| Renal cell carcinoma (RCC) is the most common kidney malignant tumor, which is not sensitive to radiotherapy and chemotherapy. Due to its insidious onset,25to30percent of patients had metastasis in the time of diagnosis and nearly30%of localized renal cell carcinoma will relapse even after radical resection. People are looking forward to a new therapeutic approach to solve this problem.With in-depth study, Von Hippel-Lindau (VHL) gene mutation is found in most renal cell carcinoma, especially clear cell carcinoma, with a high level of hypoxia-inducible factor and vascular endothelial growth factor. VEGF is closely related to tumor stage and prognosis. Therefore, inhibition or blocking of VHL-HIF-VEGF signaling pathway, in particular, blocking VEGF or its receptors VEGFR, may inhibit the growth of renal cell carcinoma.Bevacizumab is a VEGF-specific murine antibody, which is humanized and due to its biological activity of blocking VEGF, is used for the treatment of patients with solid tumors and high level of VEGF. Bevacizumab combined with IFN-a can increase the Objective Response Rate (ORR) and Progression Free Survival (PFS) of metastatic renal cell carcinoma. Thus, U.S. FDA approved bevacizumab plus IFN-alpha as first-line treatment of the targeted therapy of metastatic renal cell carcinoma in December2007.However, humanization of monoclonal antibodies obtained by hybridoma technology, on the one hand, can not completely remove antibodies’ immunogenicity. On the other hand, the antibody space configuration may be changed, the affinity tends to reduce and it is verydifficult to achieve the desired effect.In order to minimize the immunogenicity of pharmaceutical antibodies, the researchers are trying to use mammalian cell surface display technology for the screening of human antibodies. The diversity of the antibody library contructed by mammalian display technology was limited to104to106so far and can not meet the requirements of screening high affinity antibodies.We had made technical improvements of the existing mammalian cell surface display technology in order to overcome these shortcomings of it.Using total RNA isolated from peripheral blood lymphocytes of patients with autoimmune disease as starting material, whole set of antibody genes were amplified by RT-PCR and then the antibody genes were inserted into the mammalian cell expression vector pDGB-HC-TM, Three full-lenth fully human antibody gene libraries have been constructed with a diversity of1010. Using four-way ligation, the antibody heavy and light genes were simultaneously inserted into mammalian display vector pDGB4to construct the sub-libraries. After transfection of the libraries into FCHO cell, a cell library which could stably display full-length antibodies on cell surface was successfully constructed.Both heavy and light chain genes of Bevacizumab were synthesized and the codons were optimized for high expression of the antibody in mammalian cells. It will be used as a positive control during antibody screen. And it has also been used in chain exchange screen strategy to increase the success rate of screen. Replacing the heavy chain or light chain libraries in vector pDGB4with light chain or heavy chain genes of Bevacizumab, the full-length fully human anti-VEGF antibody libraries have been successfully constructed.Part Ⅰ:Construction of Full-length Fully Human primary Antibody Display Library Objective:To construct3full-length human primary antibody display libraries with large capacity.Methods:Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of selected donors by gradient centrifugation. The total RNA was isolated from the purified PBMC using RNA Easy kit. The concentration of the total RNA was measured.The amplification of heavy chain variable domain and full length kappa chain was carried out by two-step RT-PCR using specific forward and reverse primers. The vector pDGB-HC-TM and RT-PCR amplified DNA fragments were digested by proper restriction enzymes. The ligation was performed at16℃for24hours. The transformation efficiency was calculated and antibody gene libraries were constructed. The transfection of libraries into293T cells was carried out in12-well plate. The antibody expression on cell surface was detected by FACS and the data were analazed using FCS Express V3software.Results and discussion:Total6PCR reactions were carried out to amplify antibody genes,3for heavy chain variable domain (VH), and3for full length kappa chain. The sizes of these fragments are about0.45kb for VH and0.75kb for kappa chain. The PCR fragments were separated by electrophoresis and the right size fragments were purified. After digestion of VH library by BsmBI, the VH fragments were inserted into vector pDGB-HC-TM between comparable BsmBI sites. The light chain fragments were insterted into the vector pDGB-HC-TM between SfiI to replace the HC-TM in the original vector. The heavy chain library constructed has a size of1.89×105and light chain library has a size of6.54×104. Totoal3pairs of heavy and light chain liraries were constructed using this method.10heavy chain clones and10light chain clones were randomly picked up for sequence and expression analysis. Results show that8heavy chain clones and7light chain clones contain right coding regions for unique amino acid sequences. The heavy chain and light chain gen libraries were co-transfected into the293T cells. The expression of full length antibodies on293T cell surface were analyzed by FACS. The results show that63.40%cells expressed detectable antibodies.Conclusion:Using vector pDGB-HC-TM, three full-length human mammalian display antibody libraries with a combinatory diversity of1.19×10、2.0×1010and5.27×1011were successfully constructed, which allows the display of full-length antibodies on mammalian cell surface.Part Ⅱ:Construction of Full-length Fully Human secondary Antibody Display LibraryObjective:To construct three full-length human secondary antibody display libraries with large combinatory diversity.Methods:After digestion of VH library by BsmBI and light chain library with SfiI, both VH and light chain fragments were simultaneously inserted vector pDGB4by four-way ligation. Totoal3liraries were constructed using this method. After tranformation of libraries into competent bacteria cell, the transformation efficiency and library size were calculated. The stable transfection of libraries into293T cells was carried out in a12-well plate. The antibody expression on cell surface was detected by FACS and the data were analyzed using FCS Express V3software.Results and discussion:A four-way ligation library was constructed with a size of2.476x106and a background of0.30%. Another two libraries were constructed using this method, with sizes of9.22×105and1.0828×106, backgrounds of0.06%and0.17%. The transfection of one of these four-way ligation libraries to293T cells was carried out in a12-well plate with61.78%cells expressed detectable antibodies. After the four way ligation library was stably transfected into FCHO cell, a cell library which could stably display full-length antibodies was successfully constructed, with a size of3.76×106. We could detect that there were59.62%cells in the cell library could display full-length library. This cell library would be used for screening of specific antibody.Conclusion:We have successfully constructed three secondary antibody display libraries with large combinatory diversity and a stable cell library with a size of3.76×106, which could stably display full-length antibodies for antibody screen.Part Ⅲ:Construction of Anti-VEGF Antibody Display LibraryObjective:To construct a full-length human anti-VEGF antibody library by using chain exchange strategy.Methods:Both heavy and light chain genes of antibody avastin were synthesized and the codons were optimized for high expression of the antibody in mammalian cells. The appropriate restriction enzyme recognizing sequences and signal peptide sequences were added in front of the heavy chain and the light chain genes. Then both genes were simultaneously inserted into the vector pDGB4to construct the avastin expression vector. The heavy chain or light chain libraries in secondary Antibody Display Library constructed previously was replaced with avastin light chain or heavy chain. In that way, the heavy or light chain in the library was exchanged.The appropriate restriction enzyme sequence and signal peptide sequence was added in front of the heavy chain variable variable region and the light chain variable region sequence, which was then optimiazed in order to improve gene expression and meet the human codon bias. After digestion of avastin-VH by BsmBl and digestion of avastin-Vk by SfiI, the fragments were inserted into the vector pDGB4between comparable BsmBI sites and SfiI sites; a new vetor pDGB4-avastin was constructed. And then another new vector pDGB4-avastin-vh was constructed. The light chain fragments were insterted into the new vector pDGB4-avastin-vh between SfiI sites in order to construct vector pDGB4-avastin-vh-Hu-LCs. Finally, a full-length human anti-VEGF antibody library was built by the transformation of the new vector pDGB4-avastin-vh-Hu-LCs.Results and discussion:Both heavy and light chain genes of antibody avastin were synthesized and the codons were optimized for high expression of the antibody in mammalian cells. After optimization, the CAI (Codon Adaptation Index) of heavy chain was improved from0.80to0.87and the CAI of light chain from0.80to0.85.Three new expression vetors were constructed:vector pDGB4-avastin for avastin expression; vector pDGB4-avastin-vh containing avastin heavy chain and unrelated light chain; and vector pDGB4-avastin-LC containing avastin light chain and unrelated heavy chain. Finally, a full-length fully human anti-VEGF antibody library, pDGB4-avastin-vh-Hu-LCs, was constructed with a size of7.58×105and a background of2.51%by the replacement of the unrelated light chain in the vector pDGB4-avastin-vh by light chain library. Using the similar method, another full-length fully human anti-VEGF antibody library, pDGB4-avastin-vk-Hu-HCs, was constructed with a size of1.33×106and a background of1.37%by the replacement of the unrelated heavy chain in the vector pDGB4-avastin-vk by heavy chain library.Conclusion:We have successfully constructed a full-length fully human anti-VEGF antibody library by using chain exchange strategy. |
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