Tumor suppressor immune gene therapy to reverse immunotherapy resistance.

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.

Analysis of Adenoviral p53 Gene Therapy Clinical Trials in Recurrent Head and Neck Squamous Cell Carcinoma.

BACKGROUND: We 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. METHODS: The 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. RESULTS: Ad-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. CONCLUSIONS: Our 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.

Docetaxel Combined With Bavituximab in Previously Treated, Advanced Nonsquamous Non-Small-Cell Lung Cancer.

BACKGROUND: Bavituximab is a phosphatidylserine-targeting antibody with a selective tumor, vascular-directed immune response. In this phase II trial the efficacy and safety of bavituximab combined with docetaxel for previously treated, advanced nonsquamous non-small-cell lung cancer were evaluated. PATIENTS AND METHODS: Patients were randomized 1:1:1 to receive docetaxel 75 mg/m(2) every 21 days for up to 6 cycles combined with weekly, blinded infusions of placebo, bavituximab 1 mg/kg, or bavituximab 3 mg/kg until disease progression or unacceptable toxicity. The primary end point was overall response rate (ORR), with a predefined end point of 26% in the bavituximab arms. After study unblinding, vial-coding discrepancies were discovered in the placebo and bavituximab 1 mg/kg groups. In exploratory analyses, data from these groups were pooled to form the control group and compared with the 3 mg/kg group. RESULTS: Efficacy end points in the bavituximab 3 mg/kg group (n = 41) and in the placebo/bavituximab 1 mg/kg group (n = 80), respectively, were as follows: ORR, 17.1% (95% confidence interval [CI], 5.6%-28.6%) and ORR, 11.3% (95% CI, 4.3%-18.2%); median progression-free survival 4.5 and 3.3 months (hazard ratio [HR], 0.74 [95% CI, 0.45-1.21]; P = .24); median overall survival 11.7 and 7.3 months (HR, 0.66 [95% CI, 0.40-1.10]; P = .11). Toxicities were manageable and similar between arms. CONCLUSION: The combination of bavituximab and docetaxel is well tolerated. Although no firm efficacy conclusions can be drawn and the trial did not meet the predefined primary end point, exploratory analyses suggest trends favoring the combination of bavituximab 3 mg/kg with docetaxel. This regimen is being evaluated in the ongoing, global, phase III SUNRISE trial.

Biomarkers Predict p53 Gene Therapy Efficacy in Recurrent Squamous Cell Carcinoma of the Head and Neck.

PURPOSE: Most recurrent squamous cell carcinomas of the head and neck have a dysfunctional p53 tumor suppressor pathway contributing to treatment resistance. We hypothesized that tumor p53 biomarkers may predict the efficacy of normal p53 delivered by gene therapy in these patients. EXPERIMENTAL DESIGN: Tumor p53 biomarkers were evaluated in 116 patients, including 29 treated with methotrexate in a phase III randomized controlled trial. Profiles favorable for p53 gene therapy efficacy were hypothesized to have either normal p53 gene sequences or low-level p53 protein expression, whereas unfavorable p53 inhibitor profiles were predicted to have high-level expression of mutated p53 that can inhibit normal p53 protein function. RESULTS: A statistically significant increase in tumor responses was observed for patients with favorable p53 efficacy profiles compared with those with unfavorable p53 inhibitor profiles [phase I/II trials: favorable (34 of 46, 74%) versus unfavorable (1 of 5, 20%), P = 0.0290; phase III trial: favorable (17 of 24, 71%) versus unfavorable (2 of 11, 18%), P = 0.0088]. In the phase III trial, there was statistically significant increased time to progression (TTP) and survival following p53 gene therapy in patients with favorable p53 profiles compared with unfavorable p53 inhibitor profiles (median TTP, 2.7 months versus 1.4 months, P = 0.0121; median survival, 7.2 months versus 2.7 months, P < 0.0001). In contrast, the biomarker profiles predictive of p53 gene therapy efficacy did not predict methotrexate response, TTP, or survival outcomes. CONCLUSIONS: These results indicate that tumor p53 biomarker profiles may predict p53 gene therapy efficacy in recurrent squamous cell carcinoma of the head and neck. (Clin Cancer Res 2009;15(24):7719-25).

Cancer targeting using tumor suppressor genes.

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.

p53 therapy in a patient with Li-Fraumeni syndrome.

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.

A nonreplicating adenoviral vector that contains the wild-type p53 transgene combined with chemotherapy for primary breast cancer: safety, efficacy, and biologic activity of a novel gene-therapy approach.

BACKGROUND: : Primary systemic therapy (PST) is the standard approach to the management of patients with locally advanced breast cancer (LABC). The authors hypothesized that the intratumoral administration of a nonreplicating adenoviral vector (Ad5) that contains the human wild-type p53, AdCMV-p53, combined with chemotherapy, could increase the efficacy of PST as measured by pathologic complete response. METHODS: : In a prospective, open-label, Phase II trial, 13 patients with LABC were treated with 6 3-week cycles of PST, which consisted of intratumoral injections of Ad5CMV-p53 for 2 consecutive days plus docetaxel and doxorubicin followed by surgery. p53 status was determined at baseline and was assessed immediately after the first injection (up to 48 hours). Clinical response was assessed by clinical and radiologic methods. RESULTS: : The trial was terminated early, because none of the patients achieved a pathologic complete response. The median age was 56 years (range, 39-71 years), and the median tumor size was 8 cm (range, 5-11 cm). Eight patients (73%) had a p53 mutation. Serial biopsies showed an increase in p53 messenger RNA (mRNA) and p21(WAF1/Cip1) mRNA. All 12 evaluable patients achieved an objective clinical response. The surgical specimens revealed scattered tumor cells with extensive tumor-infiltrate leukocytes (predominantly T-lymphocytes). At a median follow-up of 37 months (range, 30-41 months), 4 patients (30%) developed systemic recurrence, and 2 patients died. The estimate breast cancer-specific survival rate at 3 years was 84% (95% confidence interval, 65.7-100%). There was no increase in systemic toxicity. CONCLUSIONS: : Ad5CMV-p53 combined with PST is safe, active, and associated with local immunomodulatory effects. The promising clinical activity of this combination deserves further investigation in randomized studies.

Combination of p53 cancer vaccine with chemotherapy in patients with extensive stage small cell lung cancer.

PURPOSE: The initial goal of this study was to test the immunologic and clinical effects of a new cancer vaccine consisting of dendritic cells (DC) transduced with the full-length wild-type p53 gene delivered via an adenoviral vector in patients with extensive stage small cell lung cancer. EXPERIMENTAL DESIGN: Twenty-nine patients with extensive stage small cell lung cancer were vaccinated repeatedly at 2-week intervals. Most of the patients received three immunizations. p53-specific responses were evaluated, and phenotype and function of T cells, DCs, and immature myeloid cells were analyzed and correlated with antigen-specific immune responses. Objective clinical response to vaccination as well as subsequent chemotherapy was evaluated. RESULTS: p53-specific T cell responses to vaccination were observed in 57.1% of patients. Immunologic responses to vaccination were positively associated with a moderate increase in the titer of antiadenovirus antibodies, and negatively with an accumulation of immature myeloid cells. One patient showed a clinical response to vaccination whereas most of the patients had disease progression. However, we observed a high rate of objective clinical responses to chemotherapy (61.9%) that immediately followed vaccination. Clinical response to subsequent chemotherapy was closely associated with induction of immunologic response to vaccination. CONCLUSIONS: This study provides clinical support for an emerging paradigm in cancer immunotherapy, wherein optimal use of vaccination might be more effective, not as a separate modality, but in direct combination with chemotherapy.