Clinical Validation of Automated Manufacture of Autologous CD19 CAR-T Cells for Treatment of B Neoplasia

Chimeric antigen receptor (CAR) T cells targeting the CD19 antigen effectively treat adults and children with B-cell hematologic malignancies. Centralized manufacturing may improve performance and accessibility for rapid expansion of this cellular therapy. Here we show the clinical validation of an anti-CD19 CAR (CAR19) comprised of the 4-1BB/CD137 co-stimulatory domain, and TNFRSF19-derived transmembrane region manufactured on the automated CliniMACS Prodigy. The vector and the protocol were provided by Miltenyi Biotec. CART19-HIAE cells were manufactured under current good manufacturing practices (cGMP) at Cellular Therapy Center in Hospital Albert Einstein, São Paulo, Brazil. Clinical apheresis pawn products were obtained from healthy donor undergoing stem transplantation at Hospital Albert Einstein, Sao Paulo, Brazil. Mononuclear peripheral blood cell from apheresis pawn products were processed within 24 h of receipt without cryopreservation. The study was approved by the institutional review board and all healthy donors gave written informed consent. The T cell composition at D0 was 22.14% ± 6.14% CD4 T cells and 23.55% ± 12.2% CD8 T cells. Each sample was loaded onto the CliniMACS Prodigy, and column purified using the TS520 Tubing set and CliniMACS CD4 and CD8 reagents (microbeads). After column purification, CD4 and CD8 T cells were enriched to 57.41% ± 0.05 and 20.55% ± 0.02, respectively. The CliniMACS Prodigy was loaded with 1x10⁸ CD3+ cells and the cells expanded with anti-CD3/CD28 co-stimulation and IL-7/IL-15 supplementation. After culture, we achieved an average 2.09 ± 0.69-fold expansion of CD3C cells (range 32.48% - 99.6%). To examine the transduction efficiency of manufactured CART products, flow cytometry was performed using a peptide that binds the CD19 CAR. The median transduction efficiency of CD3+ cells was 37.31 ± 3.68%, resulting in 1.36x10⁹ ± 0.26x10⁹ anti-19 CAR-T cells. Median transduction of CD4 CAR-T cells was 50.92% ± 4.54% compared to 45.35% ± 5.7% for CD8 CAR-T cells. Transduction efficiency and cell numbers were assessed on days 0, 6, and 12, and expression of CD3, CD4, CD8, and the CD19 CAR was performed by by flow cytometry. Non-T cells fraction after CD4/CD8 enrichment was below 30% (average 25.43% ± 2.89%) e drops below 16% in the final product (average 15.54% ± 7.75%), with the majority of NKT cells (average: 14.83% ± 3.19%) and some NK cells (average: 0.71% ± 0.38%). The multiplicity of infection of samples was approximately 20, leading to an average estimated vector copy number below 5 per CAR-T cell. Cells were harvested from the Prodigy® on day 12. The final product was submitted to quality control analysis including VSVG, GAG, flow cytometry, microbiological tests, endotoxin, karyotype, and mycoplasma. Samples were then diluted accordingly to the specified cohort and were cryopreserved stability control. Therefore, the data show results from three robust validations. The present study (CARTHIAE-1) was the first Phase I study with CAR-T cells approved by the National Health Surveillance Agency (ANVISA - Agência Nacional de Vigilância Sanitária) to treat patients from SUS (Unified Health System) and is scheduled to start in 2022.

No relevant conflicts of interest to declare.