Safety and efficacy of a genetic vaccine targeting telomerase plus chemotherapy for the therapy of canine B-cell lymphoma

Abstract

Client-owned pet dogs represent exceptional translational models for advancement of cancer research because they reflect the complex heterogeneity observed in human cancer. We have recently shown that a genetic vaccine targeting dog telomerase reverse transcriptase (dTERT) and based on adenovirus DNA electro-gene-transfer (Ad/DNA-EGT) technology can induce strong cell-mediated immune responses against this tumor antigen and increase overall survival of dogs affected by B-cell lymphosarcoma (LSA) in comparison with historical controls when combined with a cyclophosphamide, vincristine, and prednisone (COP) chemotherapy regimen. Here, we have conducted a double-arm clinical trial with an extended number of LSA patients, measured the antigen-specific immune response, and evaluated potential toxic effects of the immunotherapy along with a follow-up of patients survival for 3.5 years. The immune response was measured by enzyme-linked immunospot assay. The expression of dTERT was quantified by quantitative polymerase chain reaction. Changes in hematological parameters, local/systemic toxicity or organic dysfunction and fever were monitored over time during the treatment. dTERT-specific cell-mediated immune responses were induced in almost all treated animals. No adverse effects were observed in any dog patient that underwent treatment. The overall survival time of vaccine/COP-treated dogs was significantly increased over the COP-only cohort (>76.1 vs. 29.3 weeks, respectively, p<0.0001). There was a significant association between dTERT expression levels in LSA cells and overall survival among vaccinated patients. In conclusion, Ad/DNA-EGT-based cancer vaccine against dTERT in combination with COP chemotherapy is safe and significantly prolongs the survival of LSA canine patients. These data confirm the therapeutic efficacy of dTERT vaccine and support the evaluation of this approach for other cancer types as well as the translation of this approach to human clinical trials.

FIG. 1.

Vaccination schedule and electro-gene-transfer (EGT) procedure. (A) Schematic representation of the vaccination and chemotherapy combination schedule. Dark and light gray indicate the induction and maintenance phases of chemotherapy. Two weeks after the end of induction phase (week 8–10) and during the maintenance phase, dogs were vaccinated with adenovirus (Ad; triangles, 2 weeks apart) and 4–6 weeks later with DNA-EGT cycles (arrows) consisting of three injections spaced by 2-week intervals. A second cycle was repeated 2–3 months later. Black diamonds show the time points when blood was collected for detection of the immune response and other analyses. (B) Needles and electrodes. The side-hole needles were modified to inject the DNA solution in the area between them and act as electrodes, since they are connected with the pulse generator. Arrows indicate the side holes. (C) Schematic representation of the DNA injection in the tibial muscle and the DNA-EGT procedure. Needles were inserted into the muscle at an angle of approximately 45°.

FIG. 2.

Characterization of T-cell immune response. (A) Spontaneous immune response against dog telomerase reverse transcriptase (dTERT). Peripheral mononuclear cells (PBMCs) were purified from six healthy dogs (three beagles, one Labrador Retriever, and two mixed) and the 21 lymphosarcoma (LSA) dogs from the VAC cohort after the induction phase (IP). Each single dot indicates the total reactivity to dTERT peptide pools of a single subject. The bars show the geometric mean of the groups: 5.12±6.88 and 9.12±14.46 for healthy and LSA dogs, respectively. Student's t-test, p=0.35. (B) Kinetics of the dTERT-specific T-cell response in VAC arm. LSA dogs were treated as described in the text and indicated in the top scheme in Fig. 1. PBMCs were purified at the indicated time points and the response was measured by enzyme-linked immunospot (ELISPOT) with dTERT peptide pools. The graph is in log scale. *Student's t-test, p<1×10⁵ compared with prevaccination. The total reactivity for dTERT peptide pools is calculated as Total reactivity=Σ(dTertA+dTertB+dTertC+dTertD pools)−4 (dimethyl sulfoxide). (C) Distribution of the immune reactivity within dTERT. dTertA, dTertB, dTertC, and dTertD pools were used to stimulate PBMCs 2 weeks after the second Ad injection. ELISPOT assays were run in quadruplicates at the indicated time points.

FIG. 3.

dTERT vaccine augments LSA patients' survival. (A) Kaplan-Meier plot of time to first relapse. Vaccinated dogs (VAC, black line; n=21) do not show a significantly longer progression-free survival than dogs on chemotherapy alone (CTR, gray line; n=21). Median progression-free survival is 11.4 and 11.3 weeks for VAC and CTR groups, respectively (p>0.1, log-rank test). (B) Kaplan-Meier plot of overall survival. VAC arm (black line; n=21) show a significantly longer overall survival than CTR arm (gray line; n=21). Median overall survival (OS) is 76.1 and 29.3 weeks for VAC and CTR cohort, respectively (p<0.0001, log-rank test).

FIG. 4.

dTERT is a potential prognostic factor for dTERT vaccinated LSA patients' survival. RNA was extracted from six fine-needle aspirates of lymph nodes from B-cell LSA dogs as described in the text and Materials and Methods. dTERT mRNA was quantified by Taqman real-time polymerase chain reaction. There was a statistically significant association between dTERT expression levels and overall survival among vaccinated patients (Spearman's rho=0.80, p<0.05).