Production of CD19-CAR-T cells and evaluation of the co-stimulatory domains response to IL-10

Abstract

Abstract Background: Chimeric antigen receptor cell therapy is a promising methods for treating various types of cancer. To optimize CAR T cell production in terms of cost and quality, the use of aAPC-expressing anti-CD3 to activate T cells is proposed. The purpose of engineering this cell is to investigate the evaluation of CAR T cells produced by this method in terms of proliferation, phenotype, and cytotoxicity. We produced two types of second-generation CD19-CAR T cells that have shown promise in treating some blood malignancies. Another study's goal is to investigate the effect of IL-10 on the functional activities of second-generation CAR T cells expressing anti-CD19, which are FDA-approved for treating lymphoma and resistant or relapsed B-ALL patients. Methods: To generate CD19-CAR T cells, after extracting PBMCs, they were stimulated with anti-CD3/CD28 antibodies and then transduced with CD19-CAR viruses. By producing the OKT3 virus, we used it to transduce K562 cells and produce aAPC cells. We co-cultured PBMCs and aAPC cells to assess the disappearance of aAPC cells, and their viability was monitored by flow cytometry. Then, the aAPC-derived CAR T cells were tested for proliferation, CD4/CD8 ratio, activation markers, exhaustion, and memory properties and compared to standard CAR T cell generation method. To investigate the antitumor activity, CAR T cells were cultured with tumor cells, and their potency was evaluated by flow cytometry and luciferase-based assays. The IFN-γ release assay of CAR T cells was determined by the ELISA method. Additionally, the effect of IL-10 on CD19-CAR T cells was studied by exposing them to tumor cells and measuring them by flow cytometry and luciferase-based assays. Result: The findings showed that aAPC led to the generation of CAR T cells with high proliferative capacity, especially CD8+ T cells. The aAPC method did not alter the phenotype or exhaustion marker expression of T cells or CAR T cells. Additionally, CAR T cells produced with aAPC exhibited increased expression of activation markers and secreted higher levels of IFN-γ, demonstrating enhanced antitumor potential. The study involved both first- and second-generation CD19-CAR T cells, confirming their effectiveness against tumor cells. Furthermore, IL-10 was investigated, and it was found that T cells showed a good proliferative response to IL-10, and the second-generation CAR T cells' antitumor activity increased when exposed to IL-10. Conclusion: In this study, the generated aAPC cell activated and proliferated T cells and CAR T cells, specifically CD8+ T cells. The antitumor activity of CAR T cells generated using this strategy has been proven. IL-10 also boosted the proliferation of activated T cells, and its improved antitumor activity on CAR T cells was validated.