Studies On Large-scale Fermentation Of Anti-CD3 Antibody's Light Chain In Pichia Pastoris

The first anti-CD3 antibody, OKT3 was produced in 1979 which recognition site wasεchain of CD3.It was first used clinically in 1980 and became the most effective mono-antibody in clinic. The success that OKT3 could be used for the graft rejective reaction of kidney transplant impulsed OKT3's application in the transplant region.OKT3 was injected in the blood vessel and binded with T cell receptor site. Then they were removed by reticuloendothelial cell in the liver and spleen. Once OKT3's injection was stopped, the number of CD3+ cell would be recovered to normal level. It was confirmed by Hebart that the effective power of OKT3 for graft versus host reaction of hormone invalidity was 65% and annual survival rate was 25%.Anti-CD3 antibody as the new type of immunodepressant was widely used in clinic especially organ transplant region. At present, it was gotten more and more attention that the affect of anti-CD3 antibody for autoimmune disease, which had initial progress.Anti-CD3 antibody'application was restricted because initial prepared anti-CD3 antibody was all from mice. To overcome HAMA, it developed humanized antibody. The initial humanized antibody was constructed by connecting the variable region of mice monoclonal antibody with the constant region of human antibody.At present, the system of expressing anti-CD3 antibody includes prokaryotic expression and eukaryotic cell expression. E. colli as host bacterium of exogenous gene expression which grows fast is the simple expressing system of obtaining recombination protein. Although the expressing quantity of eukaryotic expression is low, the expressing protein has better biologic activity after transcription, post-translation processing and modification in eukaryotic cell and is better to research the biologic activity of expressing protein further. There is the achievement of expressing anti-CD3 antibody in Hela cell in our country.In this study, anti-CD3 human-mouse chimeric antibody was constructed by using gene engineering methods in order to decrease its immunogenic properties. To construct the anti-CD3 antibody, We connected variable region of the mouse light chain with the constant region of human light chain. Anti-CD3 human-mouse chimeric antibody could induce biological effect of Fc fragment,such as inducing immune tolerance to prevent reject reaction, prolonging the survival time of the transplant and improving the state of an illness with experimental Autoimmune disease.pPICZαIgκ, an expression vector of light chain, had been precipitated by our laboratory. On this basis, the yeast preference codes were selected to synthesize the variable region of the light chain, and then the mouse variable region was connected with the constant region of the human light chain. At last, anti-CD3 Chimeric Antibody was expressed in Pichia Pastoris. Because of its lower immunogenicity, this antibody can be immunodepressant used for organ transplant to reduce immunologic rejection and elevate the survival rate of transplanted organ.In this study, anti-CD3 human-mouse chimeric antibody was constructed by using gene engineering methods. It not only reduces immunologic rejection of mouse antibody, but also improves the expression system of anti-CD3 antibody to suit for the need of industrialized full scale operation.1.The light chain cloning of anti-CD3 chimeric antibody sequenceAnti-CD3 variable region sequence of light chain was replaced by homonymy codons which was the yeast preference codes, and then connected with pPICZαIgκvector which contains constant region of light chain to construct complete anti-CD3 Human-mouse chimeric antibody. So we can continue to do the expression of recombinant anti-pPICZαIgκ-CD3 antibody.2.Construction of recombinant anti-CD3 antibody expression systemThe recombinant plasmid of anti-pPICZαIgκ-CD3 was chosen as template and cloned to pPICZαIgκvector after being digested with corresponding endonucleases. And the correctness of our construction was demonstrated by the results of gene sequencing.Purified anti-pPICZαIgκ-CD3 vector was transformed into Pichia pastoris and smeared on YPD plates containing 100μg/ml Zeocin. After 72 hours incubation, dozens of transforming colonies appeared. Anti-pPICZαIgκ-CD3 positive strain was screened by PCR method and determination of biological activity,so as to establish eukaryotic expression system in Pichia pastoris.Experimental results indicated that we successfully established recombinant anti-pPICZαIgκ-CD3 antibody expression system in Pichia pastoris, which could product secreting type of recombinant anti-CD3 antibody by methanol inducing. In the process of fermentation, the production had been increasingfrom the first day to the sixth day, and the production reached peak on the forth day. The production decreased on the fifth day, which might be the result of proteolytic enzymes degradation. The best pH of expression is 5.5. 3.Studies on large-scale fermentation and purification process of anti-CD3 antibody's light chainPichia pastoris has many advantages as a kind of expression host, and it's very suitable for large-scale expression of the extraneous proteins. So we explored the large-scale fermentation process of antibody's light chain and found that the best pH is pH5.5±0.2, DO between 25%～30% and the supply speed of methanol is 8.5ml/h/L initial fermentation volume.A new method to purify antibody at large-scale was explored. We found the eluate could be purified by SP Sepharose XL on pH3.0.The cloning and expression of anti-CD3 antibody's light chain in improved expression system of Pichia pastoris facilitates its application in clinic. Meanwhile, it provides the source for the other basis research of the expression product.