A Novel Tgf-Beta/Il-12r Signal Conversion Platform That Protects Car T Cells From Tgf-Beta-Mediated Immune Suppression And Concurrently Amplifies Effector Function

Numerous immune-suppressive mechanisms exist within the tumor microenvironment that may hinder chimeric antigen receptor (CAR) T cell efficacy. One such mechanism is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressive immune cells that directly inhibits effector T cell activity. Effector T cells express the TGF-β receptors TGFBR1 and TGFBR2, and exposure of T cells to TGF-β induces phosphorylation of the major TGF-β signal mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins (pSMADs) induce a suppressive transcriptional program that ultimately leads to reduced cytokine production, reduced cytotoxicity, and a failure to proliferate in response to antigen stimulation. A dominant negative receptor version (DNR) of TGFBR2 that does not contain signaling domains protects T cells from the impacts of TGF-β by blocking the ability of TGF-β to induce pSMADs. Here, we report the development of a novel TGF-β signal conversion platform that provides a T cell stimulatory signal upon exposure to TGF-β. This platform utilizes co-expression of chimeric variants of TGFBR2 and TGFBR1 where the TGF-β-binding domain of each receptor is fused to the transmembrane and intracellular signaling domains of the T cell simulating IL-12 receptors IL-12R-β2 and IL-12R-β1, respectively. Using a single lentiviral vector encoding both chimeric TGF-β receptors (CTBR) and a CAR, we demonstrated that CAR-CTBR T cells were completely protected from TGF-β-mediated SMAD phosphorylation. In addition, CAR-CTBR T cells generated significant amounts of pSTAT4 and pSTAT5 in response to TGF-β exposure, a response that mimics the T cell stimulation effects of IL-12. To further demonstrate successful signal conversion, we evaluated the impact of TGF-β exposure on the secretion of IFNγ, a major downstream target of IL-12 signaling. CAR-CTBR cells secreted significantly greater amounts of IFNγ than either control CAR T or CAR-DNR T cells following activation in the presence of TGF-β. Lastly, we utilized a serial restimulation assay to expand CAR, CAR-DNR, and CAR-CTBR T cells in the presence or absence of TGF-β. As expected, TGF-β exposure resulted in a significant inhibition of T cell proliferation in control CAR T cells. By contrast, both CAR-DNR and CAR-CTBR cells were protected from TGF-β-mediated inhibition of expansion. Gene expression analysis following 21 days of weekly antigen-driven expansion revealed specific TGF-β-mediated gene expression changes in CAR-CTBR cells consistent with increased T cell potency, including significant upregulation of IFNγ, IL10, IL18RAP, IL18R1, IL21R and CD62L transcripts. These data demonstrate the successful development of a TGF-β signal conversion platform that transforms the inhibitory effects of TGF-β exposure into an IL-12R-like T cell stimulatory signal that has the potential to produce superior CAR T cell responses in vivo. Note: This abstract was not presented at the meeting. Citation Format: Benjamin Boyerinas, Sara Miller, Ryan Murray, Stacie Seidel, Geoffrey Parsons, Kathy Seidl, Kevin Friedman, Richard Morgan. A novel TGF-β/IL-12R signal conversion platform that protects CAR T cells from TGF-β-mediated immune suppression and concurrently amplifies effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 602. doi:10.1158/1538-7445.AM2017-602