mRNA-mediated delivery of anti-HIV-1 multispecific antibodies as a passive immunisation strategy

Abstract

HIV-1 broadly neutralising antibodies (bNAbs) show great promise at both reducing viraemia and preventing HIV-1 infection, however, their application may ultimately be limited due to high manufacturing costs and the requirement for combination-based therapies. This project describes the development of in vitro transcribed (IVT)-mRNA delivery of multispecific antibodies as a potential cost-effective, passive immunisation strategy for the prevention of HIV-1 infection. Previously described bispecific (Bi-scFv and Bi-NAb) and trispecific (Tri-NAb) antibodies combining VRC01/PGT121 and VRC01/PGT121/10E8 paratopes, respectively (>95% neutralisation coverage in vitro against a 208 pseudovirus panel), with geometric mean IC50 titres <0.4 μg/mL), were selected for development. Antibody-encoding DNA plasmid constructs, engineered with T7-IVT mRNA transcription compatibility, were generated. The bispecific antibodies comprise the variable regions connected with linker peptides, both with and without an Fc domain (Bi-scFv and Bi-NAb, respectively) as previously described. The Tri-NAb DNA constructs were designed to express the Tri-NAb from co-transfection (Co-T) with separate Bi-NAb and 10E8 encoding constructs as well as from a novel single open reading frame (sORF) encoding the entire multispecific antibody sequence in a single polycistronic construct. Parental (PGT121, VRC01, and 10E8) and multispecific antibodies were expressed from DNA constructs by transient transfection in mammalian 293F cells, purified and biochemically characterised. Multispecific antibody-encoding mRNA transcripts were IVT from linearised DNA, purified of dsRNA, enzymatically capped (5’ Cap1 structure), and transfected into 293F cells. The conformation, assembly, and functionality of the purified antibodies and mRNA- transfected cell culture supernatants were assessed in vitro against a panel of 17 HIV tier 2 pseudoviruses. Purified multispecific antibodies demonstrated improved neutralisation potency and coverage compared to the parental monoclonal antibodies, as expected. Encouragingly, unpurified mRNA-expressed multispecific antibodies matched the neutralisation coverage of purified antibodies (94%), with sufficient multispecific antibody titres to generate inhibitory dilution factors conferring 80% neutralisation (median ID80) >150: Bi-scFv (1 427), Bi-NAb (655), Tri-NAb Co-T (327), and Tri-NAb sORF (166). These data provide proof-of-concept of the utility of this HIV- 1 multispecific antibody delivery strategy for passive immunisation to prevent HIV-1 infection and provide the basis for advancement into preclinical development.