In Vitro Evolution Of Anti-PD-1 Antibody Based On AID Enzyme And Mammalia Cell Display

Background: Antibody as a kind of drugs has strong specificity, high safety, good effect characteristic. Antibody plays an important role in the treatment of cancer and other diseases. Antibody library technology is one of the most commonly used techniques in the preparation of many kinds of antibody. Activation induced cytidine deaminase(AID) participates in B cell receptor affinity maturation, promotes and induces the immunoglobulin variable region mutation, AID plays an important role in thematuration process of antibody in vivo. Literature reported that the introduction of AID enzyme in the antibody library, help to improve the antibody library. It was reported that AID can be used to enlarge the library content for antibody screening in vitro. Therefore, use of AID enzyme and somatic cells display rapid screening of function antibody has become an important technology.PD-1/PD-L1 as a co-stimulatory signal pathways in the immune regulation, plays a key role in tumor immune escape. Therefore, PD-1 ? PD-L1 have become a new target for the treatment of tumors, antibodies of anti-PD-1/PD-L1 drugs have become a new hotspot.Purpose : Based on the mammalian cells display technology, In our work, PD-1as the target, combining AID inducing antibody gene evolution and mammalian cells display technology through flow cytometry, we probe into a high-throughput screening method of screening antibody.Methods: Through the IMTG obtained Nivolumab gene sequences of anti-PD-1 antibodies as the maternal antibody, we synthesized Nivolumab gene(MIL75) and the Fab fragment as MIL75 Fab, and C terminal of Fab connected to a transmembrane region sequences and assembly to the pFRT-FTMK-MIL75 carrier, it was displayed on the cell membrane. The vector was transfected into CHO-K1-FRT cells by eukaryotic cell transfection technique, then vector containing AID gene was transfeced, Fab fragment was displayed on the membrane of CHO cells. The use of labeled antigen PD-1-FITC by flow cytometry to screen high affinity antibody. The enriched cell was extracted genome, then the gene sequencing was assembly on specific expression vector to obtain antibody. Preliminary function evaluation of screened antibodies was operated in vitro.(1) We used ELISA and Biaocre evaluate specificity of antibodies and the binding of antigen.(2) We use the mixed lymphocyte reaction to evaluate the antibodies of biological activity, the levels of IFN-? secreted by T cells was detected to evaluate the function of antibodies in T cell activation.(3) Through the injection of antibody in the immunodeficiency and humanized NPG mice, the biological activity of antibody was evaluated in vivo.Results:(1) The synthetic antibody variable region genes were cloned into pFRT-IgG1 and pFRTFTMK carrier, we the successfully constructed MIL75 full-length antibody expression vector of pFRTIgG1K-MIL75 and display Fab vector pFRT-FTMK-MIL75 Fab. After pFRT-IgG1K-MIL75 was stably transfected into CHO-K1-FRT cells, stably cell line expressing the MIL75 full-length antibody was obtained by pressure screening. ELISA results showed that MIL75 specific, high affinity recognite antigen of PD-1. PFRT-FTMK-MIL75 Fab was stably transfected CHO-K1-FRT cells, after pressure filter, the stable cell lines expression MIL75-Fab was obtained. The results of flow cytometry showed that MIL75-Fab was successfully display on the surface of CHO-K1-FRTO cell, and can bind with the antigen of PD-1 and has a concentration dependent. Further transfected into AID inducing mutation.(2) We obtained three new antibody sequencesby flow cytometry screening and computer sequences analysis :Fv56, Fv60, Fv70.(3) In vitro binding assay showed that Fv56, Fv60 and Fv70 three antibodies can bind to the antigen of PD-1; Fv56, Fv60 and MIL75 have the same antigen recognition epitope; the antigen recognition epitope of Fv70 occurred drift. Fv56, Fv60 could block interaction of PD-1 and PD-L1 in vitro. Biological function experiment shows: Fv56, Fv60 and maternal antibody MIL75 have the similar biological activity, were able to activate T cells and upregulate IFN-? secretion of T cells with a certain antibody concentration dependent;, Fv70 had the activation function of T cells under high concentration.(4) In vivo biological function experiment results show that Fv56 could activate T cells in mice, increase the immune rejection, shorten the survival time of mice.Conclusion: In our study, we introduced mammalian cell display system and AID inducing antibody gene evolution, thus establishing an effective technology. By this method we screened three new antibodies: Fv56, Fv60 and Fv70. After the functional evaluation, Fv60 Fv56 and MIL75 have the similar biological function, indicating that Fv56 and Fv60 antibodies are effective anti-PD-1 agent.