Fig. 2: Murine scFv 11D5-3 versus fully human heavy-chain domain FHVH33.

a Flow cytometry results of T cells from the same donor transduced with 11D5-3-CD8BBZ or FHVH33-CD8BBZ or left untransduced are shown; cells were stained with BCMA-Fc-PE. b, d–h show mean ± s.e.m. 11D5-3 means 11D5-3-CD8BBZ. FHVH33 means FHVH33-CD8BBZ. All comparisons are two-tailed, paired t-tests. P < 0.05 was considered statistically significant. N.S. is not statistically significant. b Median fluorescence intensity of CD3⁺, BCMA-Fc-PE⁺ cells expressing 11D5-3-CD8BBZ or FHVH33-CD8BBZ is shown (n = 7, P = N.S.). c Relative affinity of 11D5-3-CD8BBZ versus FHVH33-CD8BBZ was determined by staining CAR-expressing T cells with decreasing concentrations of BCMA-Fc-PE and performing flow cytometry. Y-axis is percent maximum specific binding (% of Bmax). d Relative K_D values were determined by nonlinear regression from binding curves of 11D5-3-CD8BBZ-expressing T cells and FHVH33-CD8BBZ-expressing T cells. K_D values were calculated based on the concentration of BCMA-Fc-PE yielding half-maximal binding (n = 7; P = N.S). T cells were transduced with either 11D5-3-CD8BBZ or FHVH33-CD8BBZ and stimulated in culture for 4 h; degranulation of CD4⁺ and CD8⁺ T cells was assessed by CD107a expression. T cells were stimulated with either BCMA⁺ C17-BCMA⁻K562 cells (e and f, n = 4) or BCMA⁺ RPMI8226 cells (g and h, n = 5). CD4⁺ or CD8⁺ %CD107a⁺ events are from flow cytometry plots gated on live CD3⁺ cells. Background degranulation with BCMA-negative NGFR-K562 cell stimulation was subtracted from degranulation with BCMA⁺ cell stimulation. Results were normalized for CAR expression. i T cells expressing 11D5-3-CD8BBZ, FHVH33-CD8BBZ, or negative-control CAR SP6-CD828Z were tested in a 4-h cytotoxicity assay (one of similar experiments). Points represent mean cytotoxicity of replicate wells ±s.e.m.