Construction Of Human Bladder Carcinoma-specific Full-length Antibody Display Library And Construction Of The Dual-expression Vector PDGB4

Bladder cancer is the most common type of malignant growths of the urinary bladder.75%～85% of bladder cancers are superficial bladder cancer and can be treated by Transurethral Resection of Bladder Tumor (TURBT),but the 5 year relapse may reach 70% and 10% to 20% of the patients may develop invasive bladder cancer. After radical cystectomy, up to 50% of patients with muscle invasive bladder cancer will metastasis, and the 5-year survival rate was 36～54%。The new idea of "targeted therapy" was raised early for the treatment of tumor by interfering with specific targeted molecules needed for carcinogenesis and tumor growth, rather than by simply interfering with rapidly dividing cells.So targeted cancer therapies may be more effective than current treatments and less harmful to normal cells.Monoclonal antibodies can be produced with hybridoma technology since 1975.But a human anti-mouse antibody(HAMA) reaction is inevitably induced when the non-human antibodies are injected into patients.The immunogenicity of murine monoclonal antibody can not be completely removed after humanization which is a time-consuming, labor-intensive work,and the desired effect cannot be achieved.To minimize the immunogenicity of pharmaceutical antibodies, the ideal antibody should be full human antibody.Over the past 20 years, researchers have used the techniques of molecular biology to clone full repertoires of human antibody genes. By inserting such genes into an expression vector, a variety of antibody libraries have been constructed and numerous antigen-specific antibodies have been identified from the libraries. Currently, phage display is the most developed and widely used technology for screening and selecting specific antibodies from library through the so-called panning procedure.Whereas each of these screening platforms has its specific advantages, they cannot display a full-length antibody but only single chain antibody variable fragment or antigen binding fragments,and do not have or may have different post-transcriptional modification or protein folding from mammalian. Besides, the antibodies selected from bacteria, yeast, or cell-free systems may not be expressible or lost function when they were converted into full-length and expressed from mammalian cells.An ideal therapeutic antibody display system should base on mammalian cell which can display full-length antibodies and provide correct protein folding and post-transcriptional modification.During the past 10 years, many scientists have exercised considerable effort in the attempt to develop mammalian cell surface display technology. It is therefore desirable to develop technology that can display full-length antibodies, especially human antibodies, on mammalian cell surfaces. Some techniques have fused platelet-derived growth factor receptor (PDGFR) transmembrane domain (TM) at the 3'-end of the antibody heavy chain (HC) to form the HC-TM. Co-expressing this HC-TM with a light chain, the full-length antibody could be displayed on the cell surface. Coupled with fluorescence activated cell sorting (FACS), some antigen-specific antibodies were successfully screened and identified from mammalian antibody libraries displayed on the cell surfaces.However, the diversity of mammalian display full-length antibody library developed with these techniques is limited to the range of 104～106, which cannot meet the requirements for the screening of high-affinity antibodies with biological function.In PartⅠ,we use a universal mammalian expression vector which was reported previously for rapid construction of antibody libraries.Using this vector,we reported the construction of a bladder cancer patient-specific,full-length antibody display library with a combinatory diversity of 3.32×1011.To construct a vector with dual-expression cassettes for expression of full-length antibody, requires the insertion of both HC and LC into the vector. Currently,this can only be achieved by two separate insertions into the single dual-expression vector.However, this approach is labor intensive and time consuming, and an ideal strategy would be with only one ligation to insert both HC and LC into one dual-expression vector.In part II, A unique four-way ligation strategy was developed for rapid construction of a full-length antibody library.A mammalian expression vector was constructed that contained dual mammalian expression cassettes and sequences recognized by the unique restriction enzymes BsmBI, BstXI, and Sfil. Both full-length light-chain and variable domain of heavy-chain genes were inserted into the vector in one step by four-way ligation, and full length bivalent antibodies were displayed on mammalian cell surfaces. Using this strategy, only 2 weeks were required to successfully construct high-quality, full-length human antibody libraries.PartⅠ:Construction of a mammalian cell-based full-length fully human bladder carcinoma-specific antibody display libraryObjective:To construct a bladder cancer patient-specific full-length antibody display library wtih a large combinatory diversity.Method:Peripheral blood mononuclear cells (PBMC) were isolated from patients with bladder carcinoma. The repertoires of heavy chain variable region (VH) and kappa light chain (LCκ) of antibody were amplified by RT-PCR. Then VH was inserted into the vector pDGB-HC-TM in frame with the IgGl constant region contained in the vector to form the full-length IgG1 heavy chain library. LCκwas inserted into the vectorpDGB-HC-TM to replace the HC-TM in the vector to form the full-length kappa chain library. After transformation of these ligated mixtures into competent E.coli TOPO-10 cells, the bladder carcinoma patients-specific heavy chain (IgG1) and kappa light chain (LCκ) antibody libraries were constructed.Then the DNA of heavy and light chain clones from the libraries were co-transfected into FCHO cells and the expression of full-length fully human antibody on the surface of FCHO cells was confirmed by flow cytometry.Results:A total of 1.4×108 PBMC were isolated from the blood of 10 selected patients with bladder cancer (pathological diagnosed) before surgery.Totally 3 PCR were carried to amplify heavy chain variable domain,3 PCR to amplify full length kappa chain.The VH fragments were inserted into pDGB-HC-TM between comparable BsmBI sites.The light chain fragments were inserted into the vector pDGB-HC-TM between SiiI to replace the HC-TM in the original vector.The library size of heavy chain is 1.7×106 and the size of light chain is 3.1×105.7 out of 10 clones randomly picked from heavy chain library contained the correct coding sequence of heavy chain variable region, encoding 7 specific amino acid.9 out of 10 clones randomly picked from light chain library contained the correct coding sequence of light chain, encoding 9 specific amino acid.These correted clones were co-transfected into FCHO cells with sequence confirmed light chain gene or heavy chain gene,and all the clones expressed detectable antibodies on FHCO cell suface.Conclusion:We successfully constructed a bladder cancer specific full-length antibody mammalian display library with a combinatory diversity of 3.32×1011 [(1.7×106×70%)×(3.1×105×90%)], which can be displayed on mammalian cell surfaces.PartⅡ:Construction of a dual-expression vector pDGB4 for the construction of mammalian cell-based full-length antibody display libraryObjective:To design and construct a unique vector that contains dual-expression cassettes for simultaneous insertion of both Vh (or HC) and LC genes into the vector by a single four-way ligation.Method:A dual-expression cassette vector, pDGB4, has been constructd based on the universal single-expression cassette vector, pDGB-HC-TM. Eight primers were designed and synthesized to introduce the proper restriction enzyme-recognizing sequences into the vector pDGB4 at the desired location.Four fragments were generated first by PCR amplification using designed primers:P1 and P2 for the 5.0 kb frame fragment; P3 and P4 for the 1.6 kb HC-TM (HC fused to TM domain) fragment; P5 and P6 for the 1.8 kb frame fragment; and P7 and P8 for the 0.7 kb LC fragment.After digestion with proper enzymes and electrophoresis analysis, these four fragments were purified,and ligated together to form the vector pDGB4. The molar ratio of four fragments is 1:1:1:1 with a total amount of 91 ng in 10 ul of ligation mixture.One microliter of ligation mixture was then used in the transformation, and the colonies were analysed and comfirmed by restriction enzyme digestion. Two pairs of PCR-amplified human kappa chain library and human HC variable domain library were used in the four-way ligation to replace the LC gene and the HC variable domain in the vector pDGB4. Then the clones piced randomly from the libraries constructed were transfected into FCHO cells and the expression of full-length fully human antibody on the surface of FCHO cells was confirmed by flow cytometry.Results:Two libraries were constructed with sizes in the range of 105. The ligation and transformation efficiency is in the range of 106 clones/ug of ligated DNA with a background of less than 0.5%. Plasmid DNA was isolated from 20 colonies randomly picked up from the two libraries and transiently transfected into FCHO cells. The expression of antibodies was analyzed by FACS. Compared with negative control,8 out of 10 clones from library 1 and 6 out of 10 clones from library 2 showed detectable antibody expression on the cell surfaces. The results demonstrated that the strategy reported here was very powerful not only in regular cloning but also in construction of full-length antibody display libraries with very low background.Conclusion:We successfully constructed the dual-expression vector pDGB4,and constructed two full-length antibody display libraries with very low background.