Asokan M, et al., 89(24):12501-12, J Virol., 2015
The high antigenic diversity of human immunodeficiency virus type 1 (HIV-1) makes the development of a single-antibody that potently neutralizes nearly all strains of HIV-1 a major challenge. Therefore, the physical combination of two broadly neutralizing antibodies (bNAbs) could potentially broaden neutralization coverage against majority of viral strains. Described herein is the construction of four different bispecific immunoglobulins (IgGs) using the CrossMab format. Newly constructed bispecific IgGs were based on four parental HIV-1 bNAbs, one each to the CD4 binding site (CD4bs) , glycan-dependent site near the variable loop 3 (V3-glycan), a variable-region (V1V2) glycan-dependent site on the trimer apex (V1V2-glycan), and membrane-proximal external region (MPER) on the HIV-1 envelope glycoprotein (Env). Each bispecific IgG was designed with a different combination of two antigen binding fragments. Bio-Layer Interferometry (BLI) was used to confirm the bispecificity of the bispecific antibodies and to determine the binding affinities of such antibodies to various ligands. BLI experiments were performed on a Pall ForteBio Octet RED384 instrument. For bispecificity confirmation studies, Streptavidin (SA) Biosensor probes were immobilized with biotinylated RSC3, then immersed in the antibody, followed by probing with a ligand against the second arm of the antibody. For 10E8 x PG916RSH, Amine Reactive 2G (AR2G) Biosensor probes were captured with 1VH8 x CAP256SU and dipped in antibody followed by 3AGJ-GFP. For the determination of binding affinities, Anti-Human Fc (AHC) biosensors were immobilized with either the parental or bispecific antibodies and then immersed in a serial dilutions of corresponding ligand (RSC3 for VRC07, 3AGJ-MPER-GFP for 10E8, and BG505.SOSIP for PGT121). For assays involving PG9-16-containing antibodies, 1VH8_CAP256SU was captured onto AR2G Biosensor tips and then coupled with a serial dilution of PG9-16-Fab or MAbs. The study demonstrates that newly developed bispecific antibodies possess binding specificities of the two parental antibodies. A single bispecific antibody having the optimal combination of VRC07 x PG9-16 was found to neutralize more viruses (97%) than either parental bNAb with a high potency similar to those of the parental bNAbs. These findings suggest that bispecific antibodies are promising candidates for future development of HIV-1 therapeutics.