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.

Figure 1

Targeting a single antigen results in selective survival and proliferation of escape tumor cell variants. U373 cells expressing HER2 and IL-13Rα2 were treated, in vitro, with HER2 chimeric antigen receptor (CAR) T cells in a 1:5 T cell to tumor cell ratio (20,000 T cells: 100,000 tumor cells). Serial analysis of viable tumor cells by flow cytometry from day 1 to 14 showed survival and expansion of a distinct HER2 negative population that was resistant to HER2 CAR T cells. These surviving tumor cells maintained positivity for IL-13Rα2. Gating strategy is shown in Supplementary Figure S1. One experiment of three is shown.

Figure 2

Heterogeneous antigen expression in glioblastoma (GBM). Flow cytometric analysis of single cell suspensions of primary GBM excision samples and U373-GBM cell line costained for HER2, IL-13Rα2, and EphA2 on > 100,000 gated events (Supplementary Figure S1 describes the gating strategy) shown in (a) representative histograms and (b) dot plots.

Figure 3

Hierarchy of expression of various antigenic permutations in primary glioblastoma (GBM). Multiaxial graph depicting prevalence of target antigens in various tumor cell sub-populations studied using coimmunofluorescence for HER2, IL-13Rα2, and EphA2 of six serially diagnosed GBMs surgical specimens. Significantly, more cells fell in populations expressing any two antigen permutations (versus single antigen only) by comparing means using a one-tailed unequal variance t-test. Expression of any of the three antigens was not significantly more inclusive of the tumor bulk in the patient cohort studied. Representative IFC captures are shown in Supplementary Figures S2 and S3. Data are analyzed in a mathematical model in Table 1.

Figure 4

Bispecific T cell products efficiently offset antigen escape resulting in symmetric depletion of HER2 and IL-13Rα2 expressing glioblastoma (GBM) cells. Composite bar diagram representing the antigen expression pattern in viable U373 cells, 7 days after co-culture with various T cell products in 1:5 T cell to tumor cell ratio (20,000 T cells and 100,000 tumor cells). Table depicts the breakdown of antigen positivity (IL-13Rα2 only, HER2 only, IL-13Rα2 and HER2 positive and IL-13Rα2 and HER2 negative) as a percentage of surviving tumor cells. A dot plot of IL-13Rα2 and HER2 co-expression on tumor cells is displayed in Supplementary Figure S4. Data shown are representative of an experiment repeated three times.

Figure 5

Enhanced activation of the biCAR T cells in vitro, upon simultaneously encountering two tumor-restricted antigens. In cocultures, HER2 and IL-13Rα2 specific CAR T cells secreted Th1 cytokines (IFN-γ and IL-2) upon encountering HER2 and IL-13Rα2 expressing U373 cells (25,000 T cells: 100,000 tumor cells), as detected by ELISA. biCAR T cells secreted significantly higher amounts of (a) IFN-γ in comparison with HER2 CAR T cells (P = 0.03), IL-13Rα2 CAR T cells (P = 0.04) and the pool of equal number of unispecific T cells (50% HER2 CAR T cells and 50% IL-13Rα2 CAR T cells; P = 0.046) and (b) IL-2 in comparison with HER2 CAR T cells (P = 0.04), IL-13Rα2 CAR T cells (P = 0.02) and their pooled-product (P = 0.05). Both IFN-γ and IL-2 secretion with the pooled product were consistently higher than unispecific T cells but the difference was not statistically significant (P = 0.09–0.13). No cytokine release was detected with NT T cells from the same donor. (c) While unispecific HER2 CAR and IL-13Rα2 CAR T cells showed comparable degrees of proliferative capacity, as evidenced by ³H-thymidine uptake, biCAR T cells showed significantly higher ³H-thymidine uptake in comparison to HER2 CAR T cells (P = 0.01), IL-13Rα2 CAR T cells (P = 0.03) and their pooled product (P = 0.04) upon encountering HER2 and IL-13Rα2 plate bound proteins simultaneously. CAR T cells stimulated with OKT3 were used as positive controls and NT T cells as negative control. (d) biCAR T cells exhibited higher cytolytic function in vitro, in CCK-8 cytotoxicity assays. CAR T cells recognized and killed HER2 and IL-13Rα2 expressing U373 cells at different effector to target ratios (4:1, 2:1, 1:1, and 1:2) after 8 hour incubation. Improved tumor cell killing shown by the pooled product (50% HER2 CAR and 50% IL-13Rα2 CAR T cells) was lost at low T cell to tumor ratios. Conversely, biCAR T cells consistently showed most cytolytic activity and maintained the effect even at low T cell to tumor cell ratios. NT T cells did not induce any significant tumor cell lysis. Data are M ± SD from one of three experiments done in triplicates. Validation of the methodology used in (c) is described in Supplementary Figure 6a.

Figure 6

Primary bispecific T-cell products from glioblastoma (GBM) patients exhibit enhanced functionality against autologous glioma cells. Effector cells were generated from two GBM patients (UPN-1 and UPN-10; transduction rates are shown in Supplementary Figure S8) and were tested against autologous primary glioma cells. Supernatants from 25,000 T cells: 100,000 tumor cell co-cultures were harvested and analyzed for (a) IFN-γ and (b) IL-2, for both patients. Further, cytotoxicity assays were performed; displayed in (c). Collectively, these ex vivo results using patient material in an autologous set up confirmed, as seen with healthy donors tested against GBM-U373, that a pool of unispecific T cells exhibited some enhanced (yet not statistically significant) activity above unispecific T cell products and that biCAR T cells show enhanced activation and antitumor activity over unispecific T cells and the pooled product thereof. Data are M ± SD from one of at least two experiments done in triplicates.

Figure 7

Accentuated phosphorylation of Zap70 in biCAR T cells. CAR T cells were exposed to a nontissue culture plate coated with either HER2-Fc or IL-13Rα2-Fc or both chimeric proteins for 2 hours at 37 ^°C. Activated CAR T cells were permeated with monoclonal antibody to pZap70 and analyzed using flow cytometry. biCAR T cells showed (a) a consistently higher statistically significant degree of phosphorylated Zap70 (pZap70) in comparison with both the unispecific CAR T cell products on exposure to both chimeric proteins; (b) while we observed comparable degrees of pZap70 on exposure to either HER2-Fc or IL-13Rα2-Fc individually, the pZap70 signaling was consistently enhanced upon encountering both HER2-Fc and IL-13Rα2-Fc simultaneously. The comparison of the latter mean fluorescence intensity was not statistically significant though. Displayed histograms are representatives of three experiments. The statistical analysis of fluorescence is displayed in Table 2.

Figure 8

Adoptively transferred biCAR T cells show improved tumor control in vivo. Tumors were established by stereotactic injection of 2.5 × 10⁵ eGFP. Firefly luciferase expressing U373 cells into the right frontal cortex of SCID mice. On day 6 after tumor cell injection, mice received an intra-tumoral injection of 2 × 10⁶ unispecific HER2 CAR T cells (n = 5), IL-13Rα2 CAR T cells (n = 5), pooled product of equal number of T cells containing 50% HER2-specific and 50% IL-13Rα2-specific CAR T cells (n = 5), biCAR T cells (n = 5) or NT T cells (n = 5) from the same donor. A subset of animals were not treated (n = 5). (a) Tumors grew exponentially in untreated mice and those treated with NT T cells as shown in one representative animal from each group. Unispecific HER2 CAR and IL-13Rα2 CAR T cells induced transient tumor regression that was comparable. Both bispecific T cell products, the pool of unispecific HER2 CAR and IL-13Rα2 CAR T cells as well as biCAR T cells, induced tumor regression that was sustained for significantly longer duration. Mice treated with the pooled T cell product started relapsing after the mean of day 60; (b) quantitative bioluminescence imaging showing the group median, photons/cm²/seconds/area imaged; (c) Survival analysis on a Kaplan–Meier plotted at 160 days after the tumor was established. Mice treated with unispecific HER2 CAR or IL-13Rα2 CAR T cells had median survival of 79 and 84 days respectively in comparison to median survival of 35 days in untreated mice and those treated with NT T cells (P = 0.03). Median survival improved to 120 days in mice treated with the pooled product (P = 0.04). biCAR T cells further improved tumor control with 80% of the mice surviving >160 days (biCAR versus unispecific T cells, P = 0.0007; biCAR versus pooled product, P = 0.002).