Construction Of Full-length Fully Human Anti-VEGF Antibody Display Library

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(HIF) and vascular endothelial growth factor (VEGF).VEGF is closely related to tumor stage and prognosis. Therefore, inhibition or blocking of VHL-HIF-VEGF signaling pathway particularly blocking VEGF or its receptors 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 very difficult 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.Using total RNA isolated from peripheral blood lymphocytes of patients with renal cell tumor and 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. Full-lenth fully human antibody gene libraries have been constructed with a combinatory diversity of6.67×109.In order to increase the success rate of screen, we matched the heavy and light chain genes of Bevacizumab with the heavy and light chain genes of the libraries. After that the heavy chain genes of Bevacizumab and the light chain genes of the labraris were simultaneously inserted into mammalian display vector pDGB4to construct the sub-libraries, vice verse. After the transfection of the libraries into FCHO cells, a cell library which could stably display full-length antibodies on cell surface was successfully constructed. Coupled with high-flux fluorescence-activated cell sorting (FACS), we screened out twenty-six cell clones which could bind VEGF.Part I:Construction of Full-length Fully Human primary Antibody Display LibraryObjective:To construct2full-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 of6.54×104.10heavy 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%of the cells expressed detectable antibodies.Conclusion:Using vector pDGB-HC-TM, two full-length human mammalian display antibody libraries with a combinatory diversity of6.67×109were successfully constructed, which allows the display of full-length antibodies on mammalian cell surface.Part II:Construction of Full-length secondary Antibody Display LibraryObjective:To construct two secondary antibody display libraries with large combinatory diversity and the Bevacizumab expression vector.Methods:Digest the VH library and heavy chain of Bevacizumab by BsmBI and use the SfiI to digest light chain library and Bevacizumab. Both VH library and light chain of Bevacizumab fragments were simultaneously inserted into the vector pDGB4by four-way ligation to construct the library, and both light chain library and VH of Bevacizumab fragments were simultaneously inserted into the vector pDGB4by four-way ligation to construct the library2. Additionally, both of the heavy and light chains of Bevacizumab were simultaneously inserted into the vector pDGB4by the same way to construct the Bevacizumab expression vector. After transformation of libraries into competent bacteria cell, the transformation efficiency and library size were calculated.Results and discussion:Library1was constructed with a size of9x105and a background of0.24%. Library2was constructed with sizes of1.8×106and a background of0.35%. And we also successfully constructed a new plasmid which simultaneously contains the light and heavy chain of Bevacizumab. Conclusion:We have successfully constructed two secondary antibody display libraries with large combinatory diversity and a new vector for positive control. Part Ⅲ:Construction of FCHO-Cell Baced Antibody Display Library And Screen of Anti-VEGF Antibody Display Cells from the Cell LibraryObjective:To construct FCHO-cell-baced antibody display libraries and to screen anti-VEGF antibody display cells with FACS.Methods:Transfection was performed with FCHO cells. FCHO cells were dissociated by cell dissociation buffer, followed by one wash using staining buffer (2%FBS in PBS). Then, cells were stained on ice by PE fluorescence-labeled anti-human Kappa chain antibody (PE-K) plus Biotinylated Human VEGF kit. After staining for30min, cells were washed and resuspended in staining buffer for FACS analysis. The FACS data were analyzed using FCS express V3(De Novo Software, Los Angeles, USA). Single cells with specific binding to VEGF were sorted into96-well plated for further analysis as need.Results and discussion:By stably transfection of the second library into FCHO cells, two stably antibody display libraries were constructed with a size of4.2×105and3.8X105respectively. Two FCHO cell libraries were analyzed by FACS.3.84%and10.63%of PE-K and FITC-VEGF double positive cells were sorted out from the libraries and expanded for second round FACS analysis and sorting. After two rounds of FACS screening, the percentage of PE&FITC double positive cells were up to79.72%and83.44%respectively.0.10%double positive cells in each cell library were sorted into96well plates for single cell cloning. Further FACS analysis indicated that26single cell clones could display VEGF-specific antibodies on their cell surfaces. Conclusion:We have successfully constructed two full-length anti-VEGF antibody cell libraries. Coupled with FACS, we successfully identified26cell clones which could stably display antibodies binding VEGF.