Background Chimeric antigen receptor modified (CAR-T) cell therapy has shown tremendous clinical efficacy for hematological malignancies,1 2 and limited efficacy in solid tumor therapy.3 4 However, bottlenecks in the ability to manufacture and deliver these engineered cell products, combined with their high cost, have limited patient accessibility of CAR T therapy, especially those in underserved areas or who are uninsured. Here, we describe the development of a flexible, cost-effective CAR T manufacturing process starting with fresh whole blood that generates sufficient CAR T cells numbers with excellent functionality.
Methods and Results For T cell Enrichment, a Sepax C-Pro (Cytiva, Inc.) was used to reduce 450ml fresh whole blood volume to 100ml. Concentrated cells were labeled for negative T cell enrichment (RosetteSep, Stemcell Research, Inc.), then fractionated on the Sepax C-Pro by density gradient centrifugation. Purified T cells were harvested, washed, and analyzed by flow cytometry. A cell product with >85% T cell purity, and 200x106 total T cells was routinely recovered. For large-scale T cell transduction with CAR LV expression vector, 1x 108 T cells were suspended in 100ml TexMACSTM medium (Miltenyi Biotec) with 200 IU/ml IL-2 and transferred to a G-Rex cell culture device (Wilson-Wolf), followed by addition of T cell TransActTM reagent and HIV gp120 DuoCAR lenti-vector5 (MOI 40). On Day 2, a complete medium exchange was carried out, cells were re-suspended in 400ml of fresh medium with 200 IU/ml IL-2, and returned to the same G-Rex. The cells were cultured in a tissue culture incubator to Day 8, when the final cell product was harvested. On days 3, 6 and 8, cultures were sampled to monitor T cell growth, phenotype, CAR expression, and function. Evaluating 4 healthy donors, at least 5x108 CAR-T cells could be harvested on Day 6, with ~90% cell viability. Day 6 CAR-T showed similar transduction efficiency and increased Tscm% and Tcm% compared to the standard Day 8 manufacturing time frame.5 6 Comparable target leukemia cell cytolysis was also observed.
Conclusions This large-scale CAR T production process can: 1) replace the complicated and expensive leukapheresis process using fresh whole blood for starting material; 2) provide the flexibility of earlier culture harvest. We thereby approach our goal to generate an economical process for CAR T production that can be administered to all who would benefit. Future work will include evaluation of this process with patient-derived material.
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