Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>Background: Highly selective targeted T cell therapies using chimeric antigen receptors (CAR) are emerging as safe and effective modalities for the treatment of cancer. Downregulation and/or mutation of targeted antigens is a common tactic used by cancer cells to create antigen loss escape variants; culminating in relapse. Hence, targeting two or more antigens on tumor cells simultaneously could result in better therapeutic efficacy. However, developing two separate cellular products for clinical use as combination therapy is difficult, owing to regulatory hurdles and cost. In contrast, rendering an individual T cell bispecific could increase target cell selectivity, improve T cell activation and offset tumor escape due to antigen loss. This approach could particularly be applicable in heterogeneous tumors like HGG. We have characterized and quantified the heterogeneity of antigen expression in HGG tissues and primary cell lines using immunofluorescent staining as well as flow cytometry. While dual targeting was clearly superior to single targeting, targeting of three antigens (or more) did not predict a significantly superior advantage in our patient cohort (Hegde et al. Mol Therapy 2013).</jats:p>
<jats:p>Objective: The intent of this project is to develop an effective adoptive cell therapy for HGG using T cells genetically modified to express a bispecific CAR that incorporates two scFv antigen-recognition domains, in tandem (TanCAR), simultaneously targeting two glioma-restricted antigens, HER2 and IL-13Rα2.</jats:p>
<jats:p>Methods: A model for the bispecific TanCAR was constructed using ModWeb as described previously by us (Grada and Hegde et al., Mol Ther Nucleic Acids 2013). Extracellular-domain was assembled on Clone Manager®, then cloned into the Gateway® entry vector pDONR™221 and sequence-verified. This antigen recognition domain was then cloned inline with a signaling domain of the co-stimulatory molecule, CD28 and T cell receptor ζ-chain. We used a retroviral system to graft T cells with HER2 and IL-13Rα2-specific TanCAR and confirmed their surface expression using flow cytometry. Their functionality was tested against plate-bound HER2 and IL-13Rα2 proteins as well as against HGG cells in co-cultures and cytotoxicity assays. Cytokine release was studied using ELISA. HER2-specific, IL-13Rα2-specific CAR T cells and non transduced (NT) T cells from the same donor served as controls.</jats:p>
<jats:p>Results: The extracellular domain of the TanCAR includes an IL-13Rα2 scFv followed by a Gly-Ser linker, a HER2 specific scFv (FRP5) and another Gly-Ser tandem repeat hinge. The intracytoplasmic domain consists of a CD28/CD3ζ signaling-moiety. Fifty to 80% of T cells expressed HER2/IL-13Rα2 TanCAR on the cell surface. In Cr51 cytotoxicity assays, TanCAR-grafted bispecific T cells showed improved killing of IL-13Rα2 and HER2 positive HGG cell lines as well as autologous HGG cells over the control T cells from the same donor. In co-cultures with autologous HGG cells, HER2/IL-13Rα2 TanCAR T cells proliferated and released immunostimulatory cytokines. Though HER2/IL-13Rα2 TanCAR T cells were effectively activated and secreted IFN-γ and IL-2 on recognizing HER2 and IL-13Rα2 proteins individually, cytokine secretion was significantly higher on simultaneous exposure to both target antigens.</jats:p>
<jats:p>Conclusion: The pattern of heterogeneity in HGG favors near complete targeting of tumor subpopulations using bispecific T cell approach. Simultaneous targeting of two glioma-restricted antigens using TanCAR modified T cells could improve T cell activation and can potentially be used to offset antigen escape variants and reduce tumor recurrence.</jats:p>
<jats:p>Citation Format: Meenakshi Hegde, Amanda Corder, Zakaria Grada, Tiara T. Byrd, Vita S. Brawley, Helen E. Heslop, Stephen Gottschalk, Nabil Ahmed. Adoptive immunotherapy targeting tumor heterogeneity in high-grade glioma (HGG). [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B8.</jats:p>