Volume 26, Issue 2, Pages 435-445 (February 2018) Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting TERT  Elizabeth K. Duperret, Megan C. Wise, Aspen Trautz, Daniel O. Villarreal, Bernadette Ferraro, Jewell Walters, Jian Yan, Amir Khan, Emma Masteller, Laurent Humeau, David B. Weiner  Molecular Therapy  Volume 26, Issue 2, Pages 435-445 (February 2018) DOI: 10.1016/j.ymthe.2017.11.010 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 Delivery of αCTLA-4 or αPD-1 Post-first Vaccination Synergizes with TERT DNA Vaccine above Checkpoint Alone in Generating Anti-tumor Immune Response (A) Experimental setup. Mice were implanted with TC-1 tumor cells on day 0 and then immunized four times at 1-week intervals starting 7 days after tumor implant. Mice were given antibodies (200 μg per mouse) every 3 days starting 1 day after the first immunization. Antibody delivery was continued until 1 week after the final vaccination. (B, D, F, and H) Tumor volume measurements over time for naive mice, mTERT vaccine-treated mice, or mice treated with mTERT vaccine plus αCTLA-4 (B), αPD-1 (D), or a combination of αCTLA-4 and αPD-1 (F). (C, E, and G) Mouse survival over time for naive mice, mTERT vaccine-treated mice, or mice treated with mTERT vaccine plus αCTLA-4 (C), αPD-1 (D), or a combination of αCTLA-4 and αPD-1 (G). For (B)–(G), n = 12–13 mice per group. (H) Tumor volume measurements over time for naive mice unvaccinated mice treated with αPD-1, αCTLA-4, or both αPD-1 and αCTLA-4. (I) Mouse survival over time for naive mice or unvaccinated mice treated with αPD-1, αCTLA-4, or both αPD-1 and αCTLA-4. Mice were euthanized if they appeared sick or if the tumor diameter exceeded 1.5 cm. For (H) and (I), n = 9 mice per groiup. Each experiment was repeated at least once to verify results. Significance for tumor volume measurements was determined by two-way ANOVA followed by Tukey’s HSD test. Significance for mouse survival was determined by the Gehan-Breslow-Wilcoxon test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars indicate ± SEM. Molecular Therapy 2018 26, 435-445DOI: (10.1016/j.ymthe.2017.11.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 Antigen-Specific Intracellular Cytokine Production upon TERT DNA Vaccination and Immune Checkpoint Blockade in Tumor-Bearing Mice (A) Experimental setup. Mice were implanted with TC-1 tumor cells on day 0 and then immunized on days 7 and 14. Antibody delivery was started 1 day after the first immunization and continued every 3 days. Mice were sacrificed on day 21 for immune cell analysis. (B–F) Intracellular cytokine staining of CD8 or CD4 T cells after stimulation with native mouse TERT peptides for 5 hr. CD4 T cells were stained for IFN-γ (B) or TNF-α (C). CD8 T cells were stained for IFN-γ (D), TNF-α (E), or IFN-γ/CD107a/T-bet (F). Significance was determined by two-way ANOVA followed by Tukey’s HSD test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 7–10 mice per group. A representative of three independent experiments is shown. Error bars indicate ± SEM. Molecular Therapy 2018 26, 435-445DOI: (10.1016/j.ymthe.2017.11.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 Phenotype of Peripheral and Spleen T Cells upon TERT DNA Vaccination and Immune Checkpoint Blockade in Tumor-Bearing Mice (A–F) Staining of CD8 or CD4 T cells from spleen or peripheral blood of mice treated with the indicated therapies in accordance with the schedule in Figure 3A. CD8+ splenocytes (A) or PBMCs (B) were stained for PD-1. CD4+ splenocytes (C) or PBMCs (D) were stained for PD-1. CD4+ splenocytes (E) or PBMCs (F) were stained for CD25/FoxP3. Significance was determined by two-way ANOVA followed by Tukey’s HSD test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 7–10 mice per group. A representative of three independent experiments is shown. Error bars indicate ± SEM. Molecular Therapy 2018 26, 435-445DOI: (10.1016/j.ymthe.2017.11.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 Phenotype of Tumor-Infiltrating Lymphocytes upon TERT DNA Vaccination and Immune Checkpoint Blockade (A–F) Staining of CD8 or CD4 T cells isolated from the tumors of mice treated with the indicated therapies in accordance with the schedule in Figure 4A. mTERT-stimulated CD3+ TILs were stained for CD4/CD25/FoxP3 (A), CD8/PD-1 (B), CD8/CD44 (C), CD8/T-bet (E), or CD8/CD44/Tbet (F). PMA-stimulated CD3+ T cells were stained for CD8/CD44 (D). Significance was determined by two-way ANOVA followed by Tukey’s HSD test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 7–10 mice per group. A representative of three independent experiments is shown. Error bars indicate ± SEM. Molecular Therapy 2018 26, 435-445DOI: (10.1016/j.ymthe.2017.11.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 Delivery of αCTLA-4 in Combination with TERT DNA Vaccine Is Superior to Treg Depletion in Combination with TERT DNA Vaccine (A–C) Staining of CD8 or CD4 T cells from the peripheral blood (A), spleen (B), or tumor (C) of mice treated with the indicated therapies in accordance with the schedule in Figure 2. (D) Tumor volume measurements over time for mice with indicated treatment regimen. Mice were treated in accordance with the schedule shown in Figure 2A. (E) Mouse survival over time for mice with indicated treatment regimen. Mice were euthanized if they appeared sick or if the tumor diameter exceeded 1.5 cm. Significance for tumor volume measurements was determined by two-way ANOVA followed by Tukey’s HSD test. Significance for mouse survival was determined by the Gehan-Breslow-Wilcoxon test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 10 mice per group. Error bars indicate ± SEM. Molecular Therapy 2018 26, 435-445DOI: (10.1016/j.ymthe.2017.11.010) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions