Figure 3.

GEM identifies anti-MICA epitopes correlated with strong shedding inhibition activity. (A) Cartoon representation of the MICA*001 α3 domain crystal structure (PDB: 1HYR) shows (left) previously reported cleavage sites of MICA in yellow or (right) the location of the seven individual MICA*008 GEM variants in colored spheres with the exact substitutions indicated. (B) ELISA heat map of 59 anti-MICA antibodies and their ability to bind to the seven glycosylation-engineered MICA*008 variants. White boxes indicate binding and colored boxes represent a loss of binding of at least 60 %. Eight bins (bins A-H) are identified using the GEM method for the anti-MICA antibodies, and the number of anti-MICA antibodies per bin is indicated. Three bins (bins A, B and D) show ≥ 70% shedding inhibition of MICA*008, and the epitope bin by traditional antibody competition is also shown. (C) All glycosylation site mutations used in GEM are mapped onto the ‘front’ and ‘back’ surface of the MICA*008 α3 domain structure (from the complex with the 13A9 Fab in Figure 6B). GEM positions defining epitope bins A-H in (B) are mapped onto the surface of the structure and colored according to (A and B). Epitope bins containing antibodies demonstrating 70% or greater inhibition of shedding are highlighted with boxes.