IL-15 and -21 armored GPC3-specific CAR T cells for children with solid tumors - CAR T cells can induce remarkable antitumor responses in patients with hematologic cancers but have been largely ineffective in patients with solid tumors. Several barriers limit CAR T efficacy in solid malignancies including the limited number of safe solid tumor-specific antigens and an immunosuppressive tumor microenvironment (TME) that lacks supporting stimuli. Furthermore, it remains unknown how CAR T cells influence the evolution of immune escape mechanisms in the human TME. We developed and tested a set of chimeric antigen receptors (CARs) that 1) target glypican-3 (GPC3), a tumor antigen selectively expressed by several pediatric solid tumors but not non-malignant tissues, and 2) co-express interleukin-15 (IL15) and IL21, cytokines that are important for T cell survival, expansion, persistence, and retention of antitumor effector functionality. We found that T cells co-expressing a second generation GPC3-CAR and IL15 (15.GPC3-CAR) have significantly improved expansion, persistence, and antitumor activity compared to GPC3-CAR T cells, and that co-expression of both IL15 and IL21 (15.21.GPC3-CAR) further enhances these properties in preclinical models. Additionally, our preclinical studies showed that 15.21.GPC3-CAR T cells uniquely maintain expression of T cell factor-1, which protects CAR T cells from exhaustion and preserves proliferative capacity following repeated GPC3-mediated activation. Since the safety and efficacy parameters of these cytokine-expressing CAR T cells in humans are unknown, we propose to evaluate 15.GPC3-CAR and 15.21.GPC3-CAR T cells in sequential phase 1 studies using Bayesian Optimal Interval dose escalation in children with relapsed or refractory GPC3-positive solid tumors. Our multidisciplinary team has already generated promising safety and efficacy results treating children with T cells expressing the GPC3-CAR alone. We hypothesize that 15.GPC3-CAR and 15.21.GPC3-CAR T cells will be safe and that co-expression of IL15 with IL21 will enable GPC3-CAR T cells to overcome the inhibitory TME, resulting in robust expansion, persistence, and durable antitumor responses. In Aim 1, we will evaluate the safety of 15.GPC3-CAR and 15.21.GPC3-CAR T cells in children with GPC3-positive solid tumors. In Aim 2, we will determine the in vivo persistence and antitumor activity of CAR T cells. Finally, in Aim 3, through the evaluation of CAR T cell products, peripheral blood samples, and post-infusion tumor biopsies, we will define the transcriptomic and phenotypic profiles of GPC3-CAR T cells, cancer cells, and non- neoplastic stromal cells. We will identify survival programs in CAR T cells and define immune escape mechanisms in the TME using single-cell RNA sequencing, flow cytometry, and COdetection-by-inDEXing. Through the proposed research, we will determine the safety, in vivo expansion and persistence, and antitumor activity of our therapeutic cells. The results will identify survival programs in CAR T cells and immune escape mechanisms in the human TME to provide a blueprint for developing effective measures that boost the efficacy of CAR-based and other immunotherapies for patients with solid tumors.
