In the present study, we evaluated the effects of IL-15 and IL-15-IL-15Ra complex on CAR-T therapy. Our results demonstrated that IL-15 armored CAR-T cells resulted in the highest percentage of Tscm cells and cell viability in cell culture condition. On the aspect of IL-15 combined with IL-15Ra, it had higher percentage of Tscm despite lower than IL-15. IL-15-IL-15Ra armored CAR-T cells performed lowest toxicity with less cytokine release (IFNγ) and CD132 expression. In the xenograft mouse model, IL-15 armored CAR-T cells inhibit tumor relapse totally but with severe toxicity that all mice died within 70 days, whereas IL-15-IL-15Ra armored CAR-T cells inhibit tumor relapse totally with lower toxicity that 40% mice lived for more than 90 days, demonstrating the inhibiting effect of IL-15Ra on toxicity induced by IL-15.
CD19 specific CAR-T therapy has demonstrated significant anti-tumor effects for B cell malignancies and some types of solid tumors 21. However, various studies showed that CAR-T products generated by different methods or from different laboratories exhibit inconsistent anti-tumor efficiency. Several factors limit the anti-tumor efficient of CAR-T cells, such as antibody affinity, off-target toxicity, impressive tumor microenvironment as well as terminal differentiation 22. CAR-T cell persistence is the crucial problem and considerable progression has been made to obtain cell products that result in enhanced expansion or persistence and anti-tumor response. Studies have shown that anti-tumor benefits for using IL-15 as well as IL-7 in culture 23 or IL-15 genetically engineered into T cells with CAR synchronously 24,25. Furthermore, high serum IL-15 in patients with DLBCL and B-ALL were associated with the better outcome after CD19 CAR-T therapy 26,27, demonstrating the combined adjuvant IL-15 improved the anti-tumor efficacy of CAR-T therapy. Indeed, in this study we developed the armored CAR for leukemia treatment. Our in vitro cell cultivation result indicated that this armored CAR-targeting CD19 increased the cell viability, inhibited apoptosis and maintained CAR-T cells with a less-differentiated phenotype, demonstrating the enhanced longevity of T cells 28,29.
Despite several clinical studies reported that elevated IL-15 expression correlates with improved patient survival, the administration of IL-15 has caused considerable toxicity including hypotension, fever, thrombocytopenia, and so on in cancer patients, which may prevent further the safety approval of IL-15 18,30. In line with these studies, using the NALM-6 tumor bearing mouse, we found IL-15 armored CD19-CAR-T cells inhibit tumor relapse totally but the toxicity was serious and all mice were die within 70 days, implying the severe adverse effect of IL-15. IL-15-IL-15Ra complex has been shown to significantly stimulate CD8+ T cells, especially memory CD8+ T cells, to enhance cytotoxicity against multiple tumors, such as myeloma 31, breast cancer 32, colorectal carcinoma 33, etc., and prolonged survival of tumor-bearing mice, established a long-term immune memory against tumor re-challenge 34,35. A fusion protein of IL-15-IL-15Ra and anti-FAP caused superior targeted anti-tumor killing ability, which provided rationale for the development of antibody-IL-15-IL-15Ra fusion proteins for cancer immunotherapy in the future 36. Thus, in the present study, after using IL-15Ra as a part of CAR cassette, we found overexpression of IL-15Ra together with IL-15 enhanced cell viability, reduced apoptotic cell and retain T cell differentiation with more Tscm compared with conventional CAR-T cells, and most importantly inhibit tumor relapse totally for tumor-bearing mice with lower toxicity compared with CD19-CAR-IL-15 T cells, providing a new choice for improving CAR construct.
It is demonstrated IL-15 cis presentation by IL-15Ra expressed on the CD8+ T cells was also able to enhance the proliferation and viability of these CD8+ T lymphocytes in vivo 37 and IL-15Ra significantly increased the stability of IL-15 in serum 38. In one study, peripheral T cells stably expressing second generation CD19-CAR and IL-15-IL-15 Ra retarded leukemia development and sustained resistance after tumor clearance with long-lived T-memory stem cells 24. Even so, it did not indicate the toxicity effect of IL-15. In our study, we discovered IL-15 combined with IL-15Ra reduced the adverse events significantly during CAR-T therapy and prolonged survival rate of tumor-bearing mice, which indicates a novel function of IL-15Ra. There are two reasons that may explain. Firstly, the CD19 specific CAR we used was the third generation of CARs having CD28 and 4-1BB into the CAR intracellular structure, which may influence the intracellular signal transduction 39. Secondly, unlike previous studies, we constructed CD19-specific armored CARs connecting CAR, IL-15 and IL-15Ra together. Therefore, the co-expression of CD19-CAR and IL-15-IL-15Ra fusion protein within one cells may influence the function of IL-15Ra. Anyway, the mechanism of reduced toxicity induced by IL-15Ra need to do further research.
CD132, as a common γ chain, is the subunit for the IL receptors including IL-2, IL-4, IL-7, IL-9 and IL-15. Because levels of these cytokines were shown to be increased in the serum of patients developing acute and chronic GVHD and inhibition of CD132 could have a profound effect on GVHD 20, the CD132 expression on the armored CAR-T cells was examined and the expression level of CD132 was lowest for CAR-T cells together with IL-15 and IL-15Ra compared with CD19-CAR-T and CD19-CAR-IL-15 T. In addition, studies showed that inflammatory bowel disease was associated with increased soluble CD132 40, and increased expression of IL-7Rα together with CD132 was positively related with psoriasis-like skin inflammation 41. Therefore, for the IL-15-IL-15Ra complex armored CAR-T cells, it can be predicted the low-toxicity towards patients after application.
Toxicities caused by CAR-T cells are varied and not fully understood, and the administration of immunosuppressive agents to decrease toxicity is an evolving practice 42,43. IL-15 stimulates the proliferation and activation of various immune cells, especially CD8+ T cells, leading to increased cytotoxicity and production of cytokines, but also amply the adverse effects of CAR-T cells 44. Herein, we found IL-15 combined IL-15Ra reduced the expression of CD132 compared with conventional CAR-T and IL-15 armored CAR-T cells, leading to the highest survival rate of tumor-bearing mouse, providing a novel application of IL-15Ra. Despite the observation, the precise relationship between IL-15Ra and CD132 is unclear.
In summary, sustaining the Tscm population during ex vivo expansion prior to adoptive T-cell therapy is challenging. Here, we demonstrate that the combination of IL-15 for CAR-T cell generation preserves Tscm phenotype and results in enhanced self-renewing capacity but with higher toxicity in vivo. We next successfully constructed the CD19 specific CAR combined with IL-15-IL-15Ra fusion protein and then examined its cell viability in vitro and immunotoxicity in xenograft mouse models, which provide a candidate tool for clinical leukemia treatment.