282. A scFv-Based CAR To Redirect T Cells To IL13Ra2-Positive Glioma

BACKGROUND: Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, and is virtually incurable with conventional therapies. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes. IL13Ra2 is aberrantly expressed in GBM therefore it is a promising target for CAR T-cell immunotherapy. The antigen recognition domain of CARs normally consists of a single chain variable fragment (scFv), however current IL13Ra2-specific CARs use IL13 muteins as an antigen recognition domain. We have previously shown that IL13 mutein-based CARs also recognize IL13Ra1, raising significant safety concerns. To overcome this obstacle we have recently generated a high affinity IL13Ra2-specific scFv. The goal of this project is now to develop a scFv-based IL13Ra2-specific CAR (IL13Ra2-CAR) and evaluate the effector function of IL13Ra2-CAR T cells.METHODS: We constructed a panel of IL13Ra2-CARs that contain our new IL13Ra2-specific scFv (m47) as an ectodomain, a short hinge (SH) or long hinge (LH), a CD28 transmembrane domain, and endodomains that contain signaling domains derived from CD3ζ and co-stimulatory molecules (CD28.ζ, CD137.ζ, CD28.CD137.ζ, CD28.CD134.ζ). IL13Ra2-CAR T cells were generated by retroviral transduction, and we determined their effector function in vitro, in co-culture and cytotoxicity assays, and in vivo, in the U373 brain xenograft model.RESULTS: Expression of all CARs in T cells was similar as judged by Western blot analysis. However, cell surface expression varied depending on used hinge and endodomain. In cytotoxicity assays, T cells expressing the different IL13Ra2-CARs only killed target cells expressing IL13Ra2, but not IL13Ra1 thus confirming the specificity of receptor-CAR interaction. While all IL13Ra2-CAR T cells secreted significant levels of IFNγ in co-culture assays with the IL13Ra2+ glioma cell line U373, only short hinge CAR T cells secreted significant amounts of IL2. T cells expressing IL13Ra2-CARs with a deleted endodomain (IL13Ra2.Δ-CAR) secreted no cytokines confirming that cytokine production depends on the presence of a functional IL 13Ra2-CAR. In vivo, injection of IL13Ra2.SH.CD28.ζ-CAR T cells into U373-bearing mice resulted in regression of gliomas as judged by bioluminescence imaging. IL13Ra2.LH.CD28.ζ- or IL13Ra2.Δ-CAR T cells had no antitumor effects.CONCLUSION: For the first time we have generated a CAR that only recognizes IL13Ra2 and not IL13Ra1. Comparison of several IL13Ra2-CARs revealed so far that a CAR with a SH and a CD28.ζ endodomain results in optimal T-cell activation as judged by IL2 production and in vivo anti-glioma activity. Our results warrant further active exploration of IL13Ra2-specific scFv-based CAR T cells for the adoptive immunotherapy of high grade glioma.