Bacterial cytoplasmic display platform Retained Display (ReD) identifies stable human germline antibody frameworks

Abstract

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.

Exploration of broadly neutralizing antibody fragments produced in bacteria for the control of HIV

“…Scaffolded constructs involved the transplanting of bnAb CDRs onto a heavy-light chain scFv fusion (IGHV3-23 and IGLV3-1) previously shown to be soluble in E. coli cytoplasm. 26 108E and PGT121 were chosen as candidates for two reasons. 1) These bnAbs are well characterized as being broad and potent, respectively and 2) they are predicted to bind to their epitopes only through CDR residues.…”

“…A novel cytoplasmic display platform, termed "Retained Display" (ReD), was previously developed in order to select fully-human scFv that were both stable and soluble in the bacterial cytoplasm. 26 The human germline heavy chain gene IGHV3-23 formed the basis for all constructs in the screen, due to its intrinsic stability. 27,28 IGHV3-23 was then fused with numerous light chain families in order to produce scFv in combinations that remained soluble in bacterial cytoplasm.…”

“…27,28 IGHV3-23 was then fused with numerous light chain families in order to produce scFv in combinations that remained soluble in bacterial cytoplasm. 26 The most common useful pairing was a IGHV3-23 and IGLV3-1 fusion. Previous screens demonstrated that Figure 1.…”

“…scFvs based on a IGHV3-23 and IGLV3-1 fusion tolerated CDR3 diversification 26 , suggesting it may be a good candidate to act as a scaffold for the transplantation of CDR sequences of known bnAbs. We aimed to determine if the scFv made from the fusion of IGHV3-23 and IGLV3-1 could be used to produce an scFv with the CDRs of 2 well-characterized and potent anti-HIV bnAbs termed 10E8 29 and PGT121.…”

“…In this study, the IGHV3-23 and IGLV3-1 fusion scFv previously identified by ReD screening 26 underwent CDR transplantation in order to produce soluble scFvs with the HIV-binding capacity of the bnAbs PGT121 and 10E8. Ultimately, 4 different 10E8 scFv structures, soluble in bacterial cytoplasm, were generated: a simple scaffolded 10E8 scFv, a monospecific 10E8 diabody (10E8-10E8) and two bispecifics involving the scaffolded 10E8 scFv were also produced.…”