Monoclonal antibodies limiting viral spread upon infection are a promising approach to counteract the major health burden posed by seasonal viral endemics and world-wide pandemics e.g. Influenza A (IAV). Mechanistically, antibodies of the immunoglobulin G (IgG) class may have neutralizing activity by preventing target cell binding via their Fab fragment. Fc mediated effector functions have, in addition, increasingly been recognized to contribute to protection against Influenza A virus and other viral pathogens. The complement cascade and Fc receptors expressed on immune cells can thus enhance inactivation and clearance of opsonized viral particles by phagocytosis or antibody-dependent cellular cytotoxicity (ADCC). This was confirmed for murine antibodies against IAV Hemagglutinin (HA) for which protection was associated with enhanced with Fc activity (Bournazos et al., 2020). Nevertheless, safety concerns have arisen related to Fc-mediated antibody dependent enhancement of infection or antibody-dependent enhancement of disease. Experimental studies to this extent are so far limited or inconclusive. It remains to be seen whether altering Fc effector functions by specific changes in the IgG Fc would be beneficial regarding therapeutic activity particularly in the human immune system involving human IgG. Along those lines, we have a long-standing interest in deciphering IgG Fc effector functions. Comparing the differential capacity of human IgG to interact with Fc receptors, we identified a critical role for multivalency of immune complexes (Lux et al., 2013; Robinett et al., 2018, Tan & Lux et al. 2023), and observed strong Fc activity by IgG4 both in vitro and in vivo (Lux et al., 2013; Lux et al., 2014). Small animal models such as mice are routinely used to evaluate the protective capacity of antibodies. But despite pronounced similarity in IgG effector functions, FcγR expression is not identical in mouse and man limiting transferability of results to the human immune system (Lux et al., 2013). We have thus established a humanized mouse model in which a human immune system develops upon human hematopoietic stem cell transplantation. These mice are suitable to investigate human IgG activity in the human immune system in vivo (Lux et al., 2014; Schwab et al., 2015; Danzer et al., 2020). As they lack expression of murine FcγRs, human IgG activity can predominantly be attributed to human immune cells providing for a superior in vivo model with respect to analyzing Fc effector functions in terms of protection against Influenza A.
Within this project we therefore aim to decipher the contribution of IgG Fc domain dependent effector functions for the protective activity of Influenza A specific IgG and identify variants with enhanced clinical efficacy. We will generate IgG variants against Type 1 Influenza A Hemagglutinin of different subclasses, Fc mutations and epitope locations. The influence of Fc activity and epitope location will be evaluated in vitro including analysis of their neutralization capacity, binding kinetics, complement and FcR dependent immune cell activation. Selected Fc-effector functions important in Influenza pathogenesis will be studied in detail. We will ultimately assess the therapeutic potential of selected IgG candidates in an in vivo challenge assay employing humanized mice.
