Combinational Targeting Offsets Antigen Escape and Enhances Effector Functions of Adoptively Transferred T Cells in Glioblastoma

Abstract

Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.

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

“…We analyzed the expression of HER2 and IL13Rα2 in recurrent tumors from all groups. As described above, tumors recurring after unispecific CAR treatment downregulated or eliminated target expression but maintained expression of the nontargeted antigen (20). Tumor recurrences after TanCAR T cell transfer were not only significantly smaller by bioluminescence imaging, they also had dim staining for both HER2 and IL13Rα2 ( Figure 9, D and E).…”

“…A mathematical model of the expression hierarchy of 3 validated glioma antigens (21,(25)(26)(27)(28), HER2, IL13Rα2, and EphA2, predicted enhanced odds of tumor elimination on targeting of any 2 of these 3 antigens (20). Specifically, while targeting HER2 or IL13Rα2 alone predicted a 60%-70% probability of near-complete tumor elimination, simultaneously targeting HER2 and IL13Rα2 was predicted to eliminate more than 90% in a cohort of 20 primary GBMs (20).…”

“…We previously reported on GBM's markedly heterogeneous antigenic landscape (20). A mathematical model of the expression hierarchy of 3 validated glioma antigens (21,(25)(26)(27)(28), HER2, IL13Rα2, and EphA2, predicted enhanced odds of tumor elimination on targeting of any 2 of these 3 antigens (20).…”

“…Explanations for this discrepancy include but are not limited to transient T cell persistence in vivo, modest T cell homing, and inadequate T cell activation and/or T cell inhibition at the tumor site (8,9). The limited spectrum of T cell specificity in the face of the heterogeneous and potentially dynamic antigen landscape is perhaps the biggest challenge for CAR T cell therapy for solid tumors (20)(21)(22)(23)(24).…”

Chimeric antigen receptor T cells: power tools to wipe out leukemia and lymphoma

“…To prevent the relapse of antigen-negative escape variants, the use of bispecific CAR T cells might be an option, either by engineering with two CARs, each of different specificity, or with one CAR with two specificities [21]. Alternatively, a mixture of two T cell populations, each expressing a CAR with a defined specificity, may be used [22].…”