Molecular Directed Evolution Of Reshaped Anti-CD3 Single Chain Fv Fragment

Anti-CD3 McAb is an important immunosuppressive agent for clinical organ allograft rejection. Conversely, T cell activation and proliferation induced by anti-CD3 Ab have been exploited for the treatment of immunity-poor and adoptive cancer immunotherapy. However, because the use of anti-CD3 Ab in therapy has been compromised by its HAMA response and the "first dose" syndrome, the McAb needs to be humanized.The anti-CD3 reshaped ScFv ReCD3 was the first anti-CD3 humanized antibody in China, which was constructed by grafting CDRs-L and CDRs-H of OKT3 into FRs region of human antibody LS1 and Nd, while with poor affinity.In this dissertation paper two methods to improve the affinity were addressed. One was to obtain the mutated variants of ReCD3 by two side megaprimer PCR based site-directed mutagenesis methods. By molecular modeling and comparing homologous sequences of anti-CD3 Ab, two sets of degenerate primer were designed to amplify DNA fragments containing mutations. Then by adopting these two mutated DNA fragments as megaprimer and "slow annealing" PCR procedure, full length mutated DNA was amplified. Finally, after a 106 ReCD3 site-directed mutagenesis secondary libraries was constructed and three rounds of panning were performed, four mutatants with higher affinity were obtained.Based on the above work, another method, DNA shuffling, was used to carry out the molecular directed evolution of ReCD3. The self-priming PCR was performed by mixing the 80-100bp fragments produced from DNase â…  digestion of ReCD3 with two directed-mutated fragments. Subsequently, a 10~7 ReCD3 random mutated secondary libraries was constructed. After five rounds of panning, three antibodies with higher affinity were obtained and the affinity of one was improved two fold. The molecular modeling results showed the structure of the higher affinity antibody was changed.