Fig. 2: Characterization of CAR products throughout the manufacturing process reveals compositional and phenotypic changes.
a, CD19-22-CD8.BB.z-CAR contained the CD19 FMC63 and CD22 M971 scFvs, CD8α hinge and transmembrane domains, a 4-1BB costimulatory domain and a CD3ζ domain. The unique bispecific structure shows FMC63 heavy chain proximal, followed by M971 light chain, M971 heavy chain and FMC63 light chain distal. b, CAR T manufacturing and clinical trial schema. The manufacturing schema shows the TransAct process change from old to new matrix. The clinical trial schema shows the screening, lymphodepletion, CAR T cell infusion and disease evaluation time points. LP, lumbar puncture. c, Improved culture expansion resulting from the new matrix manufacturing process compared to the old matrix (P < 0.0001, two-tailed t-test). d, Significant reduction in doubling time with the new matrix process compared to the old matrix (P = 0.0411, two-tailed t-test). e, No significant difference in transduction efficiency between old and new matrix (P = not significant (NS), two-tailed t-test). Overall average transduction efficiency was 60.1% (n = 39 individual products). f, Composition of apheresis, CD4/8-enriched and CD19-22.BB.z product over time, looking at T cell (CD3+CD56−), B cell or leukemic cell (CD20+), CD4+, CD8+, NKT-like (CD3+CD56+CD16+), NK, monocyte and neutrophil subsets. g, Phenotyping of CAR T cell product reveals a skewing toward CD4+ cells (n = 39 individual products). h, Comparing the fold increase from apheresis to enrichment to final product reveals the skewing toward CD4+ cells that occurred during culture between enrichment and final product (P < 0.0001, two-tailed t-test). i, Phenotyping of T cell memory subsets revealed an enrichment in TSCM (P < 0.0001, two-tailed t-test) and TCM (P < 0.0001, two-tailed t-test) cell subsets and a depletion of the TEMRA (P < 0.0001, two-tailed t-test) subset. There was no significant change in TN or TEM cell subsets between enrichment and CD19-22.BB.z product.
