High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms

Abstract

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood-brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII^- tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.

Keywords: CAR T cells; EGFR; GBM; adoptive T cell therapy; glioma; immunotherapy.

Figure 1

Construction and expression of 806 CAR and EGFR mutant cell lines. (A) Schematic diagram of vector map of 806 CAR containing the 4-1BB co-stimulatory domain. (B) CAR surface expression in primary human CD4⁺ and CD8⁺ T cells. Human T cells were simulated for 24 h with anti-CD3/anti-CD28 T-cell activating beads and transduced with CAR transgenes, and CAR expression was analyzed by flow cytometry using biotinylated goat-anti-mouse (806, C225, and CD19 CARs) and goat-anti human F(ab)2 fragment-specific antibodies (2173 CARs) followed by secondary staining with streptavidin-APC. (C) Schematic showing targeted missense mutations in the extracellular domain of EGFR, EGFR^R108K/G, EGFR^A289D/T/V, EGFR^G598V, and splice variant EGFRvIII. (D) Schematic of lentiviral vector co-expressing CFP and wtEGFR or EGFR mutant. (E) Flow-based analysis of endogenous and ectopically expressed EGFR in U87MG, GSC5077, and K562 cell lines using the cetuximab antibody. (F) U87MG, U87MG-EGFR, and GSC5077-EGFR expression of EGFRvIII. (G) U87MG-EGFR, GSC5077-EGFR, and K562 cell lines were transduced with a lentiviral vector co-expressing CFP and indicated EGFR missense mutations and sorted by CFP expression using fluorescent-activated cell sorting.

Figure 2

In vitro characterization of 806 EGFR CAR T cells. Antigen-specific cytolytic activity of 806 and 2173 CAR T cells against cell lines expressing EGFR and its variants. (A) U87MG-EGFR and GSC5077-EGFR cell lines expressing EGFRvIII, EGFR^R108K/G, EGFR^A289D/T/V, and EGFR^G598V mutant variants were stably transduced with Click Beetle Green (CBG) and cocultured with CAR T cells at indicated effector-to-target ratios for 24 h. One representative experiment from three normal donors is shown. Samples were performed in triplicates in three replicative experiments. C225-BB-ζ CAR, and CD19-BB-ζ CAR were used as positive and negative controls, respectively. (B) Antigen-specific cytolytic activity of 806 and 2173 CAR T cells in EGFR and its variants expressed in K562 cells in a 4-h chromium release assay at indicated effector-to-target ratios. (C) K562 cells expressing wtEGFR, EGFRvIII, or EGFR-mutants were cocultured with 806 CART cells for 48 h. IFN-γ, TNF-α, and IL2 secretion was measured in the supernatant by ELISA. Bar charts represent results from a single experiment, and values represent the average ± SD of triplicates. (D) CD107a upregulation on CAR T cells stimulated with K562 cells expressing wtEGFR, EGFRvIII, or EGFR-mutants for 4 h. The percentage of CD107a expression was quantified on CD3 cells (values represent the average of ± SD of two repeated experiments).

Figure 3

Antitumor efficacy of 806 CAR T cells in primary astrocytes and keratinocytes. (A) Surface expression of EGFR assessed by flow cytometry on human primary astrocytes and keratinocytes using EGFR-specific cetuximab antibody. (B) Primary astrocytes and keratinocytes were cocultured with 806 CAR T cells at indicated ratios in a 4-h chromium assay, and results are representative of a single experiment showing the average ± SD of triplicates. (C) Levels of IFN-γ measured in supernatants by ELISA 24 h after coculturing 806 and 2173 CAR T cells with primary astrocytes and keratinocytes at an effector-to-target ratio of 1:1. Results are representative of a single experiment with the average ± SD of triplicates.

Figure 4

In vivo antitumor effect of 806 CAR T cells in NSG mice bearing U87MG-EGFR/EGFRvIII⁺ xenografts. Seven days after 250,000 U87MG-EGFR/EGFRvIII cells were subcutaneously implanted into mice, 3 × 10⁶ T cells were injected intravenously with indicated CAR constructs. (A) Survival based on time to endpoint was plotted using a Kaplan–Meier curve and statistically significant differences between CAR groups were determined using the log-rank Mantel-Cox test. Tumor burden was assessed by bioluminescent imaging. Bars indicate means ± SD (n = 7 mice per group). Tumor burden was quantified as total flux (B) and in individual mice (C) in units of photons/second. Bars indicate means± SD (n = 7 mice). P = photons. ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01.

Figure 5

806 CAR T activity in heterogeneous GBOs highlights cross-reactivity of 806 binder against oncogenic EGFRs. CAR T coculture with GBOs was used to demonstrate anti-EGFR activity. (A) EGFR alterations identified in each GBO line. (B) Immunofluorescence images of CAR T cells engrafted GBOs, for four organoids per condition, 9057 (left) and 9066 (right). Blue = DAPI; red = CD3⁺; white = cleaved caspase 3⁺ (CC3⁺), scale bar = 100 µm. (C) Quantification of CD3⁺ cells (left) and CC3⁺ cells (right) showing antitumor activity from the 806 CAR T cells. (D) Immunofluorescence images of CAR T cell targets in GBOs, for 9057 (left) and 9066 (right). Blue = DAPI; red = EGFR⁺; white = EGFRvIII⁺, scale bar = 100 µm. (E) Quantification of EGFR⁺ (left) and EGFRvIII⁺ signals (right) showing antitumor activity from the 806 CAR T cells. Error bars are ± standard error. ns, p > 0.05.