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 ( A ntibody R eprogram and E xpression S ystem in E ngineered C ells), 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 >10 3 ). From these diversification libraries, we identified optimized variants including the anti‐PD‐L1 Dur1 with 2.01‐fold enhanced binding, demonstrating the system's capacity for rapid antibody optimization from minimal diversity sampling. Collectively, ARESEC establishes a synthetic platform 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.