Volume 18, Issue 8, Pages 1559-1567 (August 2010) A Vaccine Targeting Telomerase Enhances Survival of Dogs Affected by B-cell Lymphoma  Daniela Peruzzi, Alessandra Gavazza, Giuseppe Mesiti, George Lubas, Elisa Scarselli, Antonella Conforti, Claus Bendtsen, Gennaro Ciliberto, Nicola La Monica, Luigi Aurisicchio  Molecular Therapy  Volume 18, Issue 8, Pages 1559-1567 (August 2010) DOI: 10.1038/mt.2010.104 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 dTERT expression in canine B-cell malignant lymphoma (ML). RNA was extracted from fine needle aspirates of lymph nodes from B-cell ML dogs. dTERT mRNA was quantified by TaqMan real-time PCR. HeLa transfected with pV1J-dTERT plasmid or mock-treated were used as positive and negative control of the assay, respectively. The amplicon resides in the exon 3–4 boundary. The results were confirmed with an independent set of primers/probe, amplifying a region in exon 2–3 boundary. Molecular Therapy 2010 18, 1559-1567DOI: (10.1038/mt.2010.104) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Induction of cell-mediated immunity to dTERT in malignant lymphoma (ML) dogs by Ad and DNA-EP regimen. Top: schematic representation of the vaccination and chemotherapy combination schedule. Vaccination (Vax) indicates Ad6 injections (triangles) and DNA-EP (arrows). Dark and light gray indicate the induction and maintenance phases of chemotherapy. ELISPOT assay performed on PBMCs from dTERT immunized ML dogs 2 weeks after the second Ad6 injection. Four pools of 15 mer peptides covering dTERT (pool A, B, C, and D) were utilized. Reactivity and distribution of the immune response in (a) La, (b) Su, and (c) Ca ML patients is reported. The assay was run in duplicate, and the average value from the replicates is shown. DMSO, dimethyl sulfoxide; PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell. Molecular Therapy 2010 18, 1559-1567DOI: (10.1038/mt.2010.104) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 The immune response is exclusively CD8+. Dog peripheral blood mononuclear cells were analyzed by intracellular staining for IFN-γ. The gating strategy for the identification of T-cell sub-population is shown on the top: left, lymphogate; right, CD8 and CD4 surface staining. The response of Ca, Ma, and La ML canine patients following two Ad6 injections against three different pools of peptides (A, B, and C) is shown. DMSO, dimethyl sulfoxide. Molecular Therapy 2010 18, 1559-1567DOI: (10.1038/mt.2010.104) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Kinetics of the dTERT-specific T-cell response in malignant lymphoma (ML) dogs. ML dogs were treated as described in the text and indicated in the top scheme in Figure 2. PBMCs were purified at the indicated time points, and the response was measured by ELISPOT with dTERT peptide pools. The total reactivity for dTERT peptide pools is calculated as follows: total reactivity = σ (dTertA + dTertB + dTertC + dTertD pools) − 4 (DMSO). (a) Kinetics of the response during Ad6/DNA-EP regimen in four selected subjects: Pi, Bo, Ce, and Ma dogs. Pi received only two Ad6 injections due to owner's lack of consent. (b) An ML dog (Ca) was treated as shown in top scheme. Ad6 injections were performed 2 weeks after the induction phase and during maintenance chemotherapy at weeks 0 and 2. Three cycles of DNA-EP were executed at weeks 6, 26, and 74. At every cycle, the T-cell response was restored. Triangles indicate Ad6 injections; arrows represent vaccinations by DNA-EP. PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell. Molecular Therapy 2010 18, 1559-1567DOI: (10.1038/mt.2010.104) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 dTERT vaccine prolongs time to first relapse and survival in malignant lymphoma (ML) dogs. (a) Kaplan–Meier plot of time to first relapse. Vaccinated dogs (Vax/Chemo) (dashed lines; n = 14) show a significantly longer time to first relapse than dogs on chemotherapy alone (Chemo) (dashed lines; n = 8) (P = 0.014; exact log-rank test). (b) Kaplan–Meier plot of overall survival. Vax/Chemo dogs (dashed lines; n = 14) show a significantly longer overall survival than dogs on Chemo alone (dashed lines; n = 8) (P = 0.0004; exact log-rank test). Both in (a) and (b), the curves start from the diagnosis of ML, almost coincident with the beginning of the chemotherapy induction phase. Molecular Therapy 2010 18, 1559-1567DOI: (10.1038/mt.2010.104) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions