CAR-T Rapid Manufacturing: Implications on Product and Analytical Testing

Background & Aim Rapid CAR-T manufacturing processes have a lot of potential as part of the next generation of CAR-T products. A rapid CAR-T manufacturing process, shortened from 7-14 days to ≤ 4 days, simplifies the manufacturing process, decreases vein-to-vein time, and results in a product with greater proliferative potential and less differentiated T cells, potentially improving patient outcomes. The shortened manufacturing process includes activation and transduction but limits ex-vivo cell expansion. It is critical to understand the impact this process change may have on the product and the testing to release the product for patient infusion. Methodology To evaluate product and analytical differences with the shortened manufacturing process, healthy donor material was manufactured with the rapid CAR-T process. Briefly, Enriched T Cells (CD4/CD8 selected from apheresis) were activated, transduced with a lentiviral vector, and cryopreserved after a 3-, 4-, or 7-day process. Memory phenotype was evaluated by spectral flow cytometry. Transduction was evaluated at thaw and after extended culture by assessing %CAR+ T Cells by flow cytometry and VCN by ddPCR. CAR-T cells generated from patient starting material were also evaluated. Results Cells were successfully harvested on Day 3, 4, and 7. Post-thaw viability was ≥85% and expansion increased with extended culture from <2x to >10x. Cell phenotype was different between the 7-day and the rapid manufacturing process with %CD45RA+CCR7+ T cells (naïve-like) significantly higher at the 4-day (44%) compared to the 7-day harvest (28%). Transduction measurements of the 3-day and 4-day process were unstable when measured directly post-thaw. VCN decreased and stabilized from post-thaw over the short post-thaw culture period. %CAR+ similarly stabilized after the culture period. After establishment with healthy donors, material from 2 patients was evaluated. Similar trends were observed with a more naïve-like phenotype after the rapid process and stabilization of transduction measurements after post-thaw culture. Patient material, however, had up to 50% lower transduction rate than healthy donors. Conclusion We demonstrate rapid CAR-T manufacturing results in greater naïve T-cell population compared to the traditional 7-day process. Transduction measurements were unreliable at harvest and stabilized after a short culture period. This study highlights the importance of carefully considering analytical testing especially with the rapid CAR-T manufacturing process.