Automated Manufacturing of CD20.19 Bi-Specific Chimeric Antigen Receptor T (CAR-T) Cells at an Academic Center for a Phase I Clinical Trial in Relapsed, Refractory NHL

Adoptive cell therapy with autologous CAR-T cells has induced remarkable responses in patients with treatment-refractory hematologic malignancies, but limitations exist with off-site commercial CAR-T production: (1) manufacturing can take several weeks and requires shipping patient cells; (2) off-site manufacturing limits treatment options for patients with rapidly progressive disease; (3) high cost of the products may limit their availability. To address these challenges, we used the fully automated Miltenyi CliniMACS Prodigy GMP-compliant device to manufacture autologous CAR-T cells for a Phase I trial (NCT03019055) evaluating a first-in-human bi-specific CAR that targets CD19 and CD20 (CD20.19 CAR). Using reagents from Miltenyi Biotec, production was performed within the Medical College of Wisconsin (MCW) Cell Therapy Laboratory. Manufacturing time was 14 days, and production was as follows. First, peripheral blood mononuclear cells (MNC) collected from patients by apheresis were loaded onto the Prodigy, and CD4 and CD8 T cells enriched by positive immunomagnetic selection. The T cells were cultured in 200 U/mL IL-2, and TransACT reagent was added to stimulate the T cells in the Prodigy cell culture chamber. Next day, lentiviral vector expressing anti-CD19 and anti-CD20 (in tandem) with CD3z and 4-1BB stimulatory domains was added to the cells. Culture washes and feedings were done on days 5, 6, 8, 10 and 12 of manufacture, and final products harvested on day 14. Eligible patients either received fresh CAR-T cells, or cells that had been cryopreserved and administered on a later date. CAR-T cell products have been successfully generated for all 8 patients enrolled thus far on the Phase 1 clinical trial with no production failures, and all products passed release criteria for infusion. Three patients received cryopreserved product and 5 patients received fresh product. Average T cell recovery from the apheresis cell products was 66.0% (56.0-76.0%), and the enriched T cells were 94.3% CD3+ (87.8-97.4%) (Table 1). Protein L staining of the final cell products indicated 19.6% average CD20.19 CAR expression. Patient CAR-T cells were able to kill CD19+ and CD20+ target cells in vitro and produce IFNg upon antigen stimulation. An average yield of 5.27e+8 CAR T cells was obtained at harvest, exceeding the required cell dose for all patients. The CAR-T cells were comprised of both CD4 and CD8 T cells, with greater percentages of CD4 T cells. The majority of T cells (avg. 83.2%) had an effector-memory phenotype. In summary, we have successfully demonstrated feasibility for point-of-care CAR-T cell production for clinical use from patient apheresis products utilizing the CliniMACS Prodigy device. A major clinical advantage of CAR-T cells generated on-site is flexibility in treatment, with either immediate (i.e., fresh) or later (cryopreserved/thaw) infusion, dependent on patient need.