Manufacturing variability of CAR-T cells in patients with B-cell lymphoma: Findings from the carthiae-1 clinical trial

Abstract Background: Chimeric Antigen Receptor T-cell (CAR-T) therapy has emerged as a promising treatment modality for patients with relapsed or refractory B-cell lymphoma. However, intrinsic variability among donor-derived samples may significantly influence both the yield and quality of the final cellular product. Aim: To evaluate the variability in the CAR-T cell manufacturing process within the context of the CARTHIAE-1 clinical trial. Methods: Mononuclear peripheral blood cells from apheresis pawn product from seven patients diagnosed with B-cell lymphoma and enrolled in the CARTHIAE-1 trial were processed at the Cell Processing Center of the Hospital Israelita Albert Einstein under Good Manufacturing Practice (GMP) conditions. Results: The cohort included three male and four female patients, with a median age of 42 ± 14 years. All patients presented with bulky disease, with the largest lesion measuring 10 × 7.2 cm. Three patients had no prior corticosteroid exposure, while the remaining four underwent a washout period prior to apheresis. Marked variability was observed across manufacturing runs, particularly in total nucleated cell counts and cell viability. On days 5/6 (D5/D6), the high-performance group yielded 918.33×10⁶ ± 270.31×10⁶ cells, in contrast to 55.93×10⁶ ± 23.69×10⁶ in the low-performance group. By day 12 (D12), following harvesting, the high-performance group achieved 6,056×10⁶ ± 261.32×10⁶ cells, compared to 1,042.93×10⁶ ± 888.69×10⁶ in the low-performance group. Cell viability on D5/D6 was significantly higher in the high-performance group (93.42% ± 4.26%) than in the low-performance group (62.52% ± 13.62%). Apheresis products from the low-performance group exhibited a greater proportion of CD8+ T cells (68.09% ± 10.98%) compared to the high-performance group (37.83% ± 5.37%). After CD4/CD8 enrichment (D0), the CD4+/CD8+ ratio was normalized; nonetheless, the final product exhibited CD4+ T cell predominance in both groups. No adverse events were observed during viral transduction. The percentage of CD3+ CAR+ cells in the final product was similar between groups (58.99% ± 4.80% vs. 55.68% ± 8.74%); however, the CD4+/CD8+ CAR+ ratio was significantly higher in the low-performance group (7.71 ± 3.55) compared to the high-performance group (2.10 ± 1.30). Gene expression analysis included TP53 and BCL-2 (apoptosis), MYC (cell proliferation and survival), CDKN1A (cell cycle regulation), and TIMP1. Elevated TP53 expression was observed in the low-performance group, while expression of CDKN1A, MYC, and TIMP1 was reduced in this same group. During clinical follow-up, circulating CAR-T cells were quantified in five of the seven patients, with peak levels detected between days 10 and 14 post-infusion. At peak expansion, a decrease in CDKN1A expression was observed in the high-performance group, while the low-performance group showed reduced TP53 expression. Conclusion: Findings from the CARTHIAE-1 study indicate that intrinsic donor variability significantly impacts the efficiency of CAR-T cell manufacturing in patients with B-cell lymphoma. Samples characterized by a higher initial proportion of CD8+ T cells and reduced expression of genes associated with proliferation and cellular senescence (CDKN1A, MYC, and TIMP1) were correlated with lower expansion and viability. Conversely, elevated TP53 expression was associated with impaired cellular proliferation, suggesting a potential role for apoptotic regulation in manufacturing performance.