Introduction: On of the most effective bio-therapeutics for HIV to date are the therapeutic monoclonal antibodies, particularly those that bind to antigen and recruit the immune effector functions simultaneously. However, antibody-based therapeutics are often only effective in a subset of patients and a significant effort is essential to fine-tuning the properties of antibodies to achieve high efficacy. Constant fragment (Fc) engineering is one of the most efficient strategies to modulate the interaction of monoclonal antibodies with humoral and cellular components of the immune system. Human Fc gamma receptors (FcγR) bind to antibody Fc region and mediates antibody effector functions such as antibody dependent phagocytosis (ADP) and antibody-dependent cell-mediated cytotoxicity (ADCC). FcγRIIa is found on macrophages, neutrophils and eosinophils and contributes in ADP whereas FcγRIIa is mainly present on NK cells and involves in ADCC. In this study we aimed to engineer the Fc region of an anti-HIV broadly neutralising antibody to optimise its binding to human FcγRs contributing in ADP and ADCC functions.
Methods: Bovine HIV broadly neutralising antibody NC-COW1 (Sok et al., 2017) was chimerised by cloning of variable heavy and light genes into the vectors expressing human heavy and light constant regions. The human IgG1 heavy constant region was mutated in an attempt to enhance the binding of the antibody to FcγRIIa and FcγRIIa. We made 4 different formats of NC-COW1 with the following mutations in Fc region: 1- No mutation; 2- S239D, I332E, A330L; 3- G236A, S239D, I332E; 4- G236A, S239D, I332E, A330L (Lazar et al., 2006; Richards et al., 2008). Format 2 and 3 were constructed to improve ADCC and ADP activity, respectively while format 4 had both enhancing mutations.
Results: Our results showed that chimeric NC-COW-1 without any optimising mutations had some interactions with FcγRIIa and FcγRIIa. However, among all different formats, the antibody with 4 mutations showed the highest binding to FcγRIIa and FcγRIIa. Further experiments will be performed to confirm the enhanced antibody effector functions in in vitro.
Conclusion: The enhancements in human Fc gamma receptor binding described here provide the potential to improve the performance of therapeutic antibodies targeting HIV-1 viruses.