Background While immune checkpoint inhibitors are becoming a standard of care for multiple types of cancer, the majority of patients do not respond to this form of immunotherapy. New approaches are required to overcome resistance to immunotherapies. Methods We investigated the effects of adenoviral p53 (Ad-p53) gene therapy in combination with immune checkpoint inhibitors and selective IL2 or IL15 CD122/132 agonists in the aggressive B16F10 tumor model resistant to immunotherapies. To assess potential mechanisms of action, pre- and post- Ad-p53 treatment biopsies were evaluated for changes in gene-expression profiles by Nanostring IO 360 assays. Results The substantial synergy of “triplet” Ad-p53 + CD122/132 + anti-PD-1 therapy resulted in potential curative effects associated with the complete tumor remissions of both the primary and contralateral tumors. Interestingly, contralateral tumors, which were not injected with Ad-p53 showed robust abscopal effects resulting in statistically significant decreases in tumor size and increased survival (p < 0.001). None of the monotherapies or doublet treatments induced the complete tumor regressions. Ad-p53 treatment increased interferon, CD8+ T cell, immuno-proteosome antigen presentation, and tumor inflammation gene signatures. Ad-p53 treatment also decreased immune-suppressive TGF-beta, beta-catenin, macrophage, and endothelium gene signatures, which may contribute to enhanced immune checkpoint inhibitor (CPI) efficacy. Unexpectedly, a number of previously unidentified, strongly p53 downregulated genes associated with stromal pathways and IL10 expression identified novel anticancer therapeutic applications. Conclusions These results imply the ability of Ad-p53 to induce efficacious local and systemic antitumor immune responses with the potential to reverse resistance to immune checkpoint inhibitor therapy when combined with CD122/132 agonists and immune checkpoint blockade. Our findings further imply that Ad-p53 has multiple complementary immune mechanisms of action, which support future clinical evaluation of triplet Ad-p53, CD122/132 agonist, and immune checkpoint inhibitor combination treatment.

BackgroundWe conducted an analysis of previous adenoviral p53 (Ad-p53) treatment data in recurrent head and neck squamous cell carcinoma (HNSCC) patients to identify optimal Ad-p53 treatment methods for future clinical trials.MethodsThe analysis involved recurrent HNSCC patients treated with Ad-p53 for whom p53 genotyping and immunohistochemistry tumor biomarker studies had been performed (n = 70). Ad-p53 tumor treatment responses defined by RECIST 1.1 criteria were correlated with Ad-p53 dose and tumor p53 biomarkers. Gene expression profiles induced by Ad-p53 treatment were evaluated using the Nanostring IO 360 panel.ResultsAd-p53 dose based upon the injected tumor volume had a critical effect on tumor responses. All responders had received Ad-p53 doses greater than 7 × 1010 viral particles/cm3 of tumor volume. There was a statistically significant difference in tumor responses between patients treated with greater than 7 × 1010 viral particles/cm3 compared to patients treated at lower Ad-p53 doses (Tumor Response 31% (9/29) for Ad-p53 &gt; 7 × 1010 viral particles/cm3 versus 0% (0/25) for Ad-p53 &lt; 7 × 1010 viral particles/cm3; p = 0.0023). All responders were found to have favorable p53 biomarker profiles defined by less than 20% p53 positive tumor cells by immunohistochemistry (IHC), wild type p53 gene sequence or p53 deletions, truncations, or frame-shift mutations without functional p53 tetramerization domains. Preliminary gene expression profiling results revealed that Ad-p53 treatment increased interferon signaling, decreased TGF-beta and beta-catenin signaling resulting in an increased CD8+ T cell signature which are associated with increased responses to immune checkpoint blockade.ConclusionsOur findings have important implications for future p53 targeted cancer treatments and identify fundamental principles to guide Ad-p53 gene therapy. We discovered that previous Ad-p53 clinical trials were negatively impacted by the inclusion of patients with unfavorable p53 biomarker profiles and by under dosing of Ad-p53 treatment. Future Ad-p53 clinical trials should have favorable p53 biomarker profiles inclusion criteria and Ad-p53 dosing above 7 × 1010 viral particles/cm3 of injected tumor volume. Preliminary gene expression profiling identified p53 mechanisms of action associated with responses to immune checkpoint blockade supporting evaluation of Ad-p53 in combination with immune checkpoint inhibitors.

64 Background: Immune checkpoint inhibitors represent an important advance in cancer therapeutics. However, most cancer patients either do not respond or become resistant to this therapy. Methods: We evaluated the ability of tumor suppressors p53 and IL-24, delivered via adenoviral vectors, to reverse immune checkpoint inhibitor resistance and induce abscopal therapeutic effects in the highly immune therapy resistant murine B16F10 melanoma tumor model. To mimic clinical conditions of immune checkpoint inhibitor resistance, animals with established tumors were treated with anti-PD-1 before intra-tumoral delivery of adenoviral vectors encoding tumor suppressors p53 (Ad-p53) or IL-24 (Ad-IL24). Results: Anti-PD-1 had minimal or no therapeutic efficacy when compared to PBS controls. However, there was a reversal of anti-PD-1 resistance in animals treated with Ad-p53, or Ad-IL24, in combination with anti-PD-1. Evaluation of primary tumor growth using ANOVA confirmed synergistic effects of the combination treatments over either agent used as monotherapy (p = 0.0001 for p53, p = 0.002 for IL-24). We also observed statistically significant decreases in contralateral abscopal tumor growth in animals whose primary tumors were treated with either Ad- p53 or Ad-IL24 (p = 0.046, and p = 0.0021, respectively) or in combination with anti-PD-1 (p = 0.02 p53, and p < 0.0001 for IL-24) as compared to animals treated with anti-PD-1 alone. With respect to survival, combined tumor suppressor + anti-PD-1 therapy resulted in a statistically significant increase in survival compared to: a) tumor suppressor therapy alone (p = 0.01 for either Ad-p53 and or IL-24) and b) anti-PD-1 therapy alone (p < 0.001 for p53, and p = x for IL-24). Conclusions: Overall, these results indicate that IL-24 and p53 tumor suppressor immune gene therapy can reverse immune checkpoint inhibitor resistance in primary tumors, and induce abscopal effects inferring the activation of systemic anti-tumor immune responses that reverse resistance to immune checkpoint inhibitor therapy.

e14610 Background: Immune checkpoint inhibitors represent an important advance in cancer therapeutics. However, most cancer patients do not respond or become resistant to this form of immune therapy. Methods: We evaluated the ability of Ad-p53 to reverse immune checkpoint inhibitor resistance and induce abscopal effects in the immune therapy resistant murine B16F10 melanoma tumor model. To mimic clinical conditions of checkpoint inhibitor resistance, animals with established tumors were treated with anti-PD-1 before initiating Ad-p53 intra-tumoral therapy. Results: Anti-PD-1 had minimal therapeutic efficacy compared to control treatment. A statistical analysis of variance (ANOVA) comparison of tumor volumes revealed that the combined effect of Ad-p53 and anti-PD-1 treatment was synergistic and superior to either therapy alone (p = 0.0001). Surprisingly, there was a statistically significant abscopal effect with decreased growth of contralateral tumors not injected with Ad -p53. The Ad- p53 alone (p = 0.046) and Ad- p53 + anti-PD-1 (p = 0.0243) treatment groups both demonstrated a statistically significant decreased abscopal tumor growth compared to treatment with anti-PD-1 alone. Combined Ad- p53 and anti-PD-1 therapy demonstrated a statistically significant increase in survival compared to Ad- p53 therapy alone (p = 0.0167) and anti-PD-1 therapy alone (p < 0.001) by the log rank test. We have initiated a Phase 1 clinical trial of Ad-p53 intra-hepatic arterial therapy in combination with capecitabine for patients with solid tumor liver metastases. In the first cohort of patients at a dose of 2 x 1012viral particles, treatment has been well tolerated with transient fever, chills and rigors. In one patient, decreased SUV uptake on PET scans of distant lymph node metastases suggested possible abscopal effects. Conclusions: These results suggest that Ad-p53 tumor suppressor immune gene therapy may reverse immune checkpoint inhibitor resistance and induce abscopal effects supporting the planned clinical evaluation of combined Ad-p53 and anti-PD-1 therapy in patients resistant to immune checkpoint inhibitor therapy. Clinical trial information: NCT02842125.

BackgroundWhile immune checkpoint inhibitors are becoming a standard of care for multiple types of cancer, the majority of patients do not respond to this form of immunotherapy. New approaches are required overcome resistance to immunotherapies.MethodsWe investigated the effects of adenoviral p53 (Ad-p53) gene therapy in combination with immune checkpoint inhibitors and selective IL2 or IL15 CD122/132 agonists in the aggressive B16F10 tumor model resistant to immunotherapies. To assess potential mechanisms action, pre and post Ad-p53 treatment biopsies were evaluated for changes in gene expression profiles by Nanostring IO 360 assays.ResultsSubstantial synergy of “triplet” Ad-p53 + CD122/132 + anti-PD-1 therapy resulted in potential curative effects associated with complete tumor remissions of both primary and contralateral tumors. Interestingly, contralateral tumors which were not injected with Ad-p53 showed robust abscopal effects resulting in statistically significant decreases in tumor size and increased survival (p<0.001). None of the monotherapies or doublet treatments induced complete tumor regressions. Ad-p53 treatment increased Type I Interferon, CD8+ T cell, immuno-proteosome antigen presentation and tumor inflammation gene signatures. Ad-p53 treatment also decreased immune suppressive TGF-beta, beta-catenin, macrophage, and endothelium gene signatures, which may contribute to enhanced immune checkpoint inhibitor (CPI) efficacy. Unexpectedly, a number of previously unidentified, strongly p53 down regulated genes associated with stromal pathways and IL10 expression identified novel anti-cancer therapeutic applications.ConclusionsThese results imply the ability of Ad-p53 to induce efficacious local and systemic anti-tumor immune responses with the potential to reverse resistance to immune checkpoint inhibitor therapy when combined with CD122/132 agonists and immune checkpoint blockade. Our findings further imply that Ad-p53 has multiple complimentary immune mechanisms of action which support future clinical evaluation of triplet Ad-p53, CD122/132 agonist and immune checkpoint inhibitor combination treatment.