad Sci USA, 1992, 89: 4285-4289). A human anti-HER2 single-chain antibody 3E12ScFv 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 (V H ) of 3E12ScFv, 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 3E12ScFv, respectively. 
         [0000]    (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 codens TAA and EcoRI restriction enzyme sites at the 3′ end. The sequence of the signal peptide was: (ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAAT ATCCAGAGGA). 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 ). 
         [0025]    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 codens TAA and EcoRI restriction enzyme sites at the 3′ end. The sequence of the signal peptide was: (ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAAT ATCCAGAGGA). 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 ). 
         [0026]    3×10 5  CHO-K1 cells (ATCC CRL-9618) were inoculated into 3.5 cm tissue culture dishes, and transfected when the cells were cultured to 90-95% confluence: 10 μg of plasmids (4 μg of plasmids pcDNA3.1(+) (3E12V H C H ), bug of plasmids pcDNA3.1/ZEO(+) (3E12V 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) (KPL) overnight at 4° C. and blocked with 2% BSA-PBS at 37° C. for 2 h; 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 3E12 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 3E12, 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 3E12, respectively. 
       EXPERIMENTAL EXAMPLES 
       [0027]    hGH0/1 was prepared according to the method described in Chinese Patent Application No.01132225.X entitled “Humanized Anti-HER2 Monoclonal Antibody, Preparation Method and Pharmaceutical Composition Thereof” filed on Nov. 16, 2001. 
       Apoptosis Experiment of Anti-HER2 Antibody 
       [0028]    Human breast cancer cells SK-BR-3 (high HER2-expressing, ATCC: HTB-30), BT-474 (medium HER2-expressing, ATCC: HTB-20) and MCF-7 (low HER2-expressing, ATCC: HTB-22) were cultured with different dilution degrees of anti-HER2 antibodies (including Trastuzumab, hGH0/1, 3E12) at 37° C. for 20 h, respectively. After washing the cells, the percentage of early apoptotic cells was detected according to the instructions of AnnexinV/PI kit (BD). The results of anti-apoptotic experiment are shown in  FIG. 1 . The cell-killing ability of 3E12 antibody was significantly stronger than that of Trastuzumab antibody and hGH0/1 (when the antibody concentration was ≧0.025 nM), P&lt;0.05, t test), and the same results were also be demonstrated in BT-474 cells (when the antibody concentration was ≧0.025 nM, P&lt;0.05, t test). However, in low HER2 expressing MCF-7 cells, the killing ability of 3E12 antibody was close to that of Trastuzumab antibody and GH0/1 antibody. These results exhibited that 3E12 antibody had HER2 specificity in killing cells, and had a stronger ability to kill medium and high HER2 expressing cells than Trastuzumab antibody and hGH0/1 antibody. 
       Cell Growth Inhibition Experiment of Anti HER2 Antibody 
       [0029]    Human breast cancer cells SK-BR-3, BT-474 and MCF-7 cells were incubated with different dilution degrees of anti-HER2 antibodies at 37° C., respectively. On the fifth day, the growth inhibition ratio was calculated after reading by MTT staining. The results of growth inhibition experiment are shown in  FIG. 2 . The ability of 3E12 antibody to inhibit SK-BR3 cell growth was significantly stronger than that of Trastuzumab antibody and hGH0/1 (when the antibody concentration was ≧0.1 nM, P&lt;0.05, t test), and the same results were also be demonstrated in BT-474 cells (when the antibody concentration was ≧0.1 nM), P&lt;0.05, t test). However, in low HER2 expressing MCF-7 cells, the cell inhibiting ability of 3E12 antibody was close to that of Trastuzumab antibody and GH0/1 antibody. These results exhibited that 3E12 antibody had HER2 specificity in inhibiting cell growth, and had a stronger ability to inhibit medium and high HER2 expressing cells than Trastuzumab antibody and hGH0/1 antibody. 
       In vivo Antitumor Experiments of Anti HER2 Antibody 
       [0030]    Each of SCID mice (purchased from Slack, Shanghai) was subcutaneously inoculated with high HER2 expressing human breast cancer cells BT-747 on 0 th  day, and when the tumor grew to 0.3 cm 3 , the tumor-bearing mice were intraperitoneally injected with various anti-HER2 antibodies at 0.5, 5 mg/kg for twice a week and continuously treated for 3 weeks. The changes of body weight of mice and tumor size were regularly observed for a total of 120 days. The antitumor treatment effect of anti-HER2 antibodies was evaluated. The results of antitumor experiment in vivo are shown in  FIG. 3 . The ability of 3E12 antibody to inhibit the growth of high HER2 expressing breast cancer cells BT-747 was significantly stronger than that of Trastuzumab antibody and hGH0/1 (at the dose of 25 mg/kg, on the 50 th , 60 th , 70 th , 80 th , 90 th , 100 th , 110 th , 120th day, P&lt;0.05, Mann-Whitney test).