PMC9815724_01
Key manufacturing data from patient products manufactured using various expansion systems.. (A) Culture schematic with major processing steps. (B) TNC at harvest: CD30 CAR-T = 7 days culture, CD19/22 CAR-T = 7-9 days culture, E7 TCR = 21-24 days culture (only maximum dose level is shown). (C) CD30 CAR-T fold change = CD3+ TNC of Final (Day 7) / Day 1; CD19/CD22 bispecific CAR-T fold change = CD3+ TNC of Final (Day 7-9) / Day 0; E7 TCR fold change = CD3+ TNC of Final (Day 21-24) / Day 2 (only dose level 3 is shown). (D) Transduction efficiencies were measured via CAR-specific flow cytometry on the day of harvest. (E) - (G) Percent CD3 in the starting product compared to final product. PBMCs = peripheral blood mononuclear cells, TNC = total nucleated cells, TR=transduction, REP = rapid expansion phase.
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F1
The median total number of cells at harvest was 2.2x109 (range = 0.4-10.6x109, Figure 1B). Despite the variability of T cells in the initial PBMC population (11.4 - 63.7% CD3+ on Day 0), 24/25 products were >=80% CD3+ on Day 7 harvest, and 18/25 were >=95% CD3+ (Figure 1E). T cells were seeded on Day 0, followed by a 48-hour lentiviral transduction process on Day 1. The median TNC at harvest was 1.883x109 cells (Figure 1B), and with a median transduction efficiency of 68% at MOI = 40, this corresponded to 1.261x109 transduced T cells at harvest (Table 2). CD30 CAR-T process described above (Table 2 and Figure 1B, 2E, 2F). Certainly, the CD30 CAR-T process has increased heterogeneity in the starting product (using PBMCs instead of selected T cells), as well as potential variability from manual technician processing in bag cultures. However, the Prodigy process had a ceiling of approximately 2.2x109 cells at harvest on Day 7, whereas the bag culture could regularly yield over 3x109 cells over the same culture duration (9/25 cultures, Figure 1B). PBMCs enriched from an MNC apheresis via a COBE 2991 instrument (median %CD3 = 33.6%, Figure 1G). PBMCs were cultured for 48 hours in media supplemented with anti-CD3 (50 ng/mL) and IL-2 (300 IU/mL). For these 18 patients, the median TNC at harvest was 123.3x109 total T cells (Table 2, Figure 1B). The corresponding median fold-change at this highest dose level was 491-fold (Day 21-24 / Day 2, range = 135 - 2033-fold, Figure 1C).