Gordon Weng-Kit Cheung

Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

455. Automated Lentiviral Transduction of T Cells with CARS Using the CliniMACS Prodigy

T lymphocytes are exhibiting enormous potential in early phase clinical trials in patients with haematological malignancies. However, the complex procedures involved in the ex-vivo modification of T cells is labour intensive and currently limited to a small number of centres with the required infrastructure and expertise. To simplify procedures and widen applicability for clinical therapies, we have adopted the CliniMACS Prodigy platform to automate these multifaceted cell manufacturing processes. We found efficient lentiviral transduction of human T cells in a GMP compliant manner and demonstrate the feasibility of implementing this device in the manufacture of chimeric antigen receptor (CAR) based T cell immunotherapies. Eight automated T cell Transduction (TCT) processes have been performed using a self-inactivating third generation lentiviral vector encoding a CD19 specific CAR (CAR19), three of which using a clinical grade vector for final stage validation studies. Either fresh or cryopreserved peripheral blood mononuclear cells from non-mobilised leukapheresis from healthy donors were loaded onto the CliniMACS Prodigy using single use closed tubing sets. All cells were cultured in TexMACS media and activated with TransAct™. Transduction occurred 24-28hours post activation and cells were expanded for up to 8 days in the CentriCult-Unit enabling stable cell culture conditions and automated cell feeding. Finally, cells were harvested and cryopreserved to assess the functional capabilities of CAR19 T cells. Small scale comparison transductions were run in parallel to assess the efficiency of the automated T-cell modification process. The mean T cell expansion during automated cell cultivation was 16.2x (range 5.4-28.4x) with an average yield over 8 days of 14.5×108 total lymphocytes from a starting lymphocyte count of 1×108. This was comparable to cell expansion achieved in manual small scale experiments under the same activation conditions, 18.1x (range 11.5-27.5x). Successful transduction was also observed in the automated system with a mean transduction efficiency of 49.1% (range 23.9-64.9% CAR19+ T cells) which was again similar to transduction efficiencies achieved in manual small scale controls (mean of 51.8%). Flow cytometry analysis of the final product showed a high purity of CD45+CD3+ T cells (mean = 94%) with a relatively high frequency of CD8+ T cells (mean 48.9%). Further immunophenotyping revealed the bulk of the T cell product to be a mix of stem cell memory and central memory based on CD45RA, CD62L and CD95 expression with minimal expression of the T cell exhaustion marker PD-1. Additionally, CAR19 T cells generated using the automated procedure, were functional in cytotoxic activity both in vitro and in an in vivo mouse model. Importantly, these data are comparable to data generated from previous GMP manufacture of CAR19 T cells using the WAVE bioreactor with X-Vivo15 media and magnetic beads conjugated with anti-CD3/CD28 antibodies with the added advantage of increased simplicity in manufacture. In summary, we have demonstrated the feasibility of the CliniMACS Prodigy platform for the generation of CAR+ T cells for adoptive immunotherapy. Automated activation, transduction and expansion resulted in clinically relevant doses of CAR19 T cells with greatly reduced ‘hands-on’ operator time. Given the closed-system nature of the device, and automated features, the CliniMACS Prodigy should widen applicability of T-cell engineering beyond centres with highly specialised infrastructures.