Construction Of A Fully Synthetic Human Antibody Library And Preparation Of Anti-TIGIT Antibody

Compared with heterologous antibodies,human antibodies have less immune side effects on human body,so they are the first choice for drug development of human antibodies.In recent years,the technology of screening human antibody against any antigen by constructing human antibody library has been rapidly developed and applied.The fully synthetic human antibody library adopts genetic engineering method to clone the synthetic human antibody gene library into the phagemid vector,and rescure the recombinant phagemid with the helper phage.In recombinant phage antibody library,antibodies and coat protein become fusion proteins and display on the phage surface.The human antibodies can be obtained when using the human antigens to screen the recombinant phage antibody library.TIGIT(T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitor motif domains)is a class of immunomodulatory proteins with immunoreceptor tyrosine inhibition motifs,which is mainly expressed on the surface of T cells and NK cells.The main ligand of TIGIT is poliovirus receptor(PVR,CD155),and PVR is overexpressed on the surface of tumor cells.Binding of activated receptor CD226(DNAM-1)and PVR can enhance the killing effect of immune cells on tumor,but TIGIT/PVR binding with higher affinity inhibits this killing effect,and TIGIT can also directly bind CD226 and destroy its homologous dimerization.Anti-TIGIT antibody can achieve anti-tumor effect by binding to TIGIT antigen and blocking TIGIT’s binding to PVR.Up to now,although the anti-TIGIT antibody drugs which humanized by murine antibodies have entered clinical trials,there has been no literature report on the preparation of all-human anti-TIGIT antibody by using the fully synthetic human antibody library.The purpose of this thesis is to build a large library of the fully synthetic human antibodies and use it to prepare the fully human TIGIT monoclonal antibodies.The main research contents and results are as follows:1.The 14 synthesized CDR-Mix gene pools were respectively cloned into pComb-VK and pComb-VH vectors,then obtained 2 light chain sublibraries and 4 heavy chain sublibraries.The light chain sublibraries and the heavy chain sublibraries were cloned into the pComb3XF-scFv vector respectively to obtain 8 single-chain antibody fragment(scFv)libraries.Afterward,the TG1 phages containing recombinant antibody library were infected by M13EEA helper phage to obtain the recombinant phage antibody library.The results showed that 8 single-chain antibody libraries were successfully constructed,with the coverage of 1.41×1010.Monoclonal gene sequencing showed an accuracy rate of 80.9%for the ORF of the monoclonal antibody library.High-throughput sequencing results of pComb-VH3-Lib showed that 73%of the ORF was correct,basically consistent with the design results on length diversity and sequence diversity.Titer detection of recombinant phage antibody library suggested that we constructed a recombinant phage antibody library with 1012 pfu coverage.2.The TIGIT-IgV gene was synthesized by using the TIGIT nucleotide sequence reported in the NCBI database,and the pCD-TIGIT-Avi-His plasmid was constructed.The TIGIT-Avi-His protein was expressed in eukaryotic nucleus,and TIGIT-Avi-His-Biotin protein was obtained after Biotin labeling.The TIGIT-Avi-His protein was used as specific antigen in ELISA assays,and the TIGIT-Avi-His-Biotin protein was used as screenning antigen for the antibody library.The linearized GC-ID-TIGIT plasmid was transfected into CHO-DG-44 cells to construct stable TIGIT transfected cell lines which used in FACS assys for detecting the binding and blocking activities of anti-TIGIT antibodies.According to the Etigilimab antibody sequence published in WO2016191643A2,TIGIT monoclonal antibody namely Etigilimab was independently prepared as a positive control of anti-TIGIT antibody.3.The recombinant phage antibody library was screened by using the TIGIT-Avi-His-Biotin antigen and the alternative positive phage single chain antibodies were screened at the prokaryotic level.After that,the obtained positive monoclonal antibodies were expressed in eukaryotic cells,they were further optimized by testing their affinity activity and blocking activity based on ELISA and FACS.Finally,two TIGIT monoclonal antibodies,namely CB3 and CD23,were obtained.Their affinity constants(Kd)were 8.155×10-10M for CB3 and 1.128×10-9M for CD23,respectively.4.The CD23 monoclonal antibody was used as the template to saturate and mutate 29 amino acid residues in 6 CDR regions to construct a primary affinity mature mutation library.The affinity mature mutation library was subjected to enrichment panning and primary screening by using the TIGIT antigen.With 55 positive monoclonal antibodies as templates,the secondary affinity mature mutation library was constructed by random recombination of four DNA fragments.The secondary mutation library of affinity maturation was subjected to enrichment panning and monoclonal screening by using the TIGIT antigen.The affinity maturation was performed on CD23 antibody.It was increased to 2.661×10-10 M.5.The research results provide the basis for the development of fully human TIGIT monoclonal antibodies with high affinity and related antibody drugs.