Tanja Kaartinen1, Annu Luostarinen2, Pilvi Maliniemi3, Joni Keto4, Mikko Arvas4, Heini Belt5, Jonna Koponen5, Petri I Mäkinen, Angelica Loskog6, Satu Mustjoki7, Kimmo Porkka8, Seppo Ylä-Herttuala9, Matti Korhonen2. 1. Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland. Electronic address: Tanja.Kaartinen@bloodservice.fi. 2. Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland. 3. Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland; Research & Development, Finnish Red Cross Blood Service, Helsinki, Finland. 4. Research & Development, Finnish Red Cross Blood Service, Helsinki, Finland. 5. Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland. 6. Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. 7. Hematology Research Unit Helsinki, Biomedicum Helsinki, Department of Medicine, Division of Hematology, University of Helsinki, Helsinki, Finland; Department of Clinical Chemistry and Hematology, University of Helsinki, Helsinki, Finland. 8. Hematology Research Unit Helsinki, Biomedicum Helsinki, Department of Medicine, Division of Hematology, University of Helsinki, Helsinki, Finland. 9. Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland; Heart Center, Kuopio University Hospital, Kuopio, Finland.
Abstract
BACKGROUND: Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. METHODS: Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. RESULTS: High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (TSCM, CD95+CD45RO-CD45RA+CD27+) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. DISCUSSION: The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing.
BACKGROUND: Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. METHODS: Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. RESULTS: High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (TSCM, CD95+CD45RO-CD45RA+CD27+) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. DISCUSSION: The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing.
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