BCMA and TACI expression in MM and design of APRIL-based CARs. (A) Bone marrow aspirates obtained from patients with MM were stained and gated for plasma cell markers and analyzed for expression of BCMA and TACI. The percent of BCMA⁺ and TACI⁺ cells per total plasma cells is depicted (median ± SD). Results are grouped according to the number of lines of therapy the patients had received (n = 29). (B) Level of expression of BCMA and TACI on human MM cell lines, RPMI8226 and MM.1S, and K562 cells transduced to express either BCMA or TACI. (C) Construct designs for BCMA, APRIL, and TriPRIL CARs. (D) Predicted tertiary structure of BCMA CAR, APRIL CAR, and TriPRIL CAR modeled using the Phyre2 platform and visualized in PyMOL. (E) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed on whole-cell lysates from Jurkat-CD3ζ KO cells expressing the indicated CARs under reducing and nonreducing conditions. For detection of the CARs western blots were probed with an anti-CD3ζ antibody. Under nonreducing conditions, CAR bands were detected at the estimated monomer and trimer sizes. (F) Schematic drawing illustrating the multimerization pattern of APRIL and TriPRIL CARs. t, truncated; TM, transmembrane domain.