Genetically engineered T cells to target EGFRvIII expressing glioblastoma

Abstract

Glioblastoma remains a significant therapeutic challenge, warranting further investigation of novel therapies. We describe an immunotherapeutic strategy to treat glioblastoma based on adoptive transfer of genetically modified T-lymphocytes (T cells) redirected to kill EGFRvIII expressing gliomas. We constructed a chimeric immune receptor (CIR) specific to EGFRvIII, (MR1-zeta). After in vitro selection and expansion, MR1-zeta genetically modified primary human T-cells specifically recognized EGFRvIII-positive tumor cells as demonstrated by IFN-gamma secretion and efficient tumor lysis compared to control CIRs defective in EGFRvIII binding (MRB-zeta) or signaling (MR1-delzeta). MR1-zeta expressing T cells also inhibited EGFRvIII-positive tumor growth in vivo in a xenografted mouse model. Successful targeting of EGFRvIII-positive tumors via adoptive transfer of genetically modified T cells may represent a new immunotherapy strategy with great potential for clinical applications.

Figure 1

Schematic representation of the three CIR constructs. MR1-ζ (a) represents the full-length anti-EGFRvIII CIR based on the MR1 scFv linked to partial extracellular domain (hinge) and transmembrane domain (TM) of CD8 α that is fused with the cytoplasmic domain (IC) of human CD3ζ. MRB-ζ (b) is a binding mutant variant of MR1-ζ that has a 76 amino acid-deletion in the heavy chain. MR1-delζ (c) represents a mutant version of MR1-ζ in which the intracellular signaling domain of human CD3ζ has been truncated

Figure 2

RT-PCR analysis of the anti-EGFRvIII-CIRs transgene expression. RT-PCR analysis of the RNA expression of MR1-ζ (a), MRB-ζ (b), and MR1-delζ (c) in corresponding nucleofected PBMCs indicated successful transcription of the three CIRs. Total RNA was extracted from CIR-nucleofected PBMCs, and was amplified in the presence of RNase-free DNase I with and without RT-ase. RT-PCR products were analyzed on 1% agarose gel and photographed

Figure 3

Flow cytometry analysis of the surface expression of the anti-EGFRvIII-CIRs. Surface expression of MR1-ζ (a), MRB-ζ (b), and MR1-delζ (c) on corresponding nucleofected and hygromycin selected PBMCs was detectable after two stimulation cycles. Live cells were gated for analysis. Dashed lines represent cells stained with isotype control whereas solid lines illustrate cells stained with anti-c-myc [9E10] monoclonal antibody. Results indicated that 43.4% of MR1-ζ, 49.5% of MRB-ζ, and 49.7% MR1-delζ nucleofected PBMCs expressed the corresponding CIR on the cell surface

Figure 4

EGFR and EGFRvIII expression in target cell lines and antigen-mediated cytokine secretion by CIR+ T cells. Cell surface expression of EGFR and EGFRvIII was determined by flow cytometry analysis in U87MG (a), U87vIII–FFlucZeo (b) and U87vIII (data not shown) cell lines. Cells were stained with anti-EGFR monoclonal antibody for wild-type receptor expression (solid lines) or with L8A4 anti-EGFRvIII monoclonal antibody for EGFRvIII expression (dashed lines). Live cells were gated for analysis. Flow cytometry analysis showed that U87MG cells expressed wild-type EGFR but not the mutant EGFRvIII, whereas U87vIII–FFlucZeo and U87vIII cells expressed both the wild-type EGFR and the mutant EGFRvIII on the cell surface. 2 × 10⁵ irradiated target cells (U87MG, U87vIII, and U87vIII–FFlucZeo) were co-cultured with 2 × 10⁶ CIR+ T cells for 72 h (c). Culture supernatants were harvested and assayed for cytokine secretion by cytometric bead array. Only T cells expressing the full-length MR1-ζ secreted significant amounts of IFN-γ when co-cultured with EGFRvIII-positive cell lines; U87vIIIFFluc–Zeo (213 pg/ml) and U87vIII (125 pg/ml). No significant amount of IFN-γ was detectable when CIR+ T cells were co-cultured with the wild-type, EGFRvIII-negative U87MG cells. T cells expressing either forms of the mutant MR1-ζ CIR (MRB-ζ, and MR1-delζ) did not secret IFN-γ when co-cultured with target cells

Figure 5

In vitro cytolytic activity of the CIR+ T cells. MR1-ζ, MRB-ζ, or MR1-delζ CIR+ T cells were co-incubated with BATDA loaded wild-type, EGFRvIII-negative U87MG (a) or EGFRvIII-positive U87vIII–FFlucZeo (b) target cells at varying effector to target ratios for 2 h. Cytolytic activity of the CIR+ T cells was analyzed by europium release assay. Effector cells in their third stimulation cycle were used in this assay. Cytolytic capacity of the CIR+ T cells was expressed by specific lysis percentage of target cells. MR1-ζ CIR+ T cells exhibited significant cytotoxicity against EGFRvIII expressing target cells, but not against cells that only expressed the wild-type EGF receptor. MRB-ζ and MR1-delζ expressing T cells showed no significant cytolytic activity against either the wild-type or the EGFRvIII-positive target cells as indicated by one-way ANOVA (P < 0.001)

Figure 6

In vivo anti-tumor activity of anti-EGFRvIII CIR-T cells. (a) A total of 2.5 × 10⁴ U87-FFLuc–EGFRvIIIZeo tumor cells were admixed with CIR+ T cells in a ratio of 1:25 immediately prior to implantation in NOD/SCID mice at day 0. Cells were implanted intracranially (i.c.) in the right frontal lobe. Mean BLI (photons/s × 10⁶) is presented for each group of mice (n = 4 per group) at indicated time points. Mice were observed as long as 42 days; group means were calculated for the duration when no mice were lost to follow-up (through day 29) and were compared by one-way ANOVA (see results). (b) 5 × 10⁴ U87-FFLuc–EGFRvIIIZeo tumor cells were implanted i.c. in NOD/SCID mice at day 0. At day 3, a single i.c. inoculation of 1 million CIR+ T cells was performed. Mice were observed as long as 17 days; group means were calculated for the duration when no mice were lost to follow-up (through day 13) and were compared by one-way ANOVA (see results). Effector cells in their third stimulation cycle were used in both assays