A poxviral-based cancer vaccine the transcription factor twist inhibits primary tumor growth and metastases in a model of metastatic breast cancer and improves survival in a spontaneous prostate cancer model

Abstract

Several transcription factors play a role in the alteration of gene expression that occurs during cancer metastasis. Twist expression has been shown to be associated with the hallmarks of the metastatic process, as well as poor prognosis and drug resistance in many tumor types. However, primarily due to their location within the cell and the lack of a hydrophobic groove required for drug attachment, transcription factors such as Twist are difficult to target with conventional therapies. An alternative therapeutic strategy is a vaccine comprised of a Modified vaccinia Ankara (MVA), incorporating the Twist transgene and a TRIad of COstimulatory Molecules (B7-1, ICAM-1, LFA-3; TRICOM). Here we characterize an MVA-TWIST/TRICOM vaccine that induced both CD4+ and CD8+ Twist-specific T-cell responses in vivo. In addition, administration of this vaccine reduced both the primary tumor growth and metastasis in the 4T1 model of metastatic breast cancer. In the TRAMP transgenic model of spontaneous prostate cancer, MVA-TWIST/TRICOM alone significantly improved survival, and when combined with the androgen receptor antagonist enzalutamide, the vaccine further improved survival. These studies thus provide a rationale for the use of active immunotherapy targeting transcription factors involved in the metastatic process and for the combination of cancer vaccines with androgen deprivation.

Keywords: TRICOM; Twist; immunotherapy; metastasis; vaccine.

Figure 1. MVA-TWIST/TRICOM expresses Twist and a TRiad of COstimulatory Molecules (TRICOM)

Western blot and flow cytometric analysis of MC38 cells treated with A. no treatment, B. MVA-TRICOM or C. MVA-TWIST/TRICOM for 24 hours. Antibodies against Twist and GAPDH were used for western blots. Antibodies against murine B7-1, ICAM-1, LFA-3 and isotype controls were used for flow cytometry. Inserts: percent of cells positive for the indicated marker and its mean fluorescent intensity (MFI). Data are representative of two independent experiments.

Figure 2. Administration of MVA-TWIST/TRICOM induces Twist-specific T-cell responses

A. BALB/c mice and B. C57BL/6 mice were treated with three weekly doses of MVA-TWIST/TRICOM (black bars) or left untreated (open bars). The level of CD4⁺ T-cell proliferation was determined by [³H]-thymidine incorporation in the presence of irradiated antigen-presenting cells and Twist peptide. C. BALB/c mice and D. C57BL/6 mice were treated with three weekly doses of MVA-TWIST/TRICOM (black bars) or left untreated (open bars). The level of CD8⁺ T-cell activity was determined by the amount of secreted IFN-γ following 7 days of in vitro Twist peptide stimulation. The lytic ability of the CD8⁺ T cells from E. BALB/c mice and F. C57BL/6 mice treated with MVA-TWIST/TRICOM (black bars) or left untreated (open bars) was evaluated against peptide-pulsed p815 target cells. Error bars indicate mean ± S.E.M. from triplicate measurements. Statistical analyses for CD4⁺ T-cell proliferation were done by Student’s t-test, * = P < 0.05. For IFN-γ secretion, significance was determined by the Kolmogorov-Smirnov test. Data are representative of three independent experiments.

Figure 3. Treatment with MVA-TWIST/TRICOM induces Twist-specific T-cell responses in 4T1 tumor-bearing BALB/c mice

BALB/c mice (n = 5/group) were implanted s.c. with 5 × 10⁴ 4T1 cells. Four days post-tumor implantation, mice received the first of two weekly MVA-TWIST/TRICOM (black bars) or were left untreated (open bars). A. The level of CD4⁺ T-cell proliferation was determined by [³H]-thymidine incorporation in the presence of irradiated antigen-presenting cells and Twist peptide. B. The level of CD8⁺ T-cell activity was determined by the amount of secreted IFN-γ following 7 days of in vitro Twist peptide stimulation. C. The level of CD8⁺ T-cell activity was determined by the amount of secreted IFN-γ following 7 days of in vitro AH-1 peptide stimulation. Error bars indicate mean ± S.E.M. from triplicate measurements. Statistical analyses for CD4⁺ T-cell proliferation were done by Student’s t-test, * = P < 0.05. For IFN-γ secretion, significance was determined by the Kolmogorov-Smirnov test.

Figure 4. MVA-TWIST/TRICOM administration improves tumor infiltration of immune effector cells

BALB/c mice (n = 5/group) were implanted s.c. with 5 × 10⁴ 4T1 cells. Four days post-tumor implantation, mice received the first of two weekly MVA-TWIST/TRICOM vaccinations or were left untreated. Seventeen days following the last vaccination, tumors were harvested and analyzed by immunohistochemistry and flow cytometry. A. CD3⁺ lymphocyte infiltration as determined by immunohistochemistry. Inserts: Isotype control staining and percentage of tumor composed of CD3⁺ cells. B. Percentage of CD4⁺ T cells (CD3+ CD4+), CD8⁺T cells (CD3+ CD8+), Central Memory (CM, CD3+ CD8+CD44+CD62L+) and Effector Memory (EM, CD3+ CD8+CD44+CD62L-) cells present in the tumor as determined by flow cytometric analysis. Error bars indicate mean ± S.E.M. from five measurements. Statistical analyses were done by Student’s t-test, * = P < 0.05.

Figure 5. Treatment with MVA-TWIST/TRICOM inhibits primary tumor growth and spontaneous lung metastasis in the 4T1 metastatic breast cancer model

BALB/c mice (n = 10/group) were implanted s.c. with 5 × 10⁴ 4T1 cells. Four days post-tumor implantation the first of three weekly vaccinations was given, as indicated by arrows. A. Primary tumor growth in mice treated with MVA-TWIST/TRICOM (closed squares) or MVA-TRICOM (closed circles) or left untreated (open circles). Insert: Twist expression in 4T1 primary tumor and lung metastases as determined by RT-PCR (n = 3). B. Enumeration of clonogenic metastatic cells in the lungs of mice treated with MVA-TWIST/TRICOM (closed squares) or MVA-TRICOM (closed circles) or left untreated (open circles) obtained 21 days post-tumor implantation. Tumor dimensions were measured weekly. Clonogenic metastatic cells/lung were determined by plating a single cell suspension of lung cells in the presence of 6-thioguanine for 10–14 days after which colonies were enumerated. Error bars indicate mean ± S.E.M. Statistical analyses were done by Student’s t-test, * = P < 0.005 vs. either no treatment or MVA-TRICOM alone. Data are representative of three independent experiments.

Figure 6. MVA-TWIST/TRICOM administration reduces the formation of lung metastases when treatment is initiated up to 15 days post-tumor implantation

BALB/c mice (n = 15/group) were implanted s.c. with 5 × 10⁴ 4T1 cells. Graphs show primary tumor growth in mice treated with A. no treatment, B. MVA-TWIST/TRICOM beginning 7 days post-tumor implantation or C. MVA-TWIST/TRICOM beginning 15 days post-tumor implantation. Vaccinations are indicated by arrows. Inserts: Average tumor growth per group compared to group receiving no treatment. Enumeration of clonogenic metastatic cells, obtained when the group average tumor volume reached 1000mm³, in the lungs of mice treated with D. no treatment, E. MVA-TWIST/TRICOM beginning on day 7 post-tumor implantation or F. MVA-TWIST/TRICOM beginning on day 15 post-tumor implantation. Tumor dimensions were measured weekly. Clonogenic metastatic cells were determined as in previous experiment. Error bars indicate mean ± S.E.M. Statistical analyses were done by Student’s t-test, * = P < 0.01 vs. no treatment. Data are representative of two independent experiments.

Figure 7. Combining MVA-TWIST/TRICOM with anti-androgen therapy significantly increases survival in Tramp-Tg mice

Tramp-Tg mice were age-matched and randomized to receive one of the indicated treatment regimens. Indicated mice began receiving enzalutamide and received the first of three weekly vaccinations with MVA-TWIST/TRICOM simultaneously on day 0. A. Overall survival of Tramp-Tg mice receiving no treatment (open circles), MVA-TWIST/TRICOM (closed squares), enzalutamide (open squares) or the combination of MVA-TWIST/TRICOM and enzalutamide (closed circles). Insert: Twist expression in the prostates of wild-type C5/BL6 and Tramp-Tg mice as determined by RT-PCR (n = 12). Statistical analyses were done by Student’s t-test relative to indicated group.