Year: 2009
Type: article
Abstract: Recurrence of disease remains the largest cause of mortality for high-risk leukemia and lymphoma patients receiving stem cell transplant. Generation of T lymphocytes expressing a chimeric antigen receptor (CAR) targeting CD19 represents an attractive strategy to maintain remission after conventional treatment. However, the clinical benefit of this strategy is limited by poor expansion of T cells within the tumor microenvironment. To improve the expansion/survival of CAR-modified T lymphocytes, we generated a new vector encoding 3 molecules: CAR.19 incorporating the co-stimulatory CD28 endodomain, IL15 and an inducible suicide gene (iCaspase9) to ensure the safety of modified T cells. We compared the proliferative capacity, cytotoxic activity and in vivo anti-tumor effects of T lymphocytes expressing either CAR.19-28z alone or CAR.19-28z, IL15 and the suicide gene. T lymphocytes were activated with OKT3/CD28 antibodies and then transduced with retroviral supernatants. Phenotypic analysis showed 70±10% and 75±5% transduction efficiency for iCasp9/CAR19-28z/IL15 and CAR19-28z T cells, respectively. Only the iCasp9/CAR19-28z /IL15 T cells produced IL15 (>100pg/mL) after stimulation with CD19+ tumor cells. In co-culture experiments, T cells expressing CAR.19-28z alone or iCasp9/CAR19-28z/IL15 both completely eliminated CD19+ tumor cells within 72 hours. However, labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that the proliferation of the iCasp9/CAR19-28z/IL15+ T cells in response to CD19+ tumor cells was greater than that of CAR.19-28z control+ T cells. Finally, the activation of the suicide gene iCasp9 with a small-molecule dimerizer (CID 50ng/mL) rapidly induced >90% apoptosis of T cells expressing iCasp9/CAR19-28z/IL15. Hence all three transgenes were functional. To assess the antitumor effects of the modified cells in vivo, we used a xenograft SCID mouse model and an in vivo bioluminescence system. CD19+ Daudi cells (1 × 106) expressing firefly Luciferase (FL) were injected i.p., and on day 4, mice received i.p iCasp9/CAR19-28z/IL15+ or CAR19-28z+ or control T cells (10 × 106). By day 30 the tumor signal was significantly reduced in mice receiving iCasp9/CAR19-28z/IL15+ T cells (ROI < 7 × 107) compared to mice receiving CAR19-28z or control T cells (ROI > 2.0 × 109). In conclusion, our data indicate that T cells coexpressing CAR19-28z/IL15/iCasp9 can safely improve anti-leukemia/lymphoma effects.
Institution: Baylor College of Medicine
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $3,650
OpenAlex ID: W2164072884