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 7

TanCARs heterodimerize HER2 and IL13Rα2 by engaging both target molecules simultaneously.

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TanCARs heterodimerize HER2 and IL13Rα2 by engaging both target molecule...
(A) TanCAR T cells were conjugated to U373-GBM cells for 10 minutes on Silane-coated glass slides and stained for HER2 (red) and IL13Rα2 (blue) on the tumor surface. Conjugated CAR and U373 complexes were then imaged by dual-channel STED super-resolution microscopy. Shown is the same cell with colocalized HER2 and IL13Rα2 detected by bright field (left), confocal (middle), and STED (right). A region of interest is enlarged to show greater resolution of the colocalized ligands (bottom row). (B) Pie chart distribution of single HER2 or IL13Rα2 aggregates or dual aggregates in TanCAR and biCAR T cell conjugates to U373 cells imaged using STED microscopy is shown within a subdiffractive range of light (<200 nm). Full width at half maximum (FWHM) values for HER2 and IL13Rα2 denote that the resolution using STED was 183 nm and 250 nm, respectively. FWHM analysis is shown in Supplemental Figure 2. (C) Fluorescent microscopy of Duolink PLA detecting T cell–GBM interactions. U373 tumor cells were coincubated with TanCAR (top row), biCAR (middle row), and NT (bottom row) T cells. Conjugates were mounted and fixed, and the PLA was performed to detect colocalization of HER2 and IL13Rα2 at the IS with a proximity of less than 40 nm. Images were captured with fluorescent confocal microscopy. Positive signal is indicated by red fluorescence. Scale bar: 3 μm. Inset scale bar: 2.0 μm. (D) The PLA MFI per IS was significantly higher in the TanCAR T cell–U373 interactions than in both the biCAR T cell–U373 and the NT T cell–U373 interactions, suggesting that the TanCAR is simultaneously docking with HER2 and IL13Rα2. Shown are representative data from 4 independent experiments done in triplicates with more than 30 synapses surveyed. Two-tailed t test was used. ****P < 0.00005.