Antti Vuolanto

Repeated intratumoral administration of ONCOS-102 leads to systemic antitumor CD8+ T-cell response and robust cellular and transcriptional immune activation at tumor site in a patient with ovarian cancer

Adenoviruses are excellent immunotherapeutic agents with a unique ability to prime and boost immune responses. Recombinant adenoviruses cause immunogenic cancer cell death and subsequent release of tumor antigens for antigen presenting cells, resulting in the priming of potent tumor-specific immunity. This effect may be further enhanced by immune-stimulating transgenes expressed by the virus. We report a case of a 38-year-old female with Stage 3 metastatic micropapillary serous carcinoma of the ovary. She was treated in a Phase I study with a granulocyte-macrophage colony stimulating factor (GMCSF)-expressing oncolytic adenovirus, Ad5/3-D24-GMCSF (ONCOS-102). The treatment resulted in progressive infiltration of CD8+ lymphocytes into the tumor and concomitant systemic induction of several tumor-specific CD8+ T-cell populations. The patient was alive at the latest follow up more than 20 months after initiation of the study.

Local treatment of a pleural mesothelioma tumor with ONCOS-102 induces a systemic antitumor CD8 + T-cell response, prominent infiltration of CD8 + lymphocytes and Th1 type polarization

Late stage cancer is often associated with reduced immune recognition and a highly immunosuppressive tumor microenvironment. The presence of tumor infiltrating lymphocytes (TILs) and specific gene-signatures prior to treatment are linked to good prognosis, while the opposite is true for extensive immunosuppression. The use of adenoviruses as cancer vaccines is a form of active immunotherapy to initialise a tumor-specific immune response that targets the patients unique tumor antigen repertoire. We report a case of a 68-year-old male with asbestos-related malignant pleural mesothelioma who was treated in a Phase I study with a granulocyte-macrophage colony‑stimulating factor (GM-CSF)-expressing oncolytic adenovirus, Ad5/3-D24-GMCSF (ONCOS-102). The treatment resulted in prominent infiltration of CD8+ lymphocytes to tumor, marked induction of systemic antitumor CD8+ T-cells and induction of Th1-type polarization in the tumor. These results indicate that ONCOS-102 treatment sensitizes tumors to other immunotherapies by inducing a T-cell positive phenotype to an initially T-cell negative tumor.

Toxicological and bio-distribution profile of a GM-CSF-expressing, double-targeted, chimeric oncolytic adenovirus ONCOS-102 – Support for clinical studies on advanced cancer treatment

The purpose of this work was to carry out preclinical toxicity and bio-distribution studies required for regulatory approval of a clinical trial application for Phase I clinical studies of ONCOS-102 (Ad5/3-D24-GM-CSF) for therapy of advanced cancers (NCT01598129). The study design, route of administration and dosage differs from the clinical protocol and in more detail, investigate bio-distribution and toxicological profile of ONCOS-102 treatment in animal model. The study was carried out in 300 hamsters divided into nine test groups–three bio-distribution groups and six groups for analysis of toxicity. Hamsters received ONCOS-102 by intracardial, intraperitoneal or subcutaneous injections. Additionally, one group was administered twice a week with intraperitoneal injections of Cyclophosphamide. The control animals were administered with NaCl solution without ONCOS-102 in the same volume and the same way. No adverse effects of repeated administration of ONCOS-102 including body weight, food consumption, hematology and clinical chemistry parameters, histopathology and bio-accumulation were observed in the course of 6-month administration and following 3- month recovery period. All obtained findings indicate the treatment clinically safe.

Gene expression analysis of tumors demonstrates an induction of Th1 type immune response following intratumoral administration of ONCOS-102 in refractory solid tumor patients

Advanced tumors are often immunosuppressive. Intratumoral administration of adenovirus activates Toll-like receptor signalling leading to production of pro-inflammatory cytokines and activation of the innate immune system. Oncolytic adenovirus causes immunogenic cancer cell death and creates a danger signal at tumors, thus undermining immunological tolerance towards tumors. The release of tumor antigens from dying cancer cells results in priming of a potent anti-tumor immune response. This effect may be enhanced by the local production of immunostimulatory molecules coded by the virus. We present results of gene expression analysis of tumors from a Phase I study with ONCOS-102, an oncolytic adenovirus coding for GMCSF, in 12 patients with refractory solid tumors. A total of 9 intratumoral injections of ONCOS-102 were given to each patient. Biopsies were collected at baseline and 1 and 2 months after treatment initiation. RNA was extracted from fresh-frozen biopsies using standard methods. Gene expression profiling was carried out using the Illumina BeadChip platform. Data was normalized by quantile method, probes presenting the same genes were averaged, and the batch effects were adjusted using ComBat pipeline, as implemented in Chipster (http://chipster.csc.fi/). Finally, log2 fold-changes were computed by subtracting baseline data from after treatment data. Network and pathway analyses were conducted through the use of IPA (Ingenuity ®Systems, http://www.ingenuity.com). A cutoff value of 2-fold expression change was used to filter differentially expressed genes and run core analysis to identify underlying signaling networks and deregulated molecules. A significant enrichment of genes involved in immune cell trafficking, inflammatory response and antigen presentation were detected post treatment. A mesothelioma patient showed a prominent post-treatment induction of MAGE3-specific CD8+ T-cells in peripheral blood in IFNγ ELISPOT. Furthermore, a late decrease in metabolic activity was observed in PET imaging 7.5 months after treatment initiation during the follow-up period, measured as a 47% decrease in total lesion glycolysis in comparison to the imaging at 6 months. In the same patient, upstream regulators driving differentially expressed genes detected in the post treatment biopsy included cytokines (INFG, TNF, IL1B, CCL2, CXCL10, IL-17A, CD40LG), enzymes (FN1, NOS2, PTGS2, PARP9), growth factors (BMP2, LEP), kinases (CHUK, CRKL, IKBKB, IKBKG, ITK, STK11, SYK), transcriptional regulators (IRF1, NFATC2, NFKBIA, STAT1, STAT3, ZEB1, RELA), and transmembrane receptors (B2M, CD40, IL6R, TLR2-4, TNFSF1B). Likely, these events collectively induced a higher CD8+ mediated cytotoxic T cell response (GZMB, PRF1) post treatment. Detailed analysis per patient will be presented at the meeting.

665. Toxicity and Bio-Distribution of a GM-CSF-Expressing, Chimeric Oncolytic Adenovirus ONCOS-102

INTRODUCTION: ONCOS-102 is a serotype 5 adenovirus, comprising a chimeric capsid for enhanced gene delivery to cancer cells and a 24 bp deletion in Rb binding site of E1A region for cancer cell restricted replication. ONCOS-102 is armed with granulocyte-macrophage colony-stimulating factor (GM-CSF) for an enhanced immunostimulatory effect. ONCOS-102 treatment is a promising immunotherapy strategy for advanced cancer by directly recruiting antigen presenting cells (APC) at tumor site leading to an induction of adaptive tumor-specific CD8+ T cell response. Its immunological activity has already been demonstrated in Phase I clinical study. In this phase 1 study, local treatment of pleural mesothelioma with ONCOS-102 induced a systemic antitumor CD8+ T-cell response, prominent infiltration of CD8+ lymphocytes and Th1 type polarization.PURPOSE OF THE STUDY: The aim of this study was to evaluate the toxicity profile and biodistribution of ONCOS-102 after intracardial and repeated intraperitoneal or subcutaneous administration in Syrian hamsters.METHOD: This study was performed in compliance with the OECD principles of GLP. 300 Syrian hamsters were divided into nine groups, each group containing males and females in the same ratio. ONCOS-102 was administered in NaCl solution by intracardial, intraperitoneal or subcutaneous injections. The control animals were administered with NaCl solution without ONCOS-102 in the same volume and the same way. Hamsters were checked daily for mortality and clinical signs. Body weight and food consumption were recorded weekly. For bio-distribution analysis, hamsters were euthanized on days 0, 3, 29, and 183 and tissues and organs were collected for assessment of viral DNA. Gross necropsy and histological examination of tissues and organs were performed on days 29 and 190.RESULTS: Treatment with ONCOS-102 had no effects on body weight and food consumption. No treatment related premature deaths occurred. Apart from some findings that were not related to treatment, none of the hamsters showed signs of intoxication in the course of the first four weeks of the study. No considerable differences in hematology and clinical chemistry parameters were observed in any of the treatment groups when compared to control animals after 26 weeks of the study and recovery period. ONCOS-102 did not cause gross or histopathological changes in liver and kidneys of animals indicative of a toxic effect. The highest accumulation of viral DNA was seen in heart, liver, lungs, spleen, kidneys and bone marrow. An overall decrease of concentration of viral DNA was observed from Day 3 to Day 29 and Day 183. No adverse effects of repeated administration of ONCOS-102 on clinical signs, hematology and clinical chemistry parameters or histopathology were observed in the course of 6-month administration and following 3- month recovery period.CONCLUSIONS: Under the conditions of the study, administration of hamsters with ONCOS 102 was well tolerated, without significant signs indicating toxic effects

Local immunotherapy with ONCOS-102 shapes harmful tumor associated CD68+ macrophages to become beneficial cells that correlate with increased overall survival

With the increasing excitement around new immunotherapeutic approaches, there has been a shift in the way viral cancer therapy is regarded from providing mainly oncolysis towards being a cancer immunotherapy. Adenoviruses have a unique ability to prime and boost immune responses. GM-CSF coding adenovirus ONCOS-102 causes immunogenic cancer cell death whereupon tumor antigens are presented into the immunogenic environment. ONCOS-102 has been previously shown to initiate CD8+ T cell responses against tumor-derived antigens in chemotherapy refractory cancer patients. A total of 12 cancer patients were treated with repeated intratumoral injections of ONCOS-102 in a Phase I study. Sequential biopsies were collected at baseline and 1 and 2 months after treatment initiation and analyzed for the presence of immune cells by immunohistochemistry (IHC) in digitally scanned samples. Readout of the expression levels for innate immune cells (CD68, CD163, CD11c), T cells (CD3, CD4, CD8), and B cells (CD19) was performed in tumorous regions by the use of an image analysis algorithm based on color deconvolution and segmentation of the IHC stained cells. In an exploratory analysis, correlation between absolute expression levels of different immune cell markers in tumors and overall survival (OS) was assessed by Spearman´s rank correlation analysis. At baseline, the absolute expression level of macrophage marker CD68 negatively correlated with OS (correlation coefficient (r)= -0.59, p=0.04), suggesting that tumor-associated macrophages (TAMs) in untreated tumors were tumorigenic. No correlation between other immune cell markers and OS was seen at baseline. 11/12 patients showed a post-treatment increase in tumor-infiltrating innate and adaptive immune cells, with the most prominent increase seen in CD8+ cells. In contrast to baseline, post-treatment samples showed a positive correlation between the expression level of CD68+ cells and OS (r=0.71, p=0.01), suggesting that CD68+ macrophages that were attracted into tumors after ONCOS-102 injection displayed different functionality than TAMs present prior to treatment. Furthermore, absolute expression levels of T cell markers CD3 (r=0.74, p=0.006), CD4 (r=0.76, p=0.004), and CD8 (r=0.73, p=0.007) in post-treatment biopsies all positively correlated with OS. To summarize, ONCOS-102 induced infiltration of CD68+ macrophages and T cells which was associated with increased OS, while CD68+ TAMs pre-treatment was associated with shortened OS. This suggest that local immunotherapy with ONCOS-102 has the potential to activate immunologically silent tumors and reduce local immune suppression in advanced tumors.