Automated Production of Clinical-Grade CMV-Specific T Cells to Implement Immunotherapy at the Bedside

Despite curative potential of hematopoietic stem-cell transplantation (HSCT) significant morbidity, mortality, and financial cost secondary to opportunistic infections remains. Immune reconstitution prevents and treats infections.  Select academic facilities and biomedical companies infuse donor-derived T cells selectively cultured on antigen, identified based on desired T-cell receptor expression, or selected for ability to generate gamma-interferon (g-IFN) . The “capture” of T cells secreting g-IFN in response to stimulation with antigen is rapid (T cells are available within hours of collection from donor) and independent of human leukocyte antigen. The limitation of capturing these desired T cells using a magnetic bi-specific reagent (that recognizes CD45 and g-IFN) is the cumbersome and labor-intensive procedure associated with using first-generation CliniMACS device from Miltenyi Biotec. We report the first experience using second generation CliniMACS Prodigy device to automate production of T  cells captured for their ability to generate g-IFN in response to cytomegalovirus (CMV). Eight steady-state apheresis products from CMV seropositive donors were processed. Following activation with PepTivator CMV pp65, T cells secreting g-IFN were isolated in a closed, hands-free system within 12 hours. Two donors failed to produce CMV-specific T cells due to low starting number of CD3⁺g-IFN⁺ T cells (<0.01%). There was an average of 0.07% g-IFN CD4⁺ T cells (range 0.03-0.61%) prior to capture and an average of 62.2% (range 14.7-91.4%) after enrichment.  The g-IFN CD8⁺ T cells prior to enrichment was 0.43% (range 0.07-0.56%) and 89.36% (range 61.6-94.2%) after capture. This yielded an average of 3x10⁵ CD3⁺g-IFN⁺ T cells. For a 70 kg adult and a 30 kg child this yields 4x10³ and 10⁴ cells/kg, respectively, of pathogen-specific T cells, which is consistent with the numbers needed to treat chemotherapy-refractory CMV in clinical trials using the first generation technology. We are implementing a pre-screening approach to identify CMV seropositive donors that may fail to collect and extending our evaluation of the Prodigy system to capture pathogen-specific T cells with specificity other than CMV. These data demonstrate automated production of CMV-specific T cells is feasible and will enable investigators to produce clinical-grade pathogen specific allogeneic T cells for add-back after HSCT at multiple points-of-care broadening the application of this promising immunotherapy.

Table 1.

	IFN-g+CD4+ T cells among viable CD4+  T lymphocytes (n=6)	IFN-g+CD8+ T cells among viable CD8+ T lymphocytes (n=6)
Pre-enrichment (%)	0.07 (0.03 – 0.61)	0.43 (0.07 – 0.56)
Post-enrichment (%)	62.20 (14.73 – 91.39)	89.36 (61.59 – 94.20)
Mean Yield / 109 starting TNCs	1.5x105	1.5x105