Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma

Abstract

Background: Glioblastoma (GBM) is the most common primary malignant brain cancer, and is currently incurable. Chimeric antigen receptor (CAR) T cells have shown promise in GBM treatment. While we have shown that combinatorial targeting of 2 glioma antigens offsets antigen escape and enhances T-cell effector functions, the interpatient variability in surface antigen expression between patients hinders the clinical impact of targeting 2 antigen pairs. This study addresses targeting 3 antigens using a single CAR T-cell product for broader application.

Methods: We analyzed the surface expression of 3 targetable glioma antigens (human epidermal growth factor receptor 2 [HER2], interleukin-13 receptor subunit alpha-2 [IL13Rα2], and ephrin-A2 [EphA2]) in 15 primary GBM samples. Accordingly, we created a trivalent T-cell product armed with 3 CAR molecules specific for these validated targets encoded by a single universal (U) tricistronic transgene (UCAR T cells).

Results: Our data showed that co-targeting HER2, IL13Rα2, and EphA2 could overcome interpatient variability by a tendency to capture nearly 100% of tumor cells in most tumors tested in this cohort. UCAR T cells made from GBM patients' blood uniformly expressed all 3 CAR molecules with distinct antigen specificity. UCAR T cells mediated robust immune synapses with tumor targets forming more polarized microtubule organizing centers and exhibited improved cytotoxicity and cytokine release over best monospecific and bispecific CAR T cells per patient tumor profile. Lastly, low doses of UCAR T cells controlled established autologous GBM patient derived xenografts (PDXs) and improved survival of treated animals.

Conclusion: UCAR T cells can overcome antigenic heterogeneity in GBM and lead to improved treatment outcomes.

Fig. 1

Antigen expression pattern of HER2, IL13Rα2, and EphA2 for 15 primary patient GBM samples. Patient tumor samples were co-stained for all 3 antigens, and ≥100000 primary GBM cells were simultaneously interrogated using flow cytometry. (A) Sample of flow cytometry histograms for patient UPN001. (B) Euler diagrams with ellipsis representing the percentage of cells in patient tumor expressing each antigen. Areas of overlap indicate percentage of cells expressing multiple antigens.

Fig. 2

Expression of 3 separate CAR molecules simultaneously on the surface of T cells and their antigen specificity. (A) Plasmid map showing the single transgene encoding for 3 CAR molecules in tandem separated by viral 2A sequences. (B) Staining for 3 CAR molecules by flow cytometry in T cells for 2 separate donors. (C) IL-2 and IFN-gamma production by UCAR and NT T cells when exposed to plate-bound antigens HER2, IL13Rα2, and EphA2 measured by ELISA. ***P < 0.001. (D) Raji tumor force express model for humanized HER2 antigen (Raji-hHER2), IL13Rα2 antigen (Raji-hIL13Rα2), or EphA2 antigen (Raji-hEphA2) to test specificity of tumor killing by UCAR T cells. The reporter green fluorescent protein gene detection by flow cytometry is shown for each. (E) Four-hour ⁵¹Cr cytotoxicity assay demonstrating tumor killing of various CAR T-cell products with Raji cells that contain single tumor antigens and negative (Raji) and positive (U373) controls.

Fig. 3

Immune synapse (IS) imaging of CAR T-cell/tumor interface demonstrates enhanced cytolytic potential of UCAR T cells. (A) Conjugates of U373 and UCAR, HER2-CAR, and NT T cells stained and incubated with U373 cells. Co-stains performed for perforin (green), the microtubule organizing center (MTOC; α-tubulin, blue), and phalloidin (F-actin, red). Scale bar = 5 µm. (B) Distance of MTOC from IS measured for different combinations of T cells with U373.

Fig. 4

In vitro activity of UCAR, biCAR, single CAR, and NT T cells in autologous GBM patient models. (A) Staining for 3 CAR molecules by flow cytometry in T cells for UPN001 and UPN003. (B) CD4/CD8 distribution of autologous T cells after transduction and expansion. (C) IFN-gamma production and (D) IL-2 production by UCAR, biCAR, single CAR, and NT T cells derived from patient peripheral blood mononuclear cells (PBMCs) when exposed to patient primary tumor samples measured by ELISA. (E) Four-hour ⁵¹Cr cytotoxicity assay demonstrating tumor killing of UCAR, biCAR, single CAR, and NT T cells derived from patient PBMCs when exposed to patient primary tumor samples. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5

In vivo experiments demonstrate superior antitumor activity and survival in UCAR-treated mice in an autologous tumor model; 2.5 × 10⁵ patient GBM cells were stereotactically injected into the right caudate nucleus of SCID mice. On days 5 and 12 after tumor cell injection (indicated by arrows), mice received an intratumoral injection of 3 × 10⁶ autologous best single CAR (IL13Rα2), best biCAR (IL13Rα2 and EphA2), UCAR T cells, or NT T cells normalized for transduction efficiency. (A) Bioluminescence imaging to monitor tumor size for mice injected with 2 patient primary tumor samples and followed by treatment with UCAR, biCAR, single CAR, and NT T cells derived from the same patient’s peripheral blood mononuclear cells. Median bioluminescence (solid line) and individual mouse data (dashed lines) are shown for mice in each group (n = 5 for each group). (B) Representative images of the bioluminescence imaging to monitor tumor size. (C) Kaplan–Meier curves for the in vivo experiments followed to 60 days post tumor injection.

Fig. 6

(A) Characterization of UPN001 and UPN005 xenografts post treatment from frozen mouse brain sections. GBM tumor xenografts of mice (N = 3) from the NT and UCAR T-cell treated groups were characterized post treatment using immunofluorescence staining for HER2 (green), EphA2 (red), and IL13Rα2 (purple), DAPI (blue), at 40x magnification. Representative images shown. Scale bar = 20 µm. (B) Dot plots representing HER2, EphA2, and IL13Rα2 positively stained cells in brain sections of mice quantified collectively from 20 high power fields in each group using ImageJ. **P < 0.01 and ***P < 0.001.