A Synthetic Platform for Antibody Junctional Diversification Beyond Natural Constraints

Abstract

Antibody junctional diversity in jawed vertebrates arose from RAG transposon domestication, generating CDR3-focused V(D)J recombination with lymphocyte specificity--a constrained system limiting engineered antibody diversity. To overcome these limitations, we developed ARESEC (Antibody Reprogram and Expression System in Engineered Cells), a synthetic platform that enables programmable V(D)J recombination in non-lymphoid HEK293T cells. Through engineered recombination signal sequences (RSS) and optimized RAG1/2 expression, ARESEC enables RSS-guided DNA recombination across all three complementarity-determining regions (CDR1/2/3). Co-expression of terminal deoxynucleotidyl transferase (TdT) enhanced junctional sequence diversity by 41.5-86.3% across 3 CDRs. The platform enables native IgG production by coupling mammalian surface display with FACS-based functional screening. Using clinical-grade antibodies (Nivolumab and Durvalumab) as high-affinity starting scaffolds, we achieved further efficient affinity maturation through focused CDR diversification (library complexity >103). From these diversification libraries, we identified optimized variants including the anti-PD-L1 Dur1 with 2.01-fold enhanced binding, demonstrating the systems capacity for rapid antibody optimization from minimal diversity sampling. Collectively, ARESEC establishes a synthetic paradigm that transcends natural V(D)J constraints, generating multi-CDR diversification in non-lymphoid cells to enable rapid discovery of affinity-matured antibodies, effectively bridging immune evolution with modern antibody engineering demands.