A Phase 2, Single-Arm, Open-Label Clinical Trial on Adjuvant Peptide-Based Vaccination in Dogs with Aggressive Hemangiosarcoma Undergoing Surgery and Chemotherapy

Abstract

To test the antitumor effect and safety of peptide-based anticancer vaccination in dogs with hemangiosarcoma undergoing the standard of care (SOC; surgery and doxorubicin), canine hemangiosarcoma cells were infected with Salmonella typhi Ty21a to release immunogenic endoplasmic reticulum stress-related peptides into the extracellular milieu via CX43 hemichannels opening. The infected tumor cell secretome constituted the vaccine. Following the SOC, dogs with biologically aggressive hemangiosarcoma were vaccinated a total of five times, once every 3 weeks, and were followed up with serial imaging. A retrospective population of dogs undergoing the SOC alone served as controls. The primary endpoints were the time to progression (TTP) and overall survival (OS), and the secondary endpoints were toxicity and immune responses. A total of 28 dogs were vaccinated along with the SOC, and 32 received only the SOC. A tumor-specific humoral response along with a vaccine-specific T-cell response was observed. Toxicity did not occur. The TTP and OS were significantly longer in vaccinated versus unvaccinated dogs (TTP: 195 vs. 160 days, respectively; p = 0.001; OS: 276 vs. 175 days, respectively; p = 0.002). One-year survival rates were 35.7% and 6.3% for vaccinated and unvaccinated dogs, respectively. In dogs with hemangiosarcoma undergoing the SOC, the addition of a peptide-based vaccine increased the TTP and OS, while maintaining a safe profile. Moreover, vaccinated dogs developed a tumor-specific response, supporting the feasibility of future phase three studies.

Keywords: angiosarcoma; canine; immunotherapy; translational research.

Figure 1

S. typhi Ty21a infection induces upregulation and opening of CX43 hemichannels on hemangiosarcoma primary cells, allowing the release of immunogenic peptides. Representative images of untreated and Salmonella-infected hemangiosarcoma cells (A) and quantification of CX43 expression in 4 independent experiments. Mann–Whitney, p < 0.0001 (B). Quantification of ATP released upon Salmonella infection. Mann–Whitney, p = 0.0006 (C). Annexin V-7AAD staining, showing hemangiosarcoma cell viability (D).

Figure 2

Secretome derived from Salmonella-infected hemangiosarcoma cells used as a vaccine to induce an efficient immune response in dogs. Immunomonitoring was performed on sera and PBMCs of vaccinated dogs at several timepoints (T0 before the first vaccination, T1 after 1 dose, T2 after 2 doses, T3 after 5 doses). Scatterplot showing quantification of tumor-specific IgG in dogs’ sera through ELISA. Data are shown as normalized at T0. Multiple comparisons mixed model, p = 0.004 (on graph). Vaccination resulted in statistical significance, also according the mixed-effects model, p = 0.0013 (A). Quantification of IFN-γ produced by dogs’ PBMCs upon vaccine stimulation. Raw data expressed as OD at 450 nm are shown for PBMCs collected before vaccination (T0) and at the latest timepoint (Tlast). Wilcoxon matched-pairs signed-rank test, p = 0.0004 (B).

Figure 3

(A) Time to progression in 60 dogs with hemangiosarcoma treated with surgery and chemotherapy, grouped according to whether they received adjuvant peptide-based vaccination (solid line) or not (dashed line; p = 0.001). (B) Overall survival in 60 dogs with hemangiosarcoma treated with surgery and chemotherapy, grouped according to whether they received adjuvant peptide-based vaccination (solid line) or not (dashed line; p = 0.002).

Figure 4

(A) Time to progression in 38 dogs with splenic hemangiosarcoma treated with splenectomy and chemotherapy, grouped according to whether they received adjuvant peptide-based vaccination (solid line) or not (dashed line; p = 0.005). (B) Overall survival in 38 dogs with splenic hemangiosarcoma treated with splenectomy and chemotherapy, grouped according to whether they received adjuvant peptide-based vaccination (solid line) or not (dashed line; p = 0.004).

Figure 5

(A) Time to progression in 12 dogs with cardiac hemangiosarcoma treated with surgery and chemotherapy, grouped according to whether they received adjuvant peptide-based vaccination (solid line) or not (dashed line; p = 0.011). (B) Overall survival in 12 dogs with cardiac hemangiosarcoma treated with surgery and chemotherapy, grouped according to whether they received adjuvant peptide-based vaccination (solid line) or not (dashed line; p = 0.011).