A human ErbB2-specific T-cell receptor confers potent antitumor effector functions in genetically engineered primary cytotoxic lymphocytes

Abstract

The ErbB2 protein is a member of the tyrosine kinase family of growth factor receptors that is overexpressed in cancers of the breast, ovary, stomach, kidney, colon, and lung, and therefore represents an attractive candidate antigen for targeted cancer immunotherapy. Cytotoxic T lymphocytes specific for various immunogenic ErbB2 peptides have been described, but they often exhibit both poor functional avidity and tumor reactivity. In order to generate potent CD8(+) T cells with specificity for the ErbB2(369-377) peptide, we performed one round of in vitro peptide stimulation of CD8(+) T cells isolated from an HLA-A2(+) patient who was previously vaccinated with autologous dendritic cells pulsed with HLA class I ErbB2 peptides. Using this approach, we enriched highly avid ErbB2-reactive T cells with strong ErbB2-specific, antitumor effector functions. We then stimulated these ErbB2-reactive T cells with ErbB2(+) HLA-A2(+) tumor cells in vitro and sorted tumor-activated ErbB2(369-377) peptide T cells, which allowed for the isolation of a novel T-cell receptor (TCR) with ErbB2(369-377) peptide specificity. Primary human CD8(+) T cells genetically modified to express this ErbB2-specific TCR specifically bound ErbB2(369-377) peptide containing HLA-A2 tetramers, and efficiently recognized target cells pulsed with low nanomolar concentrations of ErbB2(369-377) peptide as well as nonpulsed ErbB2(+) HLA-A2(+) tumor cell lines in vitro. In a novel xenograft model, ErbB2-redirected T cells also significantly delayed progression of ErbB2(+) HLA-A2(+) human tumor in vivo. Together, these results support the notion that redirection of normal T-cell specificity by TCR gene transfer can have potential applications in the adoptive immunotherapy of ErbB2-expressing malignancies.

FIG. 1.

ErbB2-pulsed DC1 increase the frequency of ErbB2-directed T cells. CD8⁺ T cells were purified from a patient with ductal carcinoma in situ (DCIS) postadministration of the ErbB2-pulsed-DC1 vaccine and cocultured for 7 days with ErbB2_369–377 peptide-pulsed autologous dendritic cells. After 1 week, CD8⁺ T cells were harvested and analyzed via flow cytometry with labeled tetramer bound to ErbB2_369–377 or MART1_26–35. MART1 T cells served as negative control effector cells. The percentage of positive cells for CD8 and ErbB2 are indicated on the dot plot.

FIG. 2.

ErbB2_369–377-specific T cells strongly recognize peptide-pulsed T2 cells and differentially recognize HLA-A2-restricted ErbB2-expressing tumor cells. (A) IFN-γ production of ErbB2_369–377-specific T cells in response to peptide-pulsed targets. ErbB2- or MART1-specific T cells were cocultured with T2 cells loaded with HLA-A2-restricted ErbB2_369–377 or MART1_26–35 peptide for 18 hr. (B) ErbB2_369–377-specific T cells exhibit high avidity against the relevant peptide. ErbB2_369–377-specific T cells were incubated for 18 hr with T2 cells pulsed with a range of concentrations of ErbB2_369–377 peptide or 10 ug/mL control (MART-1) peptide. MART1 T cells served as negative control effector T cells. (C) ErbB2 or MART1-specific T cells were cultured alone (none) or stimulated overnight with human HLA-A2-restricted ErbB2⁺-established cancer cell lines. SKOV-3 (HLA-A2⁻ ErbB2⁺) and CEM (HLA-A2⁻ ErbB2⁻) served as negative control tumor targets. (D) APM expression of HLA-A2-restricted ErbB2-expressing tumor cell lines. The mRNA levels of human TAP1, TAP2, TAPASIN, and TAP2 were quantified by real-time PCR. mRNA levels are expressed as fold increase over the APM-negative T2 cell line. β-Actin was used as an endogenous gene control. Results depict the mean±SD of triplicate wells. For all assays, IFN-γ was quantified from cell-free supernatants by ELISA and is reported as the mean concentration (pg/ml)±SEM of duplicate wells. APM, antigen processing machinery; ELISA, enzyme-linked immunosorbent assay.

FIG. 3.

Expression of the ErbB2 TCR on retrovirally transduced SupT1 cells and CD8⁺ T cells. (A) Screening of TCR α/β pairs by retroviral transduction of SupT1 cells. Retroviruses encoding eight different TCR combinations were screened for ErbB2_369–377 specificity by transduction of SupT1 cells. HLA-A2/ErbB2_369–377 tetramer staining of the genetically modified SupT1 cells was performed 5 days after transduction and analyzed by flow cytometry. Two representative SupT1 populations are shown, each bearing different TCRs whose α and β chains were isolated from the ErbB2-specific polyclonal CD8⁺ T cells. Untransduced (NV) and MART1 SupT1 cells served as negative controls for HLA-A2/ErbB2_369–377 tetramer binding. (B) HLA-A2/ErbB2_369–377 tetramer staining of primary TCR-transduced CD8⁺ T cells. CD8⁺ T cells transduced with either the ErbB2 TCR7 or the MART1 TCR and untransduced CD8⁺ T cells (NV) were stained with the indicated HLA-A2/peptide tetramers. Numbers represent the percentage of tetramer⁺ cells. TCR, T-cell receptor.

FIG. 4.

ErbB2_369–377-specific T cells show potent IFN-γ production in response to ErbB2-peptide-loaded targets and ErbB2-expressing cancer cell lines in vitro. (A) ErbB2 or MART1 TCR-transduced T cells were cocultured with T2 cells loaded with HLA-A2-restricted ErbB2_369–377 or with MART1_26–35 for 18 hr. (B) ErbB2 or MART1 TCR-transduced T cells were cultured alone (none) or stimulated overnight with human HLA-A2-restricted ErbB2⁺-established cancer cell lines. SKOV-3 (HLA-A2⁻ ErbB2⁺) and CEM (HLA-A2⁻ ErbB2⁻) served as negative control tumor targets. (C) CD8⁺ T cells transduced with the ErbB2_369–377-specific TCR as well as the control MART1 TCR were incubated 11 days after transduction for 18 hr with T2 cells pulsed with a range of titrated concentrations of ErbB2_369–377 peptide. T2 pulsed with MART1_26–35 peptide served as negative control target T cells. For all assays, IFN-γ was quantified from cell-free supernatants by ELISA and is reported as the mean concentration (pg/ml)±SEM of duplicate wells.

FIG. 5.

T cells expressing ErbB2_369–377-specific TCR7 delay tumor growth in vivo. T cells expressing ErbB2_369–377-specific TCR7 delay tumor growth in vivo. Retrovirally transduced ErbB2 TCR7 CD8⁺ T cells and the breast cancer cell line MDA231 were co-injected subcutaneously into the flank of NSG mice on day 0. MART1-specific F5 TCR-transduced T cells co-injected with MDA231 were used as controls. (A) Tumor growth was determined by caliper measurement over time. Results are expressed as mean tumor volume (mm³±SEM) with n=5 for all groups. Statistical significance of p<0.05 is reported as *p=0.0495, **p=0.0075, and ***p=0.0029. After 35 days, tumors were resected, photographed (B), and measured for tumor weight (C). NSG, NOD/SCID/γ-chain^−/−.