Adoptive transfer of anti-CD19 CAR-engineered T cells have shown impressive and often durable clinical responses in patients with refractory and relapsed pre-B-cell acute lymphoblastic leukemia (ALL). However, clinical efficacy of anti-CD19 CAR-T cell therapy for lymphoma and chronic lymphocytic leukemia as well as CAR-T cell therapies that target solid tumors is not yet satisfactory. Optimal T cell activation and proliferation requires multiple signals including T cell receptor engagement (signal 1), costimulation (signal 2), and cytokine engagement (signal 3). To the best of our knowledge, CAR genes currently tested in the clinic contain a CD3z domain (signal 1) and costimulatory domain(s) (signal 2), but not a domain that can transmit signal 3. Although forced expression of cytokine genes in CAR-T cells can improve their persistence and antitumor effects, constitutive cytokine expression poses a risk for autonomous T cell growth causing cancers. In this study, we have developed a novel CAR design, 28-IL2RB-z (YXXQ), which can activate the JAK-STAT pathway in an antigen-dependent manner, recapitulating cytokine exposure upon TCR engagement. We incorporated a minimal cytoplasmic domain of the IL-2 receptor β (IL2RB) between CD28 and CD3z, which contains a box1 motif for the binding of JAK family protein tyrosine kinases and the tyrosine residue at position 510, an essential site for STAT5 association. To promote STAT3 recruitment, we introduced a YXXQ motif to the C-terminus end of CD3z (28-IL2RB-z (YXXQ)). T cells transduced with the novel CD19 CAR gene induced phosphorylation of both STAT3 and STAT5 proteins only upon CD19 encounter, and exhibited increased expression of JAK-STAT target genes. Moreover, upon stimulation with CD19+ target cells, these T cells, compared to 2nd and 3rd generation CAR-T cells, demonstrated superior proliferative capacity with increased cellular division and reduced activation-induced cell death. Interestingly, CD8+ T cells engineered with this novel CAR maintained a stem cell-like memory (TSCM) phenotype and polyfunctional cytokine secretion capacity even after repeated antigenic stimulation. Treatment of 28-IL2RB-z (YXXQ) CAR-T cells with a STAT3 inhibitor largely abrogated their proliferative advantage and decreased the frequency of TSCM cells, suggesting that STAT3 signaling played a predominant role in the maintenance of the TSCM phenotype. All the 2nd generation, 3rd generation, and our new generation CAR-T cells similarly demonstrated potent in vitro cytotoxicity and IFN-γ secretion upon initial exposure to CD19+ cells. However, our novel CAR-T cells were superior to the other CAR-T cells in both cytolytic activity and cytokine secretion following multiple antigen exposures. NSG mice were intravenously injected with the human CD19+ leukemia cell line, NALM6, and treated with adoptive transfer of each of the 2nd, 3rd, or new generation CAR-transduced T cells. The 28-IL2RB-z (YXXQ) new generation CAR-T cells demonstrated superior persistence compared to other generation CAR-T cells, which resulted in significantly better overall survival. Long-term persisting 28-IL2RB-z (YXXQ) CAR-T cells from treated mice secreted more IFN-γ when restimulated ex vivo, suggesting the maintenance of a functional and less-exhausted phenotype in vivo. Furthermore, the 28-IL2RB-z (YXXQ) CAR-T cells also demonstrated better in vivo persistence and therapeutic effects than the 28-z and 28-BB-z CAR-T cells in NSG mice inoculated with primary human CD19+ pre-B-ALL cells. Importantly, the 28-IL2RB-z (YXXQ) CD19 CAR-T cells did not exhibit any proliferative responses when adoptively transferred into leukemia-free mice, affirming that JAK-STAT signals are activated only when exposed to the antigen. Taken together, these results suggest that our new generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicities in the clinic. Clinical translation of this novel CAR is warranted.
This abstract is also being presented as Poster A73.
Citation Format: Yuki Kagoya, Shinya Tanaka, Marcus O. Butler, Mark Minden, Naoto Hirano. A novel chimeric antigen receptor containing JAK-STAT signaling domains mediates superior antitumor effects. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR09.