Figure S5.

Experimental evaluation of selected RBD variants for antibody escape, related to Figure 4

(A) The 46 selected synthetic variants were individually cloned and expressed for yeast display and ACE2 binding by flow cytometry. 43 variants showed ACE2 binding or non-binding that matched machine learning predictions. The ACE2-binding status for two variants (38 and 42) could not be conclusively determined, while one variant (41) showed was incorrectly predicted by machine learning for ACE2 binding.

(B) RBD sequences at chosen EDs (ED₀, ED₃, ED₅, and ED₇) from the Wu-Hu-1 RBD were predicted for ACE2 binding and escape from four therapeutic monoclonal antibodies (mAbs). Accuracy for antibody escape predictions are the following: LY-CoV16 = 31/33 (93.94%), LY-CoV555 = 30/33 (90.91%), REGN10933 = 31/33 (93.94%), and REGN10987 = 32/33 (96.97%).

(C and D) Two double mutants, and their constituent mutations, which were predicted to display epistasis were assayed individually by yeast surface display.

(E and F) Three synthetic RBD variants of ED₃ from Wu-Hu-1 RBD that were predicted to escape all four therapeutic antibodies by the consensus machine learning model were expressed as individual clones in yeast and evaluated by flow cytometry for binding to antibody or ACE2.