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 > 7 × 1010 viral particles/cm3 versus 0% (0/25) for Ad-p53 < 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 10 12 viral 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.

Meeting abstracts The underlying cause for the failure of immune checkpoint blockade is the overwhelming, persistent and multifocal immune suppression in the tumor microenvironment. This is due to the absence of pre-existing antitumor Teff because of the action of important upstream immune

Meeting abstracts Hepatocellular carcinoma (HCC) is the third leading cause of cancer worldwide. The incidence and mortality of HCC have increased three-fold in the United States over the past few years and the majority of patients present with advanced disease. Bavituximab is a novel chimeric IgG1

B36 Biomarkers of the p53 target of adenoviral p53 gene therapy (Advexin) were evaluated for their ability to predict efficacy in 186 patients with recurrent SCCHN. Biomarker profiles predictive of adenoviral p53 efficacy were based upon p53 gene configurations assessed by sequence analyses and their level of protein expression determined by immunohistochemistry. The absence of high level protein expression of “dominant negative” p53 mutations that can block p53 activity were predictive of adenoviral p53 efficacy. In a Phase 3 randomized controlled trial comparing adenoviral p53 and methotrexate, p53 biomarker profiles in the adenoviral p53 treated patients favorable for adenoviral p53 efficacy were associated with a statistically significant increased survival compared to patients with unfavorable p53 profiles (median survival 7.2 vs. 2.7 months, p

Conventional cancer treatments include cytotoxic chemotherapies and radiotherapy, which result in significant collateral toxicities. The goal for future cancer treatments is to leverage improved understanding of cancer biology mechanisms and thereby develop targeted drugs that display exquisite tumor selectivity and avoid iatrogenic damage. In this review, we discuss the potential of tumor suppressor genes for development of cancer-selective drugs using the tumor suppressor p53 as an archetype.

Extensive stage small cell lung cancer (SCLC) is an incurable, aggressive form of lung cancer with little improvement in outcome over the past 2 decades. SCLC tumor cells frequently express a mu-tant form of the p53 protein, which is far more stable than wild type p53, and therefore over-expressed. This differential over-expression as compared to normal cells provides the basis for the proposed tumor vaccine. The dependence of tumor cells on abnormal p53 expression for their sur-vival makes this an “ideal” candidate for cancer immunotherapy, as the emergence of antigen-loss variants is not possible. We have developed a new vaccine based on transduction of dendritic cells (DC) with wild-type p53 using an adenoviral construct. This vaccine demonstrated potency in pre-clinical experiments. Based on those observation we initiated a phase I/II clinical trial that was designed to test the safety and efficacy of the Ad.p53-DC vaccine in patients who have extensive stage SCLC. Between January 2003 and June 2006, 54 patients with extensive stage small cell lung cancer were enrolled into trial. Patients were treated with platinum based first-line chemotherapy to best response and those patients with stable disease or minor progression but retaining ECOG performance status of 0 or 1 underwent leukapheresis 8 weeks after the last dose of chemotherapy. DC were generated from peripheral blood precursors and then infected with adenoviral construct containing wild-type p53 (ADVEXIN). The number of p53 positive DCs was evaluated using flow cytometry. Patients received 3 doses of vaccine i.d. (2–5×106 p53+ DCs per dose). If patients had stable disease or better, they were given 3 more doses of the vaccine monthly. No serious adverse events attributable to the vaccine occurred. No hematologic nor organ toxicity occured, and no autoimmunity developed. To evaluate immunological response, samples of peripheral blood were collected before immunization, 2-3 weeks after completion of 3 rounds of immunization and 2 months after completion of immunization. p53 specific immune responses were evaluated in ELISPOT assay using canarypox virus (ALVAC) containing wild-type p53 or empty vector using an automated ELISPOT reader (CTL). Response to vaccination was considered significant if a p53-specific response after immunization (2-3 weeks after 3rd vaccine) was more than 2 SD higher than the p53-specific response before immunization and at least 2 SD higher than the response to control ALVAC. Immune responses were further evaluated in a subset of 12 HLA-A2 positive patients, using p53-derived or control peptides presented by HLA-A2. Significant p53 specific T cell responses to vaccination were found in 41.9% of all treated patients. The pre-vaccination level of p53-specific immunity was similar in patients who responded immunologically to the vaccine to those who did not respond. We also analyzed the possible link between the number of DCs injected with each vaccine and p53 specific immune re-sponse developed as the result of immunization. No correlation between the number of injected DCs and p53-specific immune response was found. The level of anti-p53 antibody did not correlate with the development of p53-specific T-cell response or clinical response to vaccination. Anti-adenovirus antibodies could potentially play a critical role in limiting the effectiveness of adenovirus based cancer vaccines. Most patients had a detectable pre-immunization level of anti-Adv IgG antibody. After three rounds of immunization with Adv-p53 DC, the titer of anti-Adv antibody increased in 16 of 28 tested patients (57.1%). A moderate in-crease (>2 and <8 fold) was observed in 14 patients and a substantial (>8-fold) increase in 2 pa-tients. Both patients with the substantial increase in anti-adenovirus antibodies did not develop p53-specific T-cell responses to immunization, wheares such were observed in 12 out of 14 patients (85.7%) who had a moderately increased titer of anti-Adv antibodies, and in only 5 out of 13 pa-tients (38.5%) who had no detectable increase in antibody titer (p = 0.018). The anti-adenovirus antibodies detected in those patients had neutralizing activity. The generation of a p53-specific T-cell response depends not only on the quality of antigen stimulation but also on the functional activity of T-cells and antigen presenting cells in the host, specifically DCs. Our experiments demonstrated that immunological response to vaccination did not correlate with functional activity of DCs, T cells or the presence of regulatory T cells. Since myeloid derived suppressor cells (MDSC) have been implicated in the immunosuppressive state of tumor-bearing hosts the SCLC patients were examined for the presence of these cells. Patients with SCLC had elevated level of MDSC prior to vaccination (p=0.01). After vaccination their presence increased even further (p=0.002). All patients with a normal level of MDSC prior to vaccination developed p53 specific immune responses to vaccination (100%), compared with only 4 out of 12 pa-tients who had an elevated level of MDSC (25.0%) (p=0.012). Thus, it appears that patients with an increased presence of MDSC were less likely to develop responses to vaccination compared to the patients with a normal level of MDSC. Two patients developed PRs that were directly attributable to the vaccine. Twelve patients had SD at re-staging performed 2 weeks after the third vaccine. Thirty-three of the patients who developed progressive disease after the vaccine were treated with salvage chemotherapy. Seventeen (52%) patients had objective responses (3 CRs and 14 PRs). Twenty-two of these patients had platinum-resistant disease (progressed within 90 days of receiving a platinum containing regimen), and 10 of these patients (45%) responded to salvage chemotherapy (2 CRs and 8 PRs). This observation is remarkable because the historic response rates to salvage chemotherapy in similar patient populations are 6%-16%. Response to salvage chemotherapy correlated with the induction of a productive anti-p53 T cell response (p=0.025 in Fisher's exact test). The median survival of all of the treated patients from the time of the first vaccine administration was 8.8 months with 34% 1-year and 11% 2-year survivals. There was a clear trend toward significance of a difference in median survivals of those patients who developed a productive anti-p53 specific T cell response (13.7 months) compared to those who did not (8.8 months) (p=0.13). Thus, Ad.p53 DC vaccine itself was safe, and produced major tumor responses in two patients. P53-specific T cell responses were induced by the vaccine in half of the treated patients. Our data demonstrated that the lack of immune response to vaccination was closely associated with accumu-lation of MDSC, previously shown to be immunosuppressive. However, the main findings from the trial were an unusually high frequency of major objective tumor regressions in patients treated with chemotherapy immediately after the vaccine. These observations were quite unexpected since the existing paradigm suggests that chemotherapy is detrimental to the maintenance of an immune re-sponse. This suggests a possible new direction in cancer treatment where the combination of im-munotherapy and chemotherapy in direct sequence may provide substantial clinical benefits.

Li-Fraumeni syndrome is an autosomal dominant disorder that greatly increases the risk of developing multiple types of cancer. The majority of Li-Fraumeni syndrome families contain germ-line mutations in the p53 tumor suppressor gene. We describe treatment of a refractory, progressive Li-Fraumeni syndrome embryonal carcinoma with a p53 therapy (Advexin) targeted to the underlying molecular defect of this syndrome. p53 treatment resulted in complete and durable remission of the injected lesion by fluorodeoxyglucose-positron emission tomography scans with improvement of tumor-related symptoms. With respect to molecular markers, the patient's tumor had abnormal p53 and expressed coxsackie adenovirus receptors with a low HDM2 and bcl-2 profile conducive for adenoviral p53 activity. p53 treatment resulted in the induction of cell cycle arrest and apoptosis documented by p21 and cleaved caspase-3 detection. Increased adenoviral antibody titers after repeated therapy did not inhibit adenoviral p53 activity or result in pathologic sequelae. Relationships between these clinical, radiographic, and molecular markers may prove useful in guiding future application of p53 tumor suppressor therapy.

Patients with recurrent SCCHN need effective therapies that do not compound tumor morbidity. We investigated the long term safety and prognostic factors influencing efficacy of adenoviral p53 gene therapy in a large series of SCCHN patients.

B52 The p53 signaling pathway integrates diverse environmental stimuli and serves as the nodal point for regulation of cell proliferation or cell death via apoptosis. This pathway is disrupted or dysfunctional in >50% of human tumors. In squamous cell carcinoma of the head and neck (SCCHN), overexpression of p53 protein (p53 + ) is associated with poor patient outcome and poor tumor response to therapy. With the goal of evaluating p53 pathway markers, we have used immunohistochemical (IHC) analysis to evaluate tumor samples from 64 individuals with SCCHN. Overexpression of p53 protein (p53 + ) was found in 47/64 (73.4%) samples examined, as defined by ≥20% of cells positive for nuclear staining using the monoclonal DO7 antibody. Additional p53 pathway proteins were also examined, including HDM2, Bcl-2, p14 ARF , and survivin. Utilizing ≥20% of cells positive as criteria for expression, 47.6% (30/63) of tumors analyzed expressed HDM2, 27.0% (17/63) Bcl-2, 21.9% (14/64) p14 ARF , and 74.6% (44/59) survivin. A subset of the individuals with recurrent SCCHN was treated in Phase II clinical trials using p53 gene therapy (Advexin; Adenovirus-p53). In order to determine whether any of these proteins could potentially be used as a predictive biomarker for p53 gene therapy efficacy, analysis was carried out on the subset of patients treated with Advexin for whom clinical outcome information was available (n=18). Of the markers evaluated, only p53 protein overexpression had a statistically significant correlation with locoregional tumor disease control (CR, PR, or SD, all of >3 months duration) and increased median survival following Advexin treatment. 72.7% of patients with p53 + tumors demonstrated locoregional disease control, compared to only 14% of patients with p53 - tumors (p=0.05). In addition, the median survival of patients with tumors overexpressing p53 protein (p53 + ) was 11.0 months (95% CI 6.5-16.0), compared to only 3.0 months (95% CI 1.5-3.5) in patients whose tumors were p53 - (p