抗体是免疫B细胞分泌的功能蛋白，是体液免疫系统中病原体识别的分子基础。抗体蛋白也被广泛地作为靶向药物、诊断试剂应用于疾病诊疗中。高效力抗体的产生依赖于V(D)J重排、类型转换、亲和力成熟等抗体多样化过程。抗体多样化尚不能在体外培养细胞中完全重现，因此抗体制备仍依赖于免疫动物或人群血样。在前期工作中，申请人研究组利用多样化碱基编辑工具和流式细胞术等方法，在体外初步实现了抗体亲和力成熟。其中科学问题以论文形式发表，关键技术已申请专利。在本项目中，申请人拟进一步解决抗体多样化体外重构中的瓶颈问题，针对(1)更多样化突变产生和(2)细胞水平自动筛选等两个方面，基于抗体多样化原理、利用合成生物学等手段在体外模拟和重构抗体亲和力成熟的整个过程，从而完善这一技术体系。本项目设计的抗体生产新技术，不仅拟解决基因编辑、药物生产中的科学问题，更能突破当前各种人为技术壁垒，在长远角度满足人民健康的重大需求。

Antigen receptor diversification is a hallmark of adaptive immunity which allows specificity of the receptor to particular antigen. B cell receptor (BCR) or its secreted form, antibody, is diversified through V(D)J recombination to assemble the variable region gene in bone marrow, somatic hypermutation (SHM) to allow affinity maturation and class switch recombination (CSR) to change the antibody effector function in periphery lymphoid tissues. In germinal centers, B cells compete for T helpers and the population with higher affinity BCR survives, which process is called affinity maturation. Antibody diversification cannot be fully recreated in ex vivo cultured cells. Thus, the production of highly potent antibody still replies on animal model immunization or human samples. We previously reported an antibody ex vivo affinity maturation method, by applying of diversifying base editor in an antibody display system. In the current proposal, we aim to solve the bottleneck problems in setting up the ex vivo system. In one way, we plan to expand the mutation patterns to transversion mutation, A/T mutation, indels etc. In the other, we design synthetic approach to recapture the germinal center reaction ex vivo with different modules, allowing the automatic selection of highly potent antibody based on its affinity and agonist/antagonist. Eventually, we hope to setup a next generation antibody production system, which could produce better and cheaper antibody-based molecules.