Mammalian Cell-based Antibody Display: Construction Of Full-length Fully Human Anti-renal Cell Carcinoma Antibody Display Library

Renal cell carcinoma (RCC) is the most common kidney malignant tumor, accounting for 3% of adult malignancies. International Union of Against Cancer reported that there are about 190,000 new RCC patients each year worldwide and more than 91,000 RCC patients died each year. Chinese relevant epidemiological survey also showed that RCC patients are increasing these years. At present, surgery is still the first choice for the treatment of RCC. Although a variety of modern diagnostic equipments have made it easier to early diagnose and timely treat RCC and five-year survival rate has improved, there are still many metastasis cases missing surgery opportunity. Therapeutic effect of radiotherapy and chemotherapy is limited, while side effect is severe. In the past decade, non-specific immunotherapy like the use of interferon or interleukin-2 has been considered the standard treatment for advanced renal cell carcinoma. But this treatment can not prevent most RCC patients from death eventually with the progress of the disease. People are looking forward to a new therapeutic approach to solve this problem.1975, Hybridoma technology has been developed, monoclonal antibodies with a high specificity and homogeneity were produced against a single epitope. A variety of monoclonal antibodies have been developed and used widely in research and clinical diagnosis. The idea swept the world to use monoclonal antibody as a drug or as a carrier of radioisotopes, toxins and drugs for the treatment of human disease, which were well known as "biological missile". The greatest advantage of "targeted therapy" is that monoclonal antibodies target the tumor specifically, and the therapeutic effect is significant. It will not damage normal tissue when killing tumor cells. It is considered the most promising research direction for the treatment of cancer. Because of human-anti mouse antibody (HAMA) activity, the effort to use mouse monoclonal antibodies to treat human disease is not successful. Human body will be induced to produce antibodies to replace mouse antibodies.Over the past 20 years, the development of antibody technology provides people with the ability to use molecular biology techniques to clone full reservoirs of antibody genes. By inserting the genes of human antibody reservoir into mammalian cell expression vector, large scale antibody libraries have been constructed. Using phage as a carrier, antibody fragments can be displayed on a phage surface (phage display). Currently, phage display is the most developed and widely used display technology. It is a technology to express scFv on the phage surface for screening and selecting for high affinity specific antibodies. Scale in 1×1010 library is currently available. Humanization of mouse monoclonal antibodies and direct selection of fully human antibodies dramatically advance the development of antibody therapeutics. Thus far,22 antibody therapeutics have been approved by FDA as therapeutics for the treatment of tumors, arthritis, or cardiovascular disease.Though phage display is widely used, it has some obvious disadvantages. Phage can only display antibody fragments. So far there are no reports of successful expression or display of full length human antibodies on the phage surface. Bacteria are prokaryotic cell, but most therapeutic antibodies are produced from mammalian cells. The antibodies selected through phage display often lost their specific binding ability and neutralizing function after conversion of scFv to full length antibodies and expressed from mammalian cells. This is really cost ineffective and time consuming with low success rates. It is desirable to have the technology which can display full length antibodies specialized for humans in mammalian cells. More than 10 years, many scientists have put a huge effort in the development of mammalian display technology. By fusing a transmembrane domain(TM) at the 3'end of constant region of antibody heavy chain(HC),a fusion gene HC-TM is formed. When co-expressed with the light chain, the full length antibody can be displayed on the cell surface. Coupled with FACS, high affinity antibodies have been successfully screened and identified.It has been known that the affinity level of antibodies which can be isolated from the library is correlated to the scale of antibody library. The bigger the library, the chance or possibility to find a antibody with high affinity is bigger. In order to isolate antibodies with high affinity and high quality, construction of a large scale library is a key factor. Until now, the library which can display full length antibodies on the surface of mammalian cells is still in small scale. The diversity of mammalian display library is in 103～105 range, which does not meet the requirements for the screening of high affinity antibodies with biological function.To facilitate the identification of tumor-specific antigen as well as tumor antigen-specific antibodies, it is desirable to have tumor-specific patient antibody library available. We have reported previously a universal mammalian expression vector for rapid construction of antibody libraries and expression of antibody proteins on mammalian cell surface. Using this vector, here we reported the construction of a renal cancer patient-specific, full-length antibody display library with a combinatory diversity of 7.5×1010. PartⅠ:A vector for rapid construction of libraries of full-length antibodies highly expressed on mammalian cell surfacesObjective:To obtain a universal mammalian expression vector for efficient construction and high expression of full-length human antibody libraries.Methods:Human peripheral blood mononuclear cells (PBMC) were isolated and the total RNA was extracted from PBMC by RNA easy-kit. A gene fragment coding for human heavy chain variable region(VH) was amplified using two-step RT-PCR(Fragment 1).Through the second PCR, NheⅠ/BstXI/SfiⅠ/BsmBⅠenzyme recognizing sequences was added before the start codon of VH and the second BsmBⅠrecognizing sequence was introduced in frame at the 3'end of the VH(Fragment 2). Human IgGl constant region(CH) was RT-PCR amplified from total RNA isolated from human PBMC(Fragment 3).The second BsmBⅠrecognizing sequence was introduced into the PCR product at 5'end of CH and a BamHⅠrecognizing sequence at the 3'end. RT-PCR was also performed to amplify PDGFR-TM.(Fragment 4).At the 5'end of TM, a BamHⅠcutting site was incorporated. Finally a fusion protein(Fragment 5) was synthesized by assembly PCR using fragment 2,3 and 4 as templates A SfiⅠ/BstⅪ/XhoⅠrecognizing sequence was incorporated at the 3'end of this fusion protein. The final PCR product(Fragment 5) contains full length human heavy chain(HC) and PDGFR-TM. After digestion with NheⅠand XhoⅠ, the fragment(HC-TM) was inserted into the vector pcDNA 5/FRT between NheⅠand XhoⅠat multiple cloning site to form the interim vector. To reduce the vector size, the hygromysin selection gene expression cassette was deleted from the interim vector to form the final expression vector pDGB-HC-TM. Vector plasmid DNA or antibody genes were digested by proper restriction enzymes and then separated by electrophoresis. The target fragments were purified as described and DNA fragments were mixed in a proper ratio for ligation and transformation. Transfection of 293T cells was carried out in 12 well culture plates. The antibody expression on cell surface was detected by FACS and the data were analyzed using FCS Express V3 software.Results and discussion:The vector pDGB-HC-TM has been constructed by inserting multiple cloning sites unique sequences recognized by restriction endonucleases BsmBI, SfiI and BstXI for the pop-in and pop-out of genes of interest and contains IgG-1 heavy chain (HC). Using BsmBI cutting sites, the gene fragment encoding a variable domain of a heavy chain can be easily inserted into the vector, and the full-length heavy chain could be replaced with light chain by using BstXI or SfiI. Cytomegalovirus promoter was used to drive expression of the inserted antibody genes and a transmembrane domain from platelet derived growth factor receptor was fused in frame to the C-terminus of heavy chain consistent region to anchor the antibody expressed on the mammalian cell surface. Results of restriction enzyme digestion analysis showed that digestion of the vector by five different enzymes in four combinations gave proper-sized DNA fragments, indicating that the vector pDGB-HC-TM contains all introduced enzyme cleavage sites at the right location. Sequence analysis confirmed that the HC-TM fusion gene had the right coding sequence. Results of re-ligation-transformation of these fragments in groups showed that the ligation-transformation efficiency of these ligation mixture could reach up to 3.2 x 106 and all of the fragments were ligated correctly. When this vector was co-trasfected into 293T cells with light chain expression vector pDGB-huKappa, heavy chain and light chain both can be effectively expressed on the cell surface. The percentage of double positive cells reaches to more than 30%. These features make this vector very useful for the construction of antibody libraries of full-length heavy chain, full length light chain, or variable domain of heavy chain.Conclusion:We reported a universal mammalian expression vector pDGB-HC-TM which can be used to construct an antibody display library rapidly and to express contained antibody genes with high efficiency.Part II:Construction of a mammalian cell-based full-length fully human anti-renal cell carcinoma antibody display libraryObjective:To construct a renal cancer patient-specific, full-length antibody display library with a large combinatory diversity.Methods:The blood were collected from the renal cancer patient three days prior to surgery. The PBMC were isolated from the blood by gradient centrifugation. The total RNA was isolated from the purified PBMC using RNA easy kit. The concentration of the total RNA was measured and electrophereses analyzed to confirm good quality. The amplification of human antibody variable domain and full length kappa chain was carried out using 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 at 16℃for 20 hours. Calculation of transformation was carried out. The transfection of 293T cells was carried out in a 12-well plate. The antibody expression on cell surface was detected by FACS and the data were analyzed using FCS Express V3 software. Results and discussion:Totally 26 PCR were carried out to amplify heavy chain variable domain,14 PCR to amplify full length kappa chain. The sizes of this fragment are about 0.45 kb for VH and 0.75 kb for kappa chain. The PCR fragments were separated by electrophoresis and right sized fragments were purified from the gel. After digestion of VH library by BsmBⅠ, the VH fragments were inserted into pDGB-HC-TM between comparable BsmBⅠsites. The light chain fragments were inserted into the vector pDGB-HC-TM between Sfi1 to replace the HC-TM in the original vector. The library size of heavy chain is 1.13×106. The size of light chain is 1.36×105.10 heavy chain clones and 10 light chain clones were randomly picked for sequence and expression analysis. Results show that 8 heavy chain clones and 9 light chain clones containing right coding regions for unique amino acid sequences. Each heavy chain clone was co-transfected into the cells with sequence confirmed light chain gene and each light chain clone was co-transfected into cells with sequence confirmed heavy chain gene. The expression of full length antibodies on 293T cell surface were analyzed by FACS. From the result it can be known that 7 out of 10 heavy chain clones expressed detectable antibodies and 7 out of 10 light chain clones expressed detectable antibodies. Each heavy chain clone was co-transfected into the cells with sequence confirmed light chain gene and each light chain clone was co-transfected into cells with sequence confirmed heavy chain gene. The expression of full length antibodies on 293T cell surface were analyzed by FACS. From the result it can be known that 7 out of 10 heavy chain clones expressed detectable antibodies and 7 out of 10 light chain clones expressed detectable antibodies.Conclusion:We have successfully constructed a renal cancer patient-specific full-length antibody mammalian display library with a combinatory diversity of 7.5×1010 [(1.36×105×70%)×(1.13×106×70%)...