Multiple mechanisms of CD22 antigen escape. (A) Protein domain plot of all the post-InO–acquired CD22 mutations (n = 10) identified in this cohort. (B) WT and predicted mutant CD22 protein structures. Group by mutation type. (C) Clinical flow cytometry plots of paired pre- and post-InO patient samples. CD22 status (WT or mutations) was labeled below each flow plot. The relative abundance of alternative splicing of CD22 e1 was depicted as stacking plots at the bottom. e1_e2 was the canonical splicing junction, and e1_e3, e1_e3alt, e1_e4, e1_e5, e1_e6, and e1_e7 were alternative splicing junctions. (D) Antibodies directed to different CD22 epitopes. InO and SHCL1 that bind the first Ig domain of CD22, and RFB4 that bind the third. (E-F) Functional characterization of CD22 p.R75T and p.C529Y. CD22 protein expression (by western blot) and binding (by flow cytometry) were performed using antibodies directed to different CD22 epitopes. All controls and CD22 mutants were run in the same experiment of western blot or flow cytometry. The results for CD22 p.R75T and CD22 p.C529Y were presented in seperate panels with the same controls (EV, CD22 WT). (G) Stack plots depicting the relative abundance of alternative splicing of CD22 e1 in pre-InO baseline samples, comparing responders (EFS >12 m and EFS <12 m) and nonresponders. (H) Stack plots depicting the relative abundance of alternative splicing of CD22 e1, comparing pre- and post-InO samples. EV, empty vector; WT, wild-type; m, month.