Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma

Abstract

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

Trial registration: ClinicalTrials.gov NCT01454596.

FIG. 1.

Development of CARs targeting EGFRvIII. NIH-3T3 cells (A) and BHK cells (B) were transduced with a retroviral vector expressing EGFRvIII and a neomycin resistance gene. Following selection in neomycin analogue G418, cells were subject to FACS analysis with anti-EGFRvIII mAb as shown (UT, untransduced). Histograms are representative of three independent determinations. (C) GSC lines were cultured in Neurobasal medium supplemented with bFGF and EGF (NBE) or in RPMI-1640 supplemented with 10% FBS for 3 days (to induce differentiation). RNA was extracted from each cell population and subject to RT-PCR using primers that span the variant III deletion in the wild-type EGFR gene, followed by gel electrophoresis of the products (M, marker). Control RNAs were from U251 cells engineered with EGFR wild-type (w.t.) or EGFRvIII (vIII) genes. Data are representative of three independent PCR reactions. (D) Based on the amino acid sequence for anti-EGFRvIII mAbs, scFv genes were synthesized and fused to T-cell signaling domains from CD28 and CD3ζ followed by insertion into γ-retroviral vector MSGV1 (diagram). Retroviral vector preparations were produced and used to engineer primary human T cells as described in Materials and Methods. Transduced cells (UT, untransduced; 3C10 scFv, anti-EGFRvIII CAR vector based on murine mAb 3C10; 139 scFv, anti-EGFRvIII CAR vector based on human mAb 139) were subject to FACS analysis using reagents to detect the scFv (shaded histograms, unstained; middle histograms, isotype controls; right-shifted histograms, anti-CAR transduced cells). The percentage of CAR-positive cells was as shown. Histograms are representative of multiple (more than four) independent transductions. Similar results were obtained with anti-EGFRvIII CAR vector based on murine mAb L8A4 (data not shown). LTR, long terminal repeat; sd, splice donor; sa, splice acceptor; Ψ, extended packaging signal.

FIG. 2.

Anti-EGFRvIII CAR vector-engineered T cells specifically recognize EGFRvIII-expressing cells. (A) γ-Retroviral vectors expressing the anti-EGFRvIII CARs (from murine mAbs 3C10 and L8A4 and human mAb 139) were used to transduced human T cells, which were cocultured with the indicated target cell lines. Target cell lines NIH-3T3 (3T3) and BHK were engineered with the wild-type EGFR (EGFRwt) or EGFR variant vIII (EGFRvIII) genes, or untransduced (UT). Shown is the resultant IFN-γ production as measured by ELISA (pg/mL, mean of triplicate determinations). (B) T cells from two donors (donors 2 and 3) were transduced with the 139-28Z vector (Bulk) and then bead sorted into CD8- and CD4-enriched (>96% positive) T-cell populations (CD8⁺ and CD4⁺). These cells were cocultured overnight with BHK target cells, EGFR wild-type (EGFRwt), or EGFRvIII engineered BHK cells (EGFRvIII) and the amount of IFN-γ produced determined (IFN-γ shown in pg/mL, mean of triplicate determinations). GFP, control vector transduced T cells. (C) CD4⁺ T cells from panel B were cocultured overnight with BHK target cells, EGFR wild-type (EGFRwt), or EGFRvIII engineered BHK cells (EGFRvIII) and the amount of IL-2 produced determined (IL-2 shown in pg/mL, mean of triplicate determinations). Data are representative of three independent experiments. Differences between recognition of EGFRvIII transduced versus control lines were statistically significant (p<0.01, Student's t test).

FIG. 3.

Equivalent function of second- and third-generation anti-EGFRvIII CAR vectors. Using anti-EGFRvIII human mAb 139, a third-generation CAR vector was assembled using CD28, 4-1BB, and CD3ζ T-cell signaling domains (139-28BBZ, diagram at top of figure). This vector was compared with the original vector design containing CD28 and CD3ζ T-cell signaling elements (139-28Z, diagram at top of figure). Primary human T cells were transduced with both vectors and evaluated for IFN-γ cytokine production following overnight coculture (imbedded tables) and in 4-hr ⁵¹Cr release assays to determine cell lysis activity (shown as percent lysis at the indicated E:T). Target cells included: U87 cells transduced with a GFP vector (GFP), wild-type EGFR (EGFRwt), or EGFR variant III (EGFRvIII); and U251 cells transduced with wild-type EGFR (EGFRwt) or EGFR variant III (EGFRvIII). A vector expressing an anti-ERBB2 CAR (ERBB2) was used as a control along with GFP and untransduced (UnTd) cells. Data are representative of three independent experiments. IFN-γ values were determined by ELISA (shown in pg/mL, mean of triplicate determinations), and percent lysis was plotted as the mean of quadruplicate determinations. Differences between recognition of EGFRvIII-transduced versus control lines were statistically significant (p<0.05, Student's t test).

FIG. 4.

Recognition of GSCs by anti-EGFRvIII CAR-transduced T cells. The third-generation anti-EGFRvIII CAR vector (139-28BBZ) was used to transduce T cells from two donors (donors 4 and 5) along with a GFP-expressing vector (GFP) and a CAR vector targeting ERBB2. Transduced T cells were cocultured with wild-type EGFR-engineered U251 cells (U251-EGFRwt) or EGFR variant III engineered U251 cells (U251-EGFRvIII) and GSC lines 1228, 308, and 822. IFN-γ values were determined by ELISA (shown in pg/mL, mean of triplicate determinations). Data are representative of three independent experiments. Differences between recognition of EGFRvIII-transduced U251 versus EGFRwt-U251 were statistically significant (p<0.01, Student's t test).

FIG. 5.

EGFRvIII CAR vector-transduced glioblastoma patient T cells recognize EGFRvIII-expressing tumors. (A) PBLs from normal donor 6 and two glioblastoma (GBM) patients were transduced with anti-EGFRvIII vector and cocultured with U87 or U87-EGFRvIII cells followed by intracellular cytokine staining for IFN-γ. Shown are the resultant FACS dot blots for IFN-γ and CD8. The expression of the anti-EGFRvIII CAR in the patients' T cells was as shown in the FACS histograms plotted on the right (shaded histograms, isotype control). (B) Transduced T cells from part A (CAR⁺) were subject to an REP and, 14 days later, evaluated by IFN-γ Elispot assay. Shown are the stained well images (left) along with the quantitation of the number of IFN-γ-reactive spots (right). Phytohemagglutinin (PHA) was used as a nonspecific T-cell activator. UT, untransduced T cells.

FIG. 6.

Development of an anti-EGFRvIII CAR vector for clinical applications. (A) DNA for anti-EGFRvIII vector 139-28BBZ was used to generate a PG13-based producer cell line clone (vIII CAR-F10), which was produced as an MCB. The MCB was then expanded and larger volumes of vector supernatant harvested over 4 consecutive days (Harvest 1–6) and stored in aliquots. Aliquots for each harvest were used to transduce T cells, which were tested for specific reactivity against EGFRvIII-expressing target cells (U251-EGFRvIII). Shown is the resultant IFN-γ production following overnight coculture (shown in pg/mL, mean of triplicate determinations) with the indicated target cells. Differences between recognition of EGFRvIII-transduced U251 versus EGFRwt-U251 were statistically significant (p<0.001, Student's t test). The percent gene transfer achieved was determined by staining with protein L followed by FACS analysis (shown as % CAR⁺). T cells transduced with the anti-ERBB2 CAR were used as control. (B) Harvests 3 and 4 were thawed and pooled to transduce a second donor's T cells to determine reactivity against a panel of normal human primary cell cultures. Shown is the resultant IFN-γ production following overnight coculture (shown in pg/mL, mean of triplicate determinations) with the indicated target cells. The percent CAR gene transfer achieved is shown in the FACS histograms to the right. T cells transduced with a GFP vector (GFP) and an anti-CD19 CAR (CD19) were used as controls. Primary cells were as follows: NHDF-AD, adult human diploid fibroblast; NHDF-Neo, neonatal human diploid fibroblast; HMVec, human microvascular endothelial cells; NHBC, normal human bronchial epithelial cells; HPrEC, human prostate epithelial cells; SAEC, human small airway epithelial cells; HRE, human renal epithelial cells; EBV-B, an EBV transformed B cell line.