Texmacs media

CD4⁺ T cells were isolated using a positive selection kit (Miltenyi). Cells were subsequently stained with flow cytometry antibodies CD4 V500 (BD), CD25 APC Cy7 (BD) and CD127 FITC (BD) before FACS sorting using the BD ARIA. CD4⁺CD25hiCD127low Treg and CD4⁺CD25⁻CD127+ conventional T cells (Tconv) were collected. Purity of CD4⁺CD25⁺CD127lowFoxp3+ cells was routinely >70%. Tregs: activated 1:1 with human anti-CD3/28 Dynabeads (Gibco) in Texmacs media (Miltenyi) with 100Units/mL penicillin (Gibco); 100 μg/mL streptomycin (Gibco) without IL-2 for two days. On day two cells were supplemented with fresh media and1000u/ml IL-2 (Roche). IL-2 and fresh media were added every two days for 10 days. If indicated, at day 10 Treg were restimulated 1:1 with anti-CD3/28 Dynabeads and cultured as described above until day 20. Tconv: were activated for 48 h by culturing 1:1 with anti-CD3 and anti-CD28 Dynabeads (Gibco) and IL-2 (Roche) at 300u/ml. Media exchange was performed on alternate days and cells were used for experiments 7 days post-transduction.

Second generation CD19-targeting CAR construct consists of an extracellular antigen-binding domain (CD19-scFV), CD8 for hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ chain signaling domain followed by EGFRt as a tag. PBMC were collected from buffy coat from healthy donors. T cells were purified on LS columns (Miltenyi Biotec, # 130-042-401) using CD4 and CD8 microbeads (Miltenyi Biotec, #130-045-101 and #130-045-201), were activated with CD3/CD28 beads (T Cell TransAct; Miltenyi Biotec, #130-111-160) and incubated for 24 h. Then, activated T cells were transduced with the lentiviral vector (pCDH-EF1a-CD19 (FMC63)-2nd(4-1BB)-EGFRt; Creative biolabs) carrying the CAR construct. Activated T cells were cultured in TexMACS media (Miltenyi Biotec, #130-097-196) with hIL-7 (155U/mL, Miltenyi Biotec, # 130-095-362) and hIL-15 (290U/mL, Miltenyi Biotec, #130-095-762). CAR positivity was confirmed by expression of EGFRt in CD45⁺CD3⁺CD19⁻ population by flow cytometry. Expanded CART19 cells were frozen and stored in vials in liquid nitrogen before use.

T-cell activation and expansion was performed in a DASbox Parallel Mini Bioreactor System equipped with Eppendorf BioBLU 300 mL single-use vessels using a fed-batch process. Each vessel was initially filled with 110 mL TexMACS media (Miltenyi Biotech GMbH) supplemented with 5% human serum (SeraLab), then fitted with dissolved oxygen (DO), pH, temperature, and Raman probes, and allowed to equilibrate overnight with stirring at 80 RPM. For the inoculation of the cell culture, frozen T-cells were thawed, washed, and re-suspended at a concentration of 1 × 10⁶ cells/mL in 10 mL of TexMACS media supplemented with 5% human serum, 1:100 dilution of research grade T-Cell TransAct (Miltenyi Biotech GMbH) and 120 U/mL of IL-2 (Proleukin^®, Novartis). Cells were maintained in the bioreactors for 12 days with addition of TexMACS media supplemented with 5% human serum and IL-2 (120 IU/mL final concentration) on days 2 (90 mL) and 5 (50 mL). A combination of sodium bicarbonate and carbon dioxide was used to control the pH at 7.2 and an overlay of 5% CO₂/air was used to maintain dissolved oxygen at 90%. In each experiment, T-cells banked from four different donors were cultured in parallel in 4 Eppendorf BioBLU single-use vessels for 12 days. A total of three experiments with the same four T-cell banks were performed.

After informed consent was obtained from normal volunteers, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque PLUS. T cells were transfected with an Easy-T kit from GeneChem. Briefly, isolated T cells/PBMCs were activated on a plate precoated with S buffer (EASY-T cell infection activation kit, catalog no. LCR6018, GeneChem) at a concentration of 0.5 × 10⁶ cells/ml in complete TexMACS media (Miltenyi) supplemented with 5% human serum and 300 IU IL-2 (Mitenyi). Two days later, the stimulated T cells were washed and resuspended at 0.5 × 10⁶ cells/mL with Trans B buffer (EASY-T cell infection activation kit, catalog no. LCR6018, GeneChem). CAR-encoding lentivirus (GD2.BBζ CAR) was thawed and added into the cells (virus titer: 2 × 108TU/ml, MOI = 3). The cells were seeded onto plates that had been coated for 2 h with Trans A buffer (EASY-T cell infection activation kit, catalog no. LCR6018, GeneChem). Then, the transduced T cells were cultured at 37 °C and 5% CO₂ and expanded to maintain a cell concentration of 0.5–1 × 106 cells/ml.