Conventional antibody surface display requires fusion protein export through at least one cellular membrane, constraining the yield and occasioning difficulties in achieving scaled production. To circumvent this limitation, we developed a novel cytoplasmic display platform, Retained Display (ReD), and used it to screen for human scFv frameworks that are highly soluble and stable in the bacterial cytoplasm. ReD, based on the retention of high-molecular weight complexes within detergent-permeabilized Escherichia coli, enabled presentation of exogenous targets to antibodies that were expressed and folded in the cytoplasm. All human λ and κ light chain family genes were expressed as IGHV3-23 fusions. Members of the λ subfamilies 1, 3 and 6 were soluble cytoplasmic partners of IGHV3-23. Contrary to previous in vivo screens for soluble reduced scFvs, the pairings identified by ReD were identical to the human germline sequences for the framework, CDR1 and CDR2 regions. Using the most soluble scFv scaffold identified, we demonstrated tolerance to CDR3 diversification and isolated a binding scFv to an exogenous protein target. This screening system has the potential to rapidly produce antibodies to target threats such as emerging infectious diseases and bioterror agents.

While broadly neutralizing antibodies (bnAbs) are a promising preventative and therapeutic tool for HIV infection, production is difficult and expensive. Production of antibody-like fragments in bacterial cytoplasm provides a cheaper alternative. This work explored the transplantation of the complementarity determining regions of the anti-HIV bnAbs PGT121 and 10E8 onto a single-chain variable fragment (scFv) scaffold, previously discovered through a novel screening platform. The scaffolded 10E8 scFv, but not the scaffolded PGT121 scFv, was soluble in bacterial cytoplasm, enabling efficient production in bacteria. Three additional multimeric constructs employing the scaffolded 10E8 scFv were also generated and soluble versions produced in bacteria. However, the constructs were found to have substantially lost anti-HIV binding function and had completely abrogated neutralizing activity. Overall, while this study provides a proof-of-concept for anti-HIV bnAb construct production in bacterial cytoplasm, future refinement of these technologies will be required to realize the goal of producing inexpensive and effective bnAb-like tools for the control of HIV.