Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape
Meenakshi Hegde, … , Jordan S. Orange, Nabil Ahmed
Meenakshi Hegde, … , Jordan S. Orange, Nabil Ahmed
Published July 18, 2016
Citation Information: J Clin Invest. 2016;126(8):3036-3052. https://doi.org/10.1172/JCI83416.
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Research Article Oncology

Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape

Abstract

In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.

Authors

Meenakshi Hegde, Malini Mukherjee, Zakaria Grada, Antonella Pignata, Daniel Landi, Shoba A. Navai, Amanda Wakefield, Kristen Fousek, Kevin Bielamowicz, Kevin K.H. Chow, Vita S. Brawley, Tiara T. Byrd, Simone Krebs, Stephen Gottschalk, Winfried S. Wels, Matthew L. Baker, Gianpietro Dotti, Maksim Mamonkin, Malcolm K. Brenner, Jordan S. Orange, Nabil Ahmed

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Figure 6

TanCARs recruit HER2 and IL13Rα2 to form bivalent cytolytic IS.

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TanCARs recruit HER2 and IL13Rα2 to form bivalent cytolytic IS. (A) Thre...
(A) Three-dimensional confocal microscopy of fixed cells showing representative images of TanCAR T cells, IL13Rα2 CAR T cells, HER2 CAR T cells, and NT T cells, conjugated with GBM cells. After 30 minutes at 37°C on Silane-coated glass slides (Thermo Fisher), conjugates were fixed and stained with anti-IL13Rα2 (blue) and anti-HER2 (red) antibodies. (B) The IS was reconstituted in 3D, and its volume was quantified. (C) HER2 (red circles) and IL13Rα2 (blue squares) show dual clustering at the TanCAR synapse along with significantly higher clustering of IL13Rα2 compared with the single-CAR and NT T cells as well as compared with the normal ligand accumulation on the tumor cell surface. Cells were imaged in Z stacks on a Zeiss Axio-Observer Z1 equipped with a Yokogawa CSU10 spinning disc, Zeiss 63× 1.43 NA objective, and Hamamatsu Orca-AG camera. Images were acquired with Volocity software. Scale bar: 10 μm. Shown are representative data from 3 independent experiments done in triplicates. Two-tailed t test and single-step Tukey’s range test were performed. *P < 0.05, ***P < 0.0005. MFI × Volume indicates the mean fluorescence intensity (MFI) of all voxels included in the Z stacks of a reconstituted IS multiplied by the volume of the IS.