Clinimacs system

After informed consent, leukapheresis was obtained from healthy adult volunteers (HLA-A*02:01) with a COBE Spectra apheresis system. CD14⁺ monocytes were enriched by immunomagnetic separation using a GMP-compliant CliniMACS system (Miltenyi Biotec). Quantitative and qualitative analyses of the selected CD14⁺ fraction and flow through were performed by flow cytometry. From the enriched CD14⁺ fraction, 2 × 10⁸ cells were resuspended in 25 ml of serum-free CellGro DC medium (CellGenix) and seeded in a 100 ml bag (CellGenix). Cells were preconditioned with 25 ml of medium containing hGM-CSF and human IL-4 cytokines (50 ng ml⁻¹ each, CellGenix) for 8 h. Cells were transduced with 1 × 10⁹ infective particles (multiplicity of infection of 5) in 50 ml medium containing Protamine sulfate (5 μg ml⁻¹). The bag was incubated at 37 °C and 5% CO₂ for 16 h. Next day, cells were washed three times with CellGro medium. After washing, cell number and viability was determined. Transduced cells were cryopreserved in aliquots of 2 × 10⁶ cell ml⁻¹ per vial. Surplus, non-transduced monocytes were cryopreserved in aliquots of 2 × 10⁶ cell ml⁻¹ per vial and 50 × 10⁶ cell ml⁻¹ per vial and were used as controls for the characterization experiments. Sterility tests were performed with the ‘Bactec' system (BD Biosciences).

CD19-CAR T cells were generated using peripheral blood mononuclear cells (PBMC) obtained via apheresis from pediatric and young adult patients with relapsed/refractory B cell ALL, treated on our institutional CD19-CAR T cell clinical study (NCT03573700; Hines et al., 2021 (link)). The protocol was approved by the Food and Drug Administration and by the institutional review boards at St. Jude Children’s Research Hospital. CD19-CAR T cell products were generated within the St Jude Children’s Research Hospital GMP facility. Briefly, T cells are selected from the PBMC product using CD8 and CD4 electromagnetic bead reagents (CliniMACS system; Miltenyi Biotech). T cells are then activated using CD3/CD28 monoclonal antibodies, transduced with the clinical grade CD19-CAR lentiviral vector, and expanded using IL-7 and IL-15 for 7 – 10 days until desired dose is achieved. The final CAR T cell product the undergoes QA/QC testing prior to release for clinical use. Patients then receive lymphodepleting chemotherapy, followed by CD19-CAR T cell infusion at protocol defined dosing. Characteristics of the manufactured product and post-infusion expansion were performed as described by A. Talleur (unpublished data).

Healthy donor-derived human peripheral blood apheresis cells were purchased from Key Biologics. The cells were labeled with anti-CD4/CD8 microbeads, and CD4⁺/CD8⁺ T cells were purified by using the CliniMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4⁺ and CD8⁺ T cells from peripheral blood mononuclear cells were suspended in X-VIVO 15 media with 5% human AB serum (Valley Biomedicals, Winchester, VA, UA) and 10 ng/mL of recombinant human IL-7 and IL-15 (Miltenyi Biotec) (X-VIVO 15–5% HSA/IL-7/IL-15, hereafter). Then, cells were activated with T Cell TransAct (Miltenyi Biotec) according to the manufacturer’s protocol in six-well plates (Thermo Fisher Scientific) at 37°C with 5% CO₂ for 24 h. The activated T cells were washed and suspended in X-VIVO 15–5% HSA/IL-7/IL-15 followed by transduction with four different batches of anti-CD123 chimeric antigen receptor-expressing vectors at a MOI of 25 in a total volume of 1 mL. After 22 h of transduction, the cells were transferred to a G-Rex plate (Wilson Wolf, St. Paul, MN, USA) and incubated in 30 mL of X-VIVO 15–5% HSA/IL-7/IL-15. CD123-specific chimeric antigen receptor-expressing T cell expression, viability, and expansion were determined 5 days post transduction.