Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape
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
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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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 3

Activity of GBM patients’ TanCAR T cells against autologous GBM and U373.

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Activity of GBM patients’ TanCAR T cells against autologous GBM and U373...
(A) Cytokine (IFN-γ and IL-2) production of TanCAR and nontransduced (NT) T cells generated from GBM patients detected in the supernatant 24 hours after coculture with autologous primary GBM cells. Shown are pooled data from 2 experiments done in triplicates. (B) Four-hour 51Cr-release assays of primary TanCAR T cells from UPN 1, 2, and 3 against autologous GBM cells compared with the unispecific CAR T cells, a pooled product of aliquots thereof (CARpool), T cells coexpressing both HER2 and IL13Rα2 CARs (biCAR), and NT T cells, all generated from the same patient. Healthy donor-derived T cells were tested against the U373-GBM line. biCAR T cell product for UPN 3 was not available. Shown are representative data from 3 independent experiments done in triplicates. Two-tailed t test was performed between TanCAR and the T cell product exhibiting the highest degree of killing. *P < 0.05. (C) Cytokine analysis (IFN-γ and IL-2) from supernatants of cocultures of primary TanCAR T cells, biCAR T cells, CARpool T cells, and unispecific CAR T cells with HER2 and IL13α2 expressing U373 GBM cells (25,000 T cells to 100,000 tumor cells), as detected by ELISA. Shown are pooled data from 3 independent experiments done in triplicates. A single-step Tukey’s range test was used. *P < 0.05, **P < 0.005.