Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays. CAR-T cells were 82-100% CD3 + with a mix of CD4 + and CD8 + cells that primarily expressed an effector-memory or central-memory phenotype. An average 30-fold expansion of 10 8 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. Vectors at multiplicity of infection 5-10 resulted in transduction averaging 37%. Processing time was 13 days.Įnrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3 + T-cell purity of 89 ± 10%.
We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing.
#Texmacs manual software#
The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients. This complex process requires skilled personnel and costly clean-room facilities and infrastructure.
Open processing steps that increase risk of contamination and production failure are required. Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion.
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