Hinge length contributes to the phagocytic activity of HIV-specific IgG1 and IgG3 antibodies

Antibody functions such as neutralization require recognition of antigen by the Fab region, while effector functions are additionally mediated by interactions of the Fc region with soluble factors and cellular receptors. The efficacy of individual antibodies varies based on Fab domain characteristics, such as affinity for antigen and epitope-specificity, and on Fc domain characteristics that include isotype, subclass, and glycosylation profile. Here, a series of HIV-specific antibody subclass and hinge variants were constructed and tested to define those properties associated with differential effector function. In the context of the broadly neutralizing CD4 binding site-specific antibody VRC01 and the variable loop (V3) binding antibody 447-52D, hinge truncation and extension had a considerable impact on the magnitude of phagocytic activity of both IgG1 and IgG3 subclasses. The improvement in phagocytic potency of antibodies with extended hinges could not be attributed to changes in either intrinsic antigen or antibody receptor affinity. This effect was specific to phagocytosis and was generalizable to different phagocytes, at different effector cell to target ratios, for target particles of different size and composition, and occurred across a range of antibody concentrations. Antibody dependent cellular cytotoxicity and neutralization were generally independent of hinge length, and complement deposition displayed variable local optima. In vivo stability testing showed that IgG molecules with altered hinges can exhibit similar biodistribution and pharmacokinetic profiles as IgG1. Overall, these results suggest that when high phagocytic activity is desirable, therapeutic antibodies may benefit from being formatted as human IgG3 or engineered IgG1 forms with elongated hinges. Author summary Target binding and receptor binding domains of antibodies are separated by a hinge region that differs among IgG subclasses. Among these, IgG3 has a uniquely elongated hinge, whose role in an immunoglobulin's ability to facilitate different immune functions is incompletely understood. We evaluated the effect of varied hinge length and composition on diverse antibody activities and found that extended hinges enhance phagocytosis, a process through which the immune system can clear potentially harmful particles such as viruses. This enhancement was observed across diverse effector cell types, target particles, and for multiple HIV-specific antibodies. Antibodies with extended hinges showed in vivo persistence and localization in a mouse model similar to those with natural hinges. Consequently, immune responses or antibody therapies expected to benefit from a strong phagocytic response, such as viral sequestration, may be enhanced naturally by the IgG3 subclass or artificially by hinge extension. This work expands knowledge of the role that IgG3 plays in immunity and provides a framework for hinge engineering to improve antibodies.