Hetero-hexamerization as a means to improve broadly neutralizing antibody activity against HIV-1

Background Broadly neutralizing antibodies (bnAbs) offer a promising approach for HIV-1 prevention and therapy. Development efforts have primarily focused on maximizing neutralization breadth and potency, with clinical trials now selecting bnAb combinations to maximize HIV envelope coverage and limit viral escape. Antibody Fc domain engineering provides an additional opportunity to improve antibody potency by enhancing Fc-mediated effector functions such as lysis mediated by activation of the classical complement cascade, which may also be enhanced in the context of antibody combinations. Methods To engineer monoclonal antibodies (mAbs) with enhanced complement activity, the E430G (EG) mutation, which promotes Fc hexamerization, resulting in enhanced engagement of the six heads of the cascade initiator C1q, was incorporated into a panel of anti-HIV-1 mAbs. The panel of modified and unmodified antibodies were tested for antigen binding and neutralization, Fc receptor binding and effector function, and complement cascade initiation and viral lysis activity in vitro. Results We demonstrate that the EG mutation selectively enhances complement activity while preserving neutralization, antibody-dependent cellular cytotoxicity and phagocytosis. Moreover, combinations of EG-modified mAb combinations further boosted complement activation relative to individual antibodies or equivalent mixtures of unmodified mAbs, consistent with formation of hetero-hexamers. Conclusions These findings show that Fc engineering and antibody combinations can synergize to improve antiviral bnAb breadth and potency, suggesting a potential path toward more effective mAb-based strategies to prevent or treat HIV-1 infection.