Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells

Abstract

Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z⁺ T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28⁺ T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.

Figures

Figure 1

Structure of ErbB-specific CARs. (A) Amino acid sequence of mature forms of TGF-α, EGF and the derived chimeric T1E peptide. (B) Junction of T1E peptide with the leader derived from colony-stimulating factor-1 receptor (CSF-1R). (C) Cartoon structure of T1E28z and two control CARs (EGF28z, targeted with EGF; T1NA, T1E coupled with a truncated endodomain).

Figure 2

Expression of ErbB-specific CARs in human T cells. Expression of ErbB targeted CARs in transduced T cells was detected by the fluorescence-activated cell sorter (FACS) method by using monoclonal (A; control shown as filled histogram) or polyclonal (B) anti-EGF antibody or by Western blotting (C; probed under reducing conditions with anti-CD3ζ). In each case, controls represent untransduced T-cells.

Figure 3

ErbB specificity of the T1E28z CAR. (A) ErbB null 32D myeloid cells were engineered to express all 10 ErbB receptor/dimer combinations. Expression of the indicated ErbB receptor was detected by FACS (filled histogram represents staining of parental 32D cells with the same antibody combination) and, in the case of ErbB3, by Western blotting (B). (C) To define targeting specificity, 1 × 106 of the indicated engineered T-cell populations was cocultivated with an equal number of 32D cells that express the specified ErbB receptor(s). Supernatants were harvested at 24 h (IL-2) and 72 h (IFN-γ) for ELISA analysis. Data shown are mean ± standard deviation (SD) of three (IL-2) or six (IFN-γ) replicates after correction for transduction efficiency between groups. ***P < 0.001, **P < 0.01, comparing T1E28z (black) or EGF28z (gray) with other control groups. (D) 32D cells that express ErbB1 (32D:1) or ErbB2 + 3 (32D:23) were cocultivated with T1E28z+ T cells in the presence of exogenous EGF (left) or HRG-1β (right). Supernatants harvested at 72 h were analyzed for IFN-γ content.

Figure 4

T1E28z engineered T cells destroy human head and neck cancer cells. (A) Twelve human SCCHN cell lines (and MDA-MB-435 control cells) were analyzed for cell surface expression of ErbB1–4 by FACS. Staining with the indicated ErbB-specific (open histogram) or isotype control (filled histogram) antibody is indicated. (B) Confluent monolayers of the indicated tumor cell lines (24-well plate, 1.9 cm2) were cocultivated with 1 × 106 T1E28z+ T cells. Control T cells expressed T1NA (a matched endodomain-truncated CAR) or were untransduced (Untrans.). After 72 h, residual monolayers were fixed and stained with crystal violet. Punctate staining in T1E28z wells represents adherent lymphocytes. (C) One million of the indicated T-cell populations were cocultivated overnight with a confluent 1.9-cm2 monolayer of luciferase (LUC)-expressing HN3 cells or Vβ6 cells. After the specified interval, T cells were removed and residual viable tumor cells were quantified using XTT (mean ± SD, n = 3). Percentage lysis was calculated as (100 – cell viability).

Figure 5

T1E28z engineered T cells are activated by human head and neck cancer cells. (A) Confluent head and neck tumor monolayers (24-well plate, 1.9 cm2) were cocultivated with 1 × 106 of the indicated T-cell populations. Supernatants were harvested after 72 h and analyzed for IFN-γ (mean ± SD). The number of independent replicate experiments is indicated: *** P < 0.001, **P < 0.01, *P < 0.05. (B) Engineered T cells were cocultivated with HN3 tumor cell monolayers where indicated by overhead arrows. T cells were enumerated at specified intervals. (C) T cells in (B) were analyzed by FACS for cell surface expression of T1E28z at the indicated time points. Untransduced T cells served as the negative control. (D) Engineered T cells were stimulated on a HN3 monolayer and cultured in the presence of IL-2. Control cultures were maintained in IL-2 alone without monolayer stimulation. Cultures were analyzed for CAR expression using monoclonal anti-EGF antibody at baseline and after 7 d. Filled histograms show staining of untransduced T cells. Proliferation/enrichment data are representative of at least three experiments.

Figure 6

In vitro activation of T4+ human T cells. Human T cells were engineered to coexpress the IL-4–responsive 4αβ chimeric cytokine receptor together with the T1E28z CAR (combination called “T4”). After expansion in IL-4, comparison was made with control T cells that were untransduced (Untrans) or that express T1NA (truncated endodomain). (A) To define targeting specificity for ErbB receptors/dimers, 1 × 106 of the indicated T-cell populations were cocultivated with an equal number of 32D cells that express the specified ErbB receptor(s). Supernatants were harvested at 72 h and analyzed for interferon (IFN)-γ (mean ± SD; ***P < 0.001; **P < 0.01; *P < 0.05). (B) 1 × 106 T cells were cocultivated with confluent monolayers of the indicated head and neck tumor cells cultured in 24-well (1.9-cm2) plates. Supernatants were harvested at 72 h and analyzed for IFN-γ (mean ± SD of three independent experiments; ***P < 0.001; LUC, luciferase). (C) T cells (number indicated above each well) were cocultivated for 72 h with the indicated head and neck tumor cell monolayers, cultured in 24-well plates. Nonadherent cells were then removed and, after fixation, residual tumor monolayers were stained using crystal violet (representative of three experiments). (D) T cells were established in triplicate cultures as described in (C). Cytokine analysis was performed on supernatants harvested after 24 h (IL-2, IL-4) and 72 h (IFN-γ). (E) T-cells (1 × 106 cells) were established in triplicate cultures with the indicated monolayers, in the presence or absence of IL-4 (30 ng/mL). Supernatants were analyzed after 72 h for IFN-γ.

Figure 7

In vivo antitumor activity of T1E28z-transduced T cells against a slowly progressive head and neck tumor xenograft. (A) HN3 tumor cells were engineered to express firefly luciferase (HN3-LUC) and were propagated as IP xenografts. In this representative example, a large established tumor was imaged by PET using 18F-fluorodeoxyglucose (tumor center indicated by crosshairs) or BLI, following administration of luciferin. (B) Five mice were inoculated intraperitoneally with 5 × 106 HN3-LUC tumor cells and analyzed by BLI at the indicated intervals. (C) HN3-LUC tumors were recovered from mice described in (B) and analyzed by FACS for expression of ErbB1 and ErbB2. Comparison was made with HN3-LUC cells cultured in vitro. Filled histograms show staining with isotype control alone. (D) Twenty-five SCID Beige mice were inoculated with 5 × 106 HN3-LUC tumor cells, administered by the IP route. Tumor take was confirmed on d 8 by BLI. On d 13, mice were treated with T cells transduced with T1E28z (n = 7), T1NA (n = 6), T4 (combination of 4αβ and T1E28z; n = 6) or PBS (n = 6). Analysis of T cells before infusion is shown in Supplementary Figure 4. Mean bioluminescence for each group is shown over the course of the study. ***P < 0.001 (T1E28z versus PBS and T4 versus PBS); *P < 0.05 (T1E28z versus PBS and T4 versus PBS); ‡P < 0.001 (T4 versus PBS) and P < 0.01 (T1E28z versus PBS); ‡‡P < 0.001 (T4 versus T1NA) and P < 0.01 (T1E28z versus T1NA). (E) Serial imaging of the same mouse (median representative of each group) is shown at selected time points over the course of the study. (F) Bioluminescence data at all time points for each mouse are shown.

Figure 8

In vitro antitumor activity of T1E28z+ T cells against breast cancer. (A) 1 × 106 human T cells engineered to express the indicated CARs were cocultivated with confluent breast cancer monolayers, cultured in 24-well (1.9-cm2) plates. After 24 h, nonadherent cells were removed and monolayers were fixed and stained with crystal violet. (B) Four-hour lactate dehydrogenase release cytotoxicity assay in which T1E28z+ or control S28z+ T cells were cultured with BT20 breast cancer cells or ERBBl° 410.4 mammary carcinoma cells. (C) A representative experiment in which T1E28z+ and S28z+ T cells were cultured with BT20 breast cancer cells. After 24 h, IL-2 was added. Restimulation on a confluent BT20 monolayer was performed where indicated by the overhead arrows. T cells were enumerated at the indicated intervals. (D) Transduced T cells were cocultivated in 24-well plates with confluent monolayers or suspension cells as follows: 1) Jurkat (control; 1 × 106 cells); 2) MDA-MB-435; 3) BT20; 4) BT474; 5) MCF7; 6) SKBR3; 7) T47D; 8) medium alone. Supernatants were analyzed for the indicated cytokines after 24 h.

Figure 9

In vivo activity of T1E28z+ T cells against an ErbB2/3-expressing aggressive tumor xenograft. (A) MDA-MB-435 cells were engineered to coexpress MUC1, ErbB2 and firefly luciferase (MUC-LUC ERBB2). FACS plots show staining with ErbB-specific (open histograms) and isotype control antibodies (filled histograms). (B) MUC-LUC ErbB2 tumor cells were inoculated intraperitoneally in 20 SCID Beige mice. After 3 d, mice (n = 5 per group) were treated with PBS, IL-4 (125 ng × 3 doses per wk), 2 × 107 T4+ T cells or the combination of T4+ T cells + IL-4. Tumor status was monitored by bioluminescence imaging. Data shown are mean ± standard error of the mean of each group. (***P < 0.0001 comparing either T4 or T4 + IL-4 versus either IL-4 or PBS; **P < 0.0001 comparing either T4 or T4 + IL-4 against PBS). (C) Serial bioluminescence emission by mice in the T4 and T4 + IL-4 groups (mean ± SEM), represented on a semi-log plot (*P = 0.015 on d 35; two-way ANOVA).