Abstract
BACKGROUND
Genetically modified T cells have enormous potential for the treatment of relapsed and refractory haematopoietic malignancies. CD19-positive B-cell malignancies including acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL) or B cell non-Hodgkin lymphomas (NHL) have been shown to be an excellent target for adoptive immunotherapy with T cells expressing CD19-specific chimeric antigen receptors (CARs). The increasing need for genetically modified T cells is hampered by the limited number of centres with the required infrastructure and expertise to produce this complex therapeutic product. Ex vivo modification of T cells requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, Miltenyi Biotec has developed the CliniMACS Prodigy platform and is automating complex cell manufacturing processes. These have now been adapted for lentiviral transduction of T cells and we show the feasibility and effectiveness of the device for adoptive immunotherapy using chimeric antigen receptors.
METHODS
A self-inactivating third generation lentiviral vector encoding a CAR specific for CD19 (CAR19) was used for automated T-cell transductions (TCT). Using closed single-use tubing sets (TS520), fresh or cryopreserved peripheral blood mononuclear cells from non-mobilised leukapheresis collected from healthy donors were loaded onto the CliniMACS Prodigy, washed and activated in TexMACS media with TransAct, a polymeric nanomatrix activation reagent agonist for CD3 and CD28. Cells were transduced 24-48h after activation and expanded in the CentriCult-Unit of the tubing set, allowing for stable culture conditions as well as automated feeding and media exchange. Small and large scale comparison transductions were run in parallel to assess the efficiency of the automated T-cell modification. Finally, cells were harvested and cryopreserved to assess the functional capabilities of CAR19 T cells.
RESULTS
Three automated TCT runs were performed and continuously monitored to assess cell expansion, transduction efficiency and the phenotype of the final cell product. On average, expansion during automated cultivation was 11.7x (range: 5.4 - 22.8x) which was comparable to the expansion achieved in small scale controls (12.3x ± 1.2x). The average yield from the automated process was 11.8x108 total lymphocytes/run (ranging between 4 - 23.2x108 lymphocytes/run). Notably, this was comparable to existing CAR19 T cell manufacturing processes using a WAVE-Bioreactor. In all three runs in the Prodigy, successful transduction was observed with an average transduction efficiency of 32% CAR19-positive cells (range: 22- 45%). Again, this was similar to transduction efficiencies (32% CAR19-positive; range: 27-40%) in previous WAVE-production campaigns using X-Vivo15 media and magnetic beads conjugated with anti-CD3/CD28 antibodies for T-cell activation (Dynabeads). Flow cytometry analysis of the final cell product showed a high purity of CD45+/CD3+ cells (90%) as well as a relatively high frequency of CD8-positive cytotoxic T cells (56%). Immunophenotyping revealed high expression of CD45RA, CD62L, CD27 and CD95 with moderate expression of CCR7. Importantly, no significant difference in PD-1 expression was observed between automatically and manually processed cells. Finally, functional analysis showed cytotoxic activity as well as IFN-γ/TNF-α production upon co-cultivation with CD19-expressing target cells.
CONCLUSION
In summary, we have demonstrated the feasibility of the CliniMACS Prodigy 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 very little '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.
Disclosures
Mock*: Miltenyi Biotec GmbH: Research Funding. Nickolay*:Miltenyi Biotec GmbH: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Johnston:Miltenyi Biotec GmbH: Employment. Kaiser:Miltenyi Biotec GmbH: Employment. Pule:CELLECTIS: Research Funding; AUTOLUS: Employment, Equity Ownership, Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties. Thrasher:Miltenyi Biotec GmbH: Research Funding; Autolus Ltd: Consultancy, Equity Ownership, Research Funding. Qasim:Cellectis: Research Funding; Miltenyi Biotec GmbH: Research Funding; Autolus Ltd: Consultancy, Equity Ownership, Research Funding; Cell Medica: Research Funding.
Adoptive immunotherapy with genetically engineered T cells: modification of the IgG1 Fc ‘spacer’ domain in the extracellular moiety of chimeric antigen receptors avoids ‘off-target’ activation and unintended initiation of an innate immune response