Construction And Screening The Fully Human Single Chain Variable Fragment Antibodies Library Specific For Lung Cancer And The Interaction Between The Selected Antibody Structure And Its Function

Lung cancer is the most common cause of cancer death. Non-small cell lung cancer (NSCLC) accounts for about 80% of all lung cancers. The vast majority of patients present with non-small cell lung cancer in advanced inoperable stages. The current first-line treatment for patients with advanced NSCLC includes chemotherapy, surgery, and palliative radiotherapy, but most patients relapse and eventually succumb to the disease. Advances in our knowledge of cancer cell biology have led to the development of specific molecular-targeted therapeutic strategy. Mutations in the vascular endothelial growth factor receptor-2 (VEGFR-2) have been identified in NSCLC cells, and overexpression of the VEGFR-2 and its ligands is a common feature of lung cancer; therefore, VEGFR-2 has become an attractive target for various anti-tumor strategies. Developments with antiangiogenesis agents hold promise as new approaches in lung cancer therapy. Much of the work to date has focused on the anti-vascular endothelial growth factor receptor (VEGFR) antibody, especially human single chain variable fragments antibody (scFv).Several display systems are used to produce human antibody, such as phage display, cellsurface display, ribosome display and mRNA display. The main principle of these strategies isthe linkage of genotype and phenotype during the selection cycles. Such in vivo methods arehampered by one or more of the following factors: the inability to select under conditionsdifferent from the cellular environment, problems with the selection of proteins that are toxic,cells circumventing selection pressure and low transformation efficiency. Ribosome display iscarried out fully in vitro, which overcomes some of the limitations of cell-based display systems.Through the formation of antibody-ribosome-mRN A (ARM) complexes in cell-free systems suchas Escherichia coli S30 or rabbit reticulocyte, individual antibodies are linked with theircorresponding mRNA molecules, permitting selection of the genetic material through thefunctional properties of the antibody, usually as a binding reaction. The mRNA can be amplifiedand recovered as DNA by reverse transcription polymerase chain reaction (RT-PCR) and furthercycled, mutated or cloned. The purpose of this study was to construct and screen the fully humansingle chain variable fragment antibodies library specific for lung cancer by ribosome display.The main results obtained are as follows:The total RNA was extracted from peripheral blood lymphocytes isolated from the blood of 30 lung cancer patient volunteers. Gene fragments encoding whole variable region of heavychain (VH) and variable region of light chain (VL) were amplified by PCR and assembled into a single chain by overlapping PCR with a linker DNA encoding the peptide GSASAPTLFPLVS. The full-length scFv DNA contains a T7 promoter and a translation initiation signal (Kozak sequence). The size of the human scFv antibody library contains 7.6 X1013 members. DNA fingerprinting and sequencing of 33 individual clones analyzed the library diversity. The results indicated that the library had a good diversity. The target antigen of human vascular endothelial growth factor receptor-2 (VEGFR-2) was used to screen the human single chain antibody library by ribosome display technology. After three selection cycles, anti-VEGFR-2 scFv DNA was obtained successfully, and then the DNA fragments containing anti-VEGFR-2 scFv gene with restriction enzyme sites at both sites was amplified using PCR method with primers containing restriction sites and pUCmT-Anti-VEGFR-2 Vector as a template. The PCR products was double digested with restriction endonucleases and a target fragment was recovered, then it was cloned to pET-30(a)+, an expression vector double digested with the same restriction enzymes. The anti-VEGFR-2 scFv gene in pET-30(a)+ was transformed into E. coli BL21. Transformants of E. coli. were incubated in LB medium at 37 °C for 12 h, then induced with isopropyl P -D-thiogalactoside (IPTG) for 6 h. After cultivation, the cell were harvested, and homogenized by sonication. The molecular weight of Anti-VEGFR-2 scFv was 31 kDa. The expression level was 27 % of total protein. We obtained the scFv antibody purified by affinity chromatography. The affinity constant of Anti-VEGFR-2 scFv was 1.3 X 109 L/mol by noncompetitive enzyme-linked immunosorbent assay, and the antibody titer was 1:3200. After analysis the sequence of Anti-VEGFR-2 scFv by DNA-PLOT, we found that VH was VH3 sub-group and VL was Vkih sub-group. At the same time, the 3-D structure of Anti-VEGFR-2 scFv was predicted by homologous modeling technology. The results indicated that the homology was 67% between the template protein and Anti-VEGFR-2 scFv. The interaction between Anti-VEGFR-2 scFv spacial structure and its function was also analyzed.In this study, we also used the atomic force microscopy (AFM) for the imaging of mica surface with pre-adsorbed antibodies or antibodies moieties before and after incubation of VEGFR-2 antigens. The results indicated that the diameter of Anti-VEGFR-2 scFv molecules was 6.78 nm and the diameter of antibody-antigen complex was 141.07 nm.