Matthew J. Atherton

Evolution of oncolytic viruses: novel strategies for cancer treatment

Many viruses have documented oncolytic activity, with the first evidence observed clinically over a decade ago. In recent years, there has been a resurgence of interest in the field of oncolytic viruses. Viruses may be innately oncotropic, lacking the ability to cause disease in people or they may require engineering to allow selective tumor targeting and attenuation of pathogenicity. Following infection of a neoplastic cell, several events may occur, including direct viral oncolysis, apoptosis, necrotic cell death and autophagic cellular demise. Of late, a large body of work has recognized the ability of oncolytic viruses (OVs) to activate the innate and adaptive immune system, as well as directly killing tumors. The production of viruses expressing transgenes encoding for cytokines, colony-stimulating factors, costimulatory molecules and tumor-associated antigens has been able to further incite immune responses against target tumors. Multiple OVs are now in the advanced stages of clinical trials, with several individual viruses having completed their respective trials with positive results. This review introduces the multiple mechanisms by which OVs are able to act as an antineoplastic therapy, either on their own or in combination with other more traditional treatment modalities. The full benefit and the place where OVs will be integrated into standard-of-care therapies will be determined with ongoing studies ranging from the laboratory to the patient. With various different viruses now in the clinic this therapeutic option is beginning to prove its worth, and the versatility of these agents means further innovative and novel applications will continue to be developed.

S6K-STING interaction regulates cytosolic DNA–mediated activation of the transcription factor IRF3

Cytosolic DNA–mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.

Customized Viral Immunotherapy for HPV-Associated Cancer

Oncolytic Maraba virus can selectively infect HPV+ human cancers as well as generate substantial antitumor immunity. This resulted in complete destruction of advanced HPV+ tumors in mice, providing a promising immunological approach to combat HPV-associated cancer. The viral-transforming proteins E6 and E7 make human papillomavirus–positive (HPV+) malignancies an attractive target for cancer immunotherapy. However, therapeutic vaccination exerts limited efficacy in the setting of advanced disease. We designed a strategy to induce substantial specific immune responses against multiple epitopes of E6 and E7 proteins based on an attenuated transgene from HPV serotypes 16 and 18 that is incorporated into MG1-Maraba virotherapy (MG1-E6E7). Mutations introduced to the transgene abrogate the ability of E6 and E7 to perturb p53 and retinoblastoma, respectively, while maintaining the ability to invoke tumor-specific, multifunctional CD8+ T-cell responses. Boosting with MG1-E6E7 significantly increased the magnitude of T-cell responses compared with mice treated with a priming vaccine alone (greater than 50 × 106 E7-specific CD8+ T cells per mouse was observed, representing a 39-fold mean increase in boosted animals). MG1-E6E7 vaccination in the HPV+ murine model TC1 clears large tumors in a CD8+-dependent manner and results in durable immunologic memory. MG1-Maraba can acutely alter the tumor microenvironment in vivo and exploit molecular hallmarks of HPV+ cancer, as demonstrated by marked infection of HPV+ patient tumor biopsies and is, therefore, ideally suited as an oncolytic treatment against clinical HPV+ cancer. This approach has the potential to be directly translatable to human clinical oncology to tackle a variety of HPV-associated neoplasms that cause significant morbidity and mortality globally. Cancer Immunol Res; 5(10); 847–59. ©2017 AACR.

Preclinical development of peptide vaccination combined with oncolytic MG1-E6E7 for HPV-associated cancer

Human papilloma virus (HPV)-associated cancer is a significant global health burden and despite the presence of viral transforming antigens within neoplastic cells, therapeutic vaccinations are ineffective for advanced disease. HPV positive TC1 cells are susceptible to viral oncolysis by MG1-E6E7, a custom designed oncolytic Maraba virus. Epitope mapping of mice vaccinated with MG1-E6E7 enabled the rational design of synthetic long peptide (SLP) vaccines against HPV16 and HPV18 antigens. SLPs were able to induce specific CD8+ immune responses and the magnitude of these responses significantly increased when boosted by MG1-E6E7. Logically designed vaccination induced multi-functional CD8+ T cells and provided complete sterilising immunity of mice challenged with TC1 cells. In mice bearing large HPV-positive tumours, SLP vaccination combined with MG1-E6E7 was able to clear tumours in 60% of mice and these mice were completely protected against a long term aggressive re-challenge with the TC1 tumour model. Combining conventional SLPs with the multi-functional oncolytic MG1-E6E7 represents a promising approach against advanced HPV positive neoplasia.

Transforming the prostatic tumor microenvironment with oncolytic virotherapy

ABSTRACT Prostate cancer (PCa) was estimated to have the second highest global incidence rate for male non-skin tumors and is the fifth most deadly in men thus mandating the need for novel treatment options. MG1-Maraba is a potent and versatile oncolytic virus capable of lethally infecting a variety of prostatic tumor cell lines alongside primary PCa biopsies and exerts direct oncolytic effects against large TRAMP-C2 tumors in vivo. An oncolytic immunotherapeutic strategy utilizing a priming vaccine and intravenously administered MG1-Maraba both expressing the human six-transmembrane antigen of the prostate (STEAP) protein generated specific CD8+ T-cell responses against multiple STEAP epitopes and resulted in functional breach of tolerance. Treatment of mice with bulky TRAMP-C2 tumors using oncolytic STEAP immunotherapy induced an overt delay in tumor progression, marked intratumoral lymphocytic infiltration with an active transcriptional profile and up-regulation of MHC class I. The preclinical data generated here offers clear rationale for clinically evaluating this approach for men with advanced PCa.

Preclinical evaluation of a MAGE-A3 vaccination utilizing the oncolytic Maraba virus currently in first-in-human trials

ABSTRACT Multiple immunotherapeutics have been approved for cancer patients, however advanced solid tumors are frequently refractory to treatment. We evaluated the safety and immunogenicity of a vaccination approach with multimodal oncolytic potential in non-human primates (NHP) (Macaca fascicularis). Primates received a replication-deficient adenoviral prime, boosted by the oncolytic Maraba MG1 rhabdovirus. Both vectors expressed the human MAGE-A3. No severe adverse events were observed. Boosting with MG1-MAGEA3 induced an expansion of hMAGE-A3-specific CD4+ and CD8+ T-cells with the latter peaking at remarkable levels and persisting for several months. T-cells reacting against epitopes fully conserved between simian and human MAGE-A3 were identified. Humoral immunity was demonstrated by the detection of circulating MAGE-A3 antibodies. These preclinical data establish the capacity for the Ad:MG1 vaccination to engage multiple effector immune cell populations without causing significant toxicity in outbred NHPs. Clinical investigations utilizing this program for the treatment of MAGE-A3-positive solid malignancies are underway (NCT02285816, NCT02879760).

Abstract B077: Evaluating oncolytic vaccines targeting mutated epitopes in a murine fibrosarcoma model CMS-5

Oncolytic viruses have traditionally been used as stand alone therapy delivered to animals and patients to lytically destroy tumor tissue. However, it has recently become clear that involvement of the immune system is an extremely important aspect of these therapies. To further enhance anti-tumor immune responses we have utilized an oncolytic vaccine approach. This approach involves priming the immune system to a specific tumor associated antigen (TAA) with an Adenovirus and then subsequently delivering an oncolytic Rhabdovirus expressing the same tumor antigen leading to robust anti-tumor immune responses and improved efficacy. For the studies presented herein we have developed oncolytic vaccines targeting mutated epitopes expressed in CMS-5. Immune responses generated towards these epitopes through oncolytic vaccination will be characterized and their ability to control tumor growth will be evaluated. We hypothesize that the foreign nature of these mutated epitopes will allow for the generation of substantial immune responses following oncolytic vaccination in tumor bearing mice. In preliminary studies, oncolytic vaccination has been able to generate robust immune responses leading to as many as 60% of circulating CD8+ T cells responding to a single mutated epitope in tumor free animals. Therapeutic oncolytic vaccination of CMS-5 tumor bearing animals was still able to generate large anti-tumor immune responses, however these responses were cut in half compared to tumor free animals. The resultant anti-tumor immune response was able to significantly control tumor growth, and the magnitude of these responses correlated with survival. Even though there was a delay in tumor growth following therapy, the responses were not durable and all mice ultimately failed the therapy. We are currently engaged in studies to further characterize the immune responses generated and determine what, if any, immunosuppression led to failure of our therapy; even in the presence of anti-tumor immune responses exceeding 20% of peripheral CD8+ T cells. Nanostring analysis of the tumor microenvironment are being conducted focusing on gene signatures for immune cells and inflammation and these data will be confirmed by histology and flow cytometry. These studies will help shed light on potential mechanisms of immune evasion/suppression following oncolytic vaccination and point to potential targets for modulation of the oncolytic vaccine induced immune response. Citation Format: Kyle Stephenson, Matthew Atherton, John Bell, Brian Lichty. Evaluating oncolytic vaccines targeting mutated epitopes in a murine fibrosarcoma model CMS-5. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B077.

Abstract B096: Oncolytic vaccination for HPV induced cancer

Oncogenic human papilloma virus (HPV) is directly responsible for 5% of the world9s cancer burden. HPV has been etiologically implicated in virtually all cases of cervical carcinoma and is increasingly responsible for head and neck cancers in the developed world with strains 16 and 18 considered high risk. Recently it was estimated that there were over half a million new cases of cervical cancer worldwide and in the same year this disease was projected to claim over 250,00 lives, making it the third most prevalent and fourth most lethal non- skin tumor in women. Current standard of care for advanced cervical carcinoma and head and neck cancers involve chemo-radiation with or without prior surgical resection resulting in significant morbidity without guaranteed cure. Screening programs and prophylactic vaccination are reducing the incidence of some HPV induced tumors, however lack of compliance and unvaccinated populations mean that significant illness and mortality still exists. We therefore sought to develop a novel pre-clinical immunotherapeutic for HPV induced cancers. An oncolytic vaccination strategy utilizing an attenuated Maraba MG1 rhabdovirus expressing inactive HPV antigens based on the E6 and E7 transforming proteins of strains 16 and 18 has been designed (MG1 E6E7). The TC1 cell line, which is a C57BL/6 syngeneic murine HPV cancer model, was acquired. Specific anti-E7 CD8+ T cell responses have been induced after oncolytic vaccination of mice generating sterilizing immunity against future engraftment with TC1 cells. Oncolytic vaccination of tumor free mice has generated between 24 and 50 million, combined blood and splenic CD8+ T cells per mouse, determined by interferon gamma production in response to a single E7 peptide quantified using intracellular staining and flow cytometry. Primed mice, boosted with MG1 E6E7 develop long lasting, marked and specific immunity against E7 and to a lesser extent E6. The prime: boost regimen has therapeutic efficacy against established subcutaneous TC1 tumors (mean volumes of 250 mm3) resulting in durable cures in this model. Depletion of CD8+ T cells impairs the activity of the vaccination protocol supporting the key role of cytotoxic lymphocytes in our treatment regimen. The data generated here support the pre-clinical activity of this oncolytic vaccination and pave the way for future clinical trials for the treatment of advanced metastatic HPV induced carcinomas. Citation Format: Matthew J. Atherton, Kyle B. Stephenson, Yonghong Wan, David F. Stojdl, Brian D. Lichty. Oncolytic vaccination for HPV induced cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B096.