Ultrapotent Broadly Neutralizing Human-llama Bispecific Antibodies against HIV-1

Broadly neutralizing antibodies are proposed as therapeutic and prophylactic agents against HIV-1, but their potency and breadth are less than optimal. This study describes the immunization of a llama with the prefusion-stabilized HIV-1 envelope (Env) trimer, BG505 DS-SOSIP, and the identification and improvement of potent neutralizing nanobodies recognizing the CD4-binding site (CD4bs) of vulnerability. Two of the vaccine-elicited CD4bs-targeting nanobodies, G36 and R27, when engineered into a triple tandem format with llama IgG2a-hinge region and human IgG1-constant region (G36×3-IgG2a and R27×3-IgG2a), neutralized 96% of a multiclade 208-strain panel at geometric mean IC₈₀s of 0.314 and 0.033 µg mL^-1, respectively. Cryo-EM structures of these nanobodies in complex with Env trimer revealed the two nanobodies to neutralize HIV-1 by mimicking the recognition of the CD4 receptor. To enhance their neutralizing potency and breadth, nanobodies are linked to the light chain of the V2-apex-targeting broadly neutralizing antibody, CAP256V2LS. The resultant human-llama bispecific antibody CAP256L-R27×3LS exhibited ultrapotent neutralization and breadth exceeding other published HIV-1 broadly neutralizing antibodies, with pharmacokinetics determined in FcRn-Fc mice similar to the parent CAP256V2LS. Vaccine-elicited llama nanobodies, when combined with V2-apex broadly neutralizing antibodies, may therefore be able to fulfill anti-HIV-1 therapeutic and prophylactic clinical goals.