Inhibition of the angiopoietin/Tie2 axis induces immunogenic modulation, which sensitizes human tumor cells to immune attack

Abstract

Background: The angiopoietin/Tie2 pathway is an attractive target for cancer therapy due to its well-known role in regulating angiogenesis. Trebananib, a recombinant peptide-Fc fusion protein, or peptibody, that binds to angiopoietin-1 (Ang1) and Ang2 to block their interaction with the Tie2 receptor, is under active clinical investigation. We investigated whether suppressing the angiopoietin/Tie2 pathway, using the preclinical version of Trebananib (mL4-3 and L1-7(N)), could increase the sensitivity of human tumor cells to immune-mediated lysis through immunogenic modulation, which would make Trebananib a promising candidate for combination with immunotherapy.

Methods: We assessed human carcinoma cells for expression and activation of Ang1 and Ang2 and their receptor tyrosine kinase Tie2. In vitro, we exposed tumor cell lines expressing Tie2 to the peptibodies mL4-3 and L1-7(N), which inhibit the binding of Ang1 and Ang2 to Tie2, and assessed the cells for changes in viability, proliferation, surface phenotype, and sensitivity to attack by antigen-specific cytotoxic T lymphocytes (CTLs).

Results: Suppression of the angiopoietin/Tie2 pathway using mL4-3 and L1-7(N) had no effect on the proliferation or viability of tumor cells. However, these inhibitors markedly altered tumor cell phenotype, rendering tumor cells significantly more sensitive to antigen-specific CTL killing. ICAM-1 was shown to be mechanistically involved in these inhibitors' ability to sensitize tumor cells to immune-mediated attack by functional blocking studies.

Conclusion: Our findings provide a rationale for the combination of agents targeting the angiopoietin/Tie2 pathway with cancer immunotherapies.

Keywords: Angiopoietin; Immunogenic modulation; Immunotherapy; Tie2.

Fig. 1

RNA expression of Ang1, Ang2, and Tie2 in human tumor cell lines. Seven human tumor cell lines were evaluated by RT-PCR for RNA expression of Ang1, Ang2, and Tie2. Data were normalized to GAPDH. Bars indicate mean ± SEM

Fig. 2

Activation of Tie2 pathway in human tumor cell lines. Tie2 phosphorylation at sites py992 and py1102 and the ability of Ang1 (mL4-3) and Ang2 (L1-7(N) inhibitors to reduce activation of the Tie2 pathway were evaluated by flow cytometry in OV17-1 (a) and MDA-MB-231 (b) tumor cell lines. A range of concentrations of mL4-3 (0.017–170 μg/mL) and L1-7(N) (0.0407–407 μg/mL) were tested, with no dose-dependent effects noted. Data are shown at 17 μg/mL and 4.07 μg/mL for mL-4-3 and L1-7(N), respectively. Exogenous recombinant Ang1 or Ang2 (2 μg/mL) was added where indicated. Tumor cells were treated for 30 min at 37 °C, then harvested and stained to assess Tie2 phosphorylation

Fig. 3

Ang1 and Ang2 inhibitors did not affect tumor-cell number or viability. Human ovarian cancer cells (OV17-1; a), breast cancer cells (MDA-MB-231; b) and prostate cancer cells (LNCaP; c) were treated with the Cmax of mL4-3 and L1-7(N) (16 and 10 μg/mL, respectively) or control (human IgG1-Fc, 26 μg/mL) for 3 days. Cells were then harvested, counted, and assessed for viability via trypan blue exclusion. Cell number is reported as a percentage of untreated cells. Data were analyzed with an unpaired t test. p values are indicated

Fig. 4

Ang1 and Ang2 inhibitors increased CTL-mediated lysis of human tumor cells. Human ovarian cancer cells (OV17-1; a), breast cancer cells (MDA-MB-231; b) and prostate cancer cells (LNCaP; c) were treated with the Cmax of mL4-3 and L1-7(N) (16 and 10 μg/mL, respectively) or control (human IgG1-Fc at 26 μg/mL or no treatment) for 3 days. Cells were labeled with ¹¹¹In and co-incubated with CEA-specific CD8⁺ T cells at an effector: target ratio of 30:1 or MUC-1-specific CD8⁺ T cells at an effector:target ratio of 15:1 for 18 h. Effectors are indicated in the graphs. Bars indicate mean ± SEM. Statistical analyses were performed with an unpaired t test. p values < 0.05 were considered significant

Fig. 5

ICAM-1 blockade reduced the enhanced CTL lysis noted with Ang1 and Ang2 inhibitors. Human ovarian cancer cells (OV17-1) were treated with the Cmax of mL4-3 and L1-7(N) (16 and 10 μg/mL, respectively) or control (no treatment) for 3 days. Tumor cells treated for 3 days with Ang1 and Ang2 inhibitor were then pretreated for 1 h and co-incubated during the CTL assay with anti-ICAM-1 antibody (10 μg/mL) or the corresponding isotype control. Cells were labeled with ¹¹¹In and co-incubated with CEA-specific CD8⁺ T cells at an effector: target ratio of 30:1 for 18 h. Bars indicate mean ± SEM. Statistical analyses were performed with an unpaired t test. p values < 0.05 were considered significant