Neutralization Breadth and Potency of Single-Chain Variable Fragments Derived from Broadly Neutralizing Antibodies Targeting Multiple Epitopes on the HIV-1 Envelope

Abstract

Passive administration of HIV-directed broadly neutralizing antibodies (bNAbs) can prevent infection in animal models, and human efficacy trials are under way. Single-chain variable fragments (scFv), comprised of only the variable regions of antibody heavy and light chains, are smaller molecules that may offer advantages over full-length IgG. We designed and expressed scFv of HIV bNAbs prioritized for clinical testing that target the V2-apex (CAP256-VRC26.25), V3-glycan supersite (PGT121), CD4 binding site (3BNC117), and MPER (10E8v4). The use of either a 15- or 18-amino-acid glycine-serine linker between the heavy- and light-chain fragments provided adequate levels of scFv expression. When tested against a 45-multisubtype virus panel, all four scFv retained good neutralizing activity, although there was variable loss of function compared to the parental IgG antibodies. For CAP256-VRC26.25, there was a significant 138-fold loss of potency that was in part related to differential interaction with charged amino acids at positions 169 and 170 in the V2 epitope. Potency was reduced for the 3BNC117 (13-fold) and PGT121 (4-fold) scFv among viruses lacking the N276 and N332 glycans, respectively, and in viruses with a longer V1 loop for PGT121. This suggested that scFv interacted with their epitopes in subtly different ways, with variation at key residues affecting scFv neutralization more than the matched IgGs. Remarkably, the scFv of 10E8v4 maintained breadth of 100% with only a minor reduction in potency. Overall, scFv of clinically relevant bNAbs had significant neutralizing activity, indicating that they are suitable for passive immunization to prevent HIV-1 infection. IMPORTANCE Monoclonal antibodies have been isolated against conserved epitopes on the HIV trimer and are being investigated for passive immunization. Some of the challenges associated with full-sized antibody proteins may be overcome by using single-chain variable fragments (scFv). These smaller forms of antibodies can be produced more efficiently, may show fewer off-target effects with increased tissue penetration, and are more adaptable to vectored-mediated expression than IgG. Here, we demonstrate that scFv of four HIV-directed bNAbs (CAP256-VRC26.25, PGT121, 3BNC117, and 10E8v4) had significant neutralizing activity against diverse global strains of HIV. Loss of potency and/or breadth was shown to be due to increased dependence of the scFv on key residues within the epitope. These smaller antibody molecules with functional activity in the therapeutic range may be suitable for further development as passive immunity for HIV prevention.

Development and characterization of specific anti‐Usutu virus chicken‐derived single chain variable fragment antibodies

“…Besides this optimization, a conversion of scFv to either Fab or IgG versions could also enhance their usefulness. It has previously been described that a conversion of IgGs to scFv resulted in a loss of neutralizing function 33 . Therefore, we hypothesize that a conversion from scFv to IgG might beneficially influence the neutralizing potency.…”

“…It has previously been described that a conversion of IgGs to scFv resulted in a loss of neutralizing function. 33 Therefore, we hypothesize that a conversion from scFv to IgG might beneficially influence the neutralizing potency. Analogously, a humanization of the scFv might change their overall behavior; it remains to be determined whether the scFvs will retain antigen specificity, avidity, and neutralizing activity upon conversion or humanization.…”

Combinations of Single Chain Variable Fragments From HIV Broadly Neutralizing Antibodies Demonstrate High Potency and Breadth

“…All scFv were expressed and purified by size exclusion columns and, with size and purity confirmed SDS-PAGE gels. scFv were stored in buffers containing 5% glycerol as determined previously to prevent aggregation (38). Combinations of scFv were tested against a panel of 17 pseudoviruses from subtypes A, B, and C (n=3, 4 and 10, respectively).…”

“…Similarly, only a few individual viral titers showed a potential loss of potency for the IC 50 (n=8, 4.7%) and IC 80 (n=11, 6.5%) or a potential gain of potency (IC 50 and IC 80 : n=5, 3%) for triple combinations compared to the model (Figures 4C, D). None of the outliers in dual or triple combinations could be linked to In order to assess their breadth-potency on a larger virus panel, we used the IC 50 and IC 80 data of single scFv to predict neutralization titers for combinations of two and three scFv, now including 8ANC195, on a 45-virus panel described in our previous study (38) (see Supplementary Figure 1 for single IC 50 in nM (A) and IC 80 in nM (B) or mg/mL (C, D) titers) (38). These theoretical scFv combinations were ranked based on their geometric mean potency using both IC 50 and IC 80 data for dual and triple combinations (Figures 5A, B, E, F).…”

Mutational networks of escape from transmitted HIV-1 infection

“…This pair was incident to the glycan commencing at N276. Loss of the N276 glycan can significantly reduce clearance by broadly neutralising antibodies [19], and so it is reasonable that its presence would differentiate founder from chronic sequences. The majority (117 of 143) of the features formed a connected network with most nodes being of low degree.…”