Contributions
Abstract: EP720
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
Use of Chimeric Antigen Receptor (CAR) T-cells have demonstrated excellent results in B-lymphoid malignancies. In myeloid malignancies, several other targets are investigated as the interleukin-1 receptor accessory protein (IL-1RAP). A limitation of CAR T-cells success is the persistence in patients over time without relapse. A central memory T-cells (TCM) phenotype seems to be the best candidate for improving CAR T-cells efficiency.
Aims
The aim of this work is characterizing our preclinical “GMP-like” IL-1RAP CAR T-cells production, transferable for the clinic, in comparison to our experimental production.
Methods
Two arms were compared: CD4+/CD8+ cells were sorted from peripheral blood mononucleated cells of healthy donors on a ClinMACS Prodigy® device. Sorted CD4+/CD8+ cells were then activated by CD3/CD28 + IL-7/IL-15 before transduction, at day(D)1, using a IL-1RAP CAR lentiviral supernatant. In Comparison, for the experimental conditions, CD4+/CD8+ cells were activated using CD3/CD28 Beads + IL-2 before transduction at day 2. Cells were cultured for 9 days either in flask or in GMP approved device. Transduction efficiency was measure 4 days after the transduction by ΔCD19 reported gene labeling using flow cytometry. T-cells subsets phenotyping was perform on the fresh or thawed CAR T-cells final product (D9) and compared with untransduced T-cells (D0). Cytotoxicity was assessed by CD107a staining or IFNγ quantification by ELISA, after coculture with leukemic cell lines and IL-1RAP-coated recombinant protein. CAR T-cells metabolism was also assessed by Seahorse® analysis after IL-1RAP stimulation.
Results
Cells proliferation was similar between the two procedures of IL-1RAP CAR T-cells. However, the transduction efficiency was higher in the preclinical (64.6%±15.6%) than in the experimental process (45.6% ±10.4%). The CD4/CD8 ratio was maintained between D0 and D9 in the preclinical process in comparison to the experimental process (4.0±0.9, 4.0±1.9, and 1.7±0.7 respectively). At D9, the CAR T-cells product was mostly composed of stem cell-like memory T-cells (TSCM) and TCM in the preclinical GMP-like process in comparison with the experimental procedure (48.8%±0.8% and 30.9%±2.6% vs 30.1%±19,1% and 18.5%±6.5% respectively).
Better functionality was observed in the GMP-like process compare to the experimental one characterize by a higher CD107a positive CAR T-cells percentage, after coculturing against MonoMac-6 IL-1RAP tumor cell lines (34.1% ±9.8% vs 3.4% ±2.0% respectively, p=0.006%) or in presence of a IL-1RAP coated recombinant protein (55.7% ±4.6% vs 1.1% ±0.4% respectively, p<0.0001). These results were confirmed by IFNγ secretion quantitation (3708 pg/mL ±2176 pg/ml and 2535 pg/mL ± 1352 pg/mL respectively). Moreover, IL-1RAP coated recombinant protein stimulation show a basal glycolytic activation of CAR T-cells (fold rate at 2.0 ±0.2, p<0.001), but without difference of maximal glycolytic activity, suggesting a specific activation without exhaustion. Oxidative metabolism was similar with and without activation.
Conclusion
We have demonstrated that our IL-1RAP CAR T-cells preclinical GMP-like process meets standard requirements of production in term of T-cells subpopulation phenotype for anti-leukemia immunologic memory and cytotoxic functionality. It is ready for transfer in clean room in order to produce for AML patients in first in man phase I clinical trial.
Keyword(s): CAR-T
Abstract: EP720
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
Use of Chimeric Antigen Receptor (CAR) T-cells have demonstrated excellent results in B-lymphoid malignancies. In myeloid malignancies, several other targets are investigated as the interleukin-1 receptor accessory protein (IL-1RAP). A limitation of CAR T-cells success is the persistence in patients over time without relapse. A central memory T-cells (TCM) phenotype seems to be the best candidate for improving CAR T-cells efficiency.
Aims
The aim of this work is characterizing our preclinical “GMP-like” IL-1RAP CAR T-cells production, transferable for the clinic, in comparison to our experimental production.
Methods
Two arms were compared: CD4+/CD8+ cells were sorted from peripheral blood mononucleated cells of healthy donors on a ClinMACS Prodigy® device. Sorted CD4+/CD8+ cells were then activated by CD3/CD28 + IL-7/IL-15 before transduction, at day(D)1, using a IL-1RAP CAR lentiviral supernatant. In Comparison, for the experimental conditions, CD4+/CD8+ cells were activated using CD3/CD28 Beads + IL-2 before transduction at day 2. Cells were cultured for 9 days either in flask or in GMP approved device. Transduction efficiency was measure 4 days after the transduction by ΔCD19 reported gene labeling using flow cytometry. T-cells subsets phenotyping was perform on the fresh or thawed CAR T-cells final product (D9) and compared with untransduced T-cells (D0). Cytotoxicity was assessed by CD107a staining or IFNγ quantification by ELISA, after coculture with leukemic cell lines and IL-1RAP-coated recombinant protein. CAR T-cells metabolism was also assessed by Seahorse® analysis after IL-1RAP stimulation.
Results
Cells proliferation was similar between the two procedures of IL-1RAP CAR T-cells. However, the transduction efficiency was higher in the preclinical (64.6%±15.6%) than in the experimental process (45.6% ±10.4%). The CD4/CD8 ratio was maintained between D0 and D9 in the preclinical process in comparison to the experimental process (4.0±0.9, 4.0±1.9, and 1.7±0.7 respectively). At D9, the CAR T-cells product was mostly composed of stem cell-like memory T-cells (TSCM) and TCM in the preclinical GMP-like process in comparison with the experimental procedure (48.8%±0.8% and 30.9%±2.6% vs 30.1%±19,1% and 18.5%±6.5% respectively).
Better functionality was observed in the GMP-like process compare to the experimental one characterize by a higher CD107a positive CAR T-cells percentage, after coculturing against MonoMac-6 IL-1RAP tumor cell lines (34.1% ±9.8% vs 3.4% ±2.0% respectively, p=0.006%) or in presence of a IL-1RAP coated recombinant protein (55.7% ±4.6% vs 1.1% ±0.4% respectively, p<0.0001). These results were confirmed by IFNγ secretion quantitation (3708 pg/mL ±2176 pg/ml and 2535 pg/mL ± 1352 pg/mL respectively). Moreover, IL-1RAP coated recombinant protein stimulation show a basal glycolytic activation of CAR T-cells (fold rate at 2.0 ±0.2, p<0.001), but without difference of maximal glycolytic activity, suggesting a specific activation without exhaustion. Oxidative metabolism was similar with and without activation.
Conclusion
We have demonstrated that our IL-1RAP CAR T-cells preclinical GMP-like process meets standard requirements of production in term of T-cells subpopulation phenotype for anti-leukemia immunologic memory and cytotoxic functionality. It is ready for transfer in clean room in order to produce for AML patients in first in man phase I clinical trial.
Keyword(s): CAR-T