Thursday, November 21, 2024
11/21/2024
SUMMARY Structural studies have established that naturally elicited antibodies can bind to HIV envelope (Env) over a wide range of epitopes and angles mediated by their Fab arm (i.e. “immune complex geometry”). How this affects antibody dependent cellular cytotoxicity (ADCC) activity, especially by Natural Killer (NK) cells is unknown. Our knowledge is limited by a poor understanding of how antibody immune complexes orchestrate antibody receptor based signaling (specifically for Fc gamma RIIIa, FcRIIIa). The long-term goal is to acquire a more detailed understanding of how antibodies recruit Fc mediated cellular activity and to develop novel strategies to engineer antibodies, drugs, and vaccines that can recruit specific effector functions with maximal potency in vivo. The objective of this proposal is to determine how immune complex geometry impacts NK cell ADCC against HIV and IgG receptor spatiotemporal dynamics within the NK cell immune synapse (NKIS). Our central hypothesis is that antibody geometry will modulate FcRIIIa interaction and ADCC activity in relation to immune complex geometry. The rationale for this work is that cutting-edge microscopy observations will provide new insight into ADCC function with immediate impacts on HIV antibody therapeutic design with applications to a broader range of human diseases. Our central hypothesis will be tested in three specific aims: 1) Determine how immune complex geometry influences FcgRIIIa interaction during ADCC; 2) Perform single molecule tracking of FcRIIIa within the NKIS during ADCC; 3) Determine nanometer-scale localization of FcRIIIa and signaling kinases within the NKIS during ADCC. We will pursue these aims using the innovative technique of MINFLUX nanoscopy, a super-resolution fluorescence microscopy technique that is capable of 1- to 3-nm spatial resolutions in both 2- and 3-dimensions as well as sub-millisecond tracking of single molecules in live cells. Carefully measured in vitro ADCC activity and Förster resonance energy transfer (FRET) measurements will also complement our MINFLUX observations and broaden the interpretation of our results. These studies are significant because they will establish a molecular basis for antibody effector function, especially in relation to NK cell ADCC, that could improve therapeutics for HIV. The techniques established in this proposal will also be useful for interrogating antibody ADCC function for other viral pathogens. The expected outcome of our studies is the characterization of biophysical principles that alter NK cell ADCC activity as well as the molecular mechanics that form the basis for such activity. These findings will have an important impact on human health by offering a rational basis for designing improved antibody therapeutics for HIV, as well as other viruses and diseases, and will increase our basic understanding of NK cell ADCC.
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