Chimeric Oncolytic Adenovirus Research Articles

Abstract CT139: Immunological activity of oncolytic adenovirus encoding TNFα and IL-2 (TILT-123) in combination with pembrolizumab in platinum resistant or refractory ovarian cancer patients

Abstract Background TILT-123 (Ad5/3-E2F-D24-hTNFα-IRES-hIL-2) is a serotype chimeric dual safety device oncolytic adenovirus armed with tumor necrosis factor alpha and interleukin-2. It was designed to improve efficacy of T-cell therapies such as immune checkpoint blockade or adoptive cell transfer. We present biological and immunological endpoints from an open-label phase 1 dose escalation study of TILT-123 in combination with anti-PD-1 pembrolizumab in platinum resistant or refractory ovarian cancer patients. Experimental procedures In this dose escalation trial (NCT05271318), patients were first injected with an intravenous (i.v.) dose of TILT-123 ranging from 3x1011 to 4x1012 virus particles (VP), followed by five intratumoural (i.t.) or intraperitoneal (i.p.) injections ranging from 1x1011 to 5x1011 VP. Patients also received intravenous injections of 200 mg pembrolizumab every three weeks starting 36 days after the first dose of TILT-123. The primary endpoint was safety, while efficacy by RECIST1.1 was a secondary endpoint. Collection of tumor biopsies, circulating immune cells, serum, saliva, urine and feces enabled multi-omic evaluation of mechanism of action and identification of predictive biomarkers. Summary of data As of January 2nd 2024, 15 patients received trial therapy. No dose-limiting toxicities were observed and the most frequent AEs associated with therapy included chills and nausea (>10% of patients). Stable disease by RECIST 1.1 was seen in 67% in 12 evaluable patients. TILT-123 replication was detected in both injected and non-injected tumors as assessed by qPCR and anti-hexon IHC. Multiplex immunofluorescence demonstrated induction of robust immunological activity in both injected and non-injected tumors, including significant increase in granzyme B+, PD-1+ CD4+ and CD8+ T cells as well as PD-1+ NK cells. Baseline intratumoral CD4+, FOXP3+ regulatory T cells were not associated with clinical response. Tertiary lymphoid structures and lymphoid aggregates were identified in patients showing long term disease control. Low to medium titers of neutralizing antibodies against 5/3 chimeric adenovirus were detected in 57% of patients at baseline and were not a predictive factor of disease control. No patients had high titers at baseline. Conclusion TILT-123 in combination with anti-PD-1 pembrolizumab was safe and resulted in disease control in both platinum resistant and refractory ovarian cancer patients. Analysis of biological samples revealed insights into mechanism of action, including an immune profile predictive of clinical response. These results, together with those from four other trials (NCT04695327, NCT04217473, NCT06125197 and NCT05222932) set the stage for continued clinical evaluation of TILT-123. Citation Format: James H. Clubb, Matthew S. Block, Johanna Mäenpää, Santeri Pakola, Lyna Haybout, Dafne C. Quixabeira, Tatiana Kudling, Mirte van der Heijden, Elise Jirovec, Tuomo Alanko, Daniel A. Adamo, Susan Ramadan, Jorma Sormunen, Juha Kononen, Julia W. Cohen, Nadena Singh, John Goldfinch, Suvi Sorsa, Riikka Havunen, Claudia Kistler, João M. Santos, Víctor Cervera-Carrascon, Akseli Hemminki. Immunological activity of oncolytic adenovirus encoding TNFα and IL-2 (TILT-123) in combination with pembrolizumab in platinum resistant or refractory ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT139.

Chimeric oncolytic adenovirus to break away from neutralizing antibodies

Chimeric oncolytic adenovirus to break away from neutralizing antibodies

Chimeric oncolytic adenovirus evades neutralizing antibodies from human patients and exhibits enhanced anti-glioma efficacy in immunized mice

Oncolytic viruses are a promising treatment for patients with high-grade gliomas, but neutralizing antibodies can limit their efficacy in patients with prior virus exposure or upon repeated virus injections. Data from a previous clinical trial using the oncolytic adenovirus Delta-24-RGD showed that generation of anti-viral neutralizing antibodies may affect the long-term survival of glioma patients. Past studies have examined the effects of neutralizing antibodies during systemic virus injections, but largely overlooked their impact during local virus injections into the brain. We found that immunoglobulins colocalized with viral proteins upon local oncolytic virotherapy of brain tumors, warranting a strategy to prevent virus neutralization and maximize oncolysis. Thus, we generated a chimeric virus, Delta-24-RGD-H43m, by replacing the capsid protein HVRs from the serotype 5-based Delta-24-RGD with those from the rare serotype 43. Delta-24-RGD-H43m evaded neutralizing anti-Ad5 antibodies and conferred a higher rate of long-term survival than Delta-24-RGD in glioma-bearing mice. Importantly, Delta-24-RGD-H43m activity was significantly more resistant to neutralizing antibodies present in sera of glioma patients treated with Delta-24-RGD during a phase 1 clinical trial. These findings provide a framework for a novel treatment of glioma patients that have developed immunity against Delta-24-RGD.

Development of a novel ddPCR assay for nonclinical and clinical pharmacokinetic characterization of ICVB-1042.

e14693 Background: ICVB-1042 is a novel chimeric oncolytic Adenovirus (Ad) with potential for intravenous administration. A key component of the clinical assessment of ICVB-1042 will be pharmacokinetic (PK) viral genome analysis by Polymerase Chain Reaction (PCR) to understand plasma viral kinetics and tumor replication. Droplet Digital PCR (ddPCR) is more sensitive and more accurately quantifies a lower template concentration compared to traditional quantitative PCR. Our ddPCR assay targets ICVB-1042 and will not amplify other Ad5 or Ad34 viruses. The aim is to show that this assay specifically measures ICVB-1042 and may be used as a clinical biomarker. Methods: PCR primers and probe were designed to target the Ad5/Ad34 chimeric fiber of ICVB-1042. Template ICVB-1042 was spiked into representative human and murine sample matrices. Preclinical studies tested assay performance in vitro using patient derived disassociated tumor cells (DTCs) and in vivo using tumor bearing and non-tumor bearing mouse models. Viral replication and PK were assessed in vitro by analyzing supernatant of infected cells at multiple timepoints and in vivo with serial bleeds from different animals. Viral distribution was assessed by isolating viral DNA from homogenized tissue samples taken at multiple timepoints. Results: Analysis of ICVB-1042 by ddPCR showed linear quantification over a wide range of viral concentrations (5E5-5E12 viral genomes/mL) with sensitivity to low concentrations of virus (lower limit of quantification = 1.11E2 total viral genomes). Viral genomes were detected and quantified in human plasma samples spiked with ICVB-1042. Non-spiked samples were negative for signal. ICVB-1042 viral genomes were also detectible in tumor sections from ICVB-1042-treated NSG mice. Increasing viral genomes could be detected in vitro at days 6 and 9 post ICVB-1042 infection in the supernatants of several DTCs (2 donors each). The measured increase in viral genomes was accompanied by viral mediated cell death. ICVB-1042 could be detected in vivo in the plasma of non-tumor bearing CD-1 mice 1 and 24 hours post ICVB-1042 dosing but was cleared and not detected out to 90 days post dose. ICVB-1042 was detected post dose in the plasma of tumor bearing NSG mice and was again cleared. ICVB-1042 was detectable in plasma samples and accompanied by anti-tumor efficacy at later timepoints. Studies directly comparing tumor bearing to non-tumor bearing NSG mice have shown that the virus present in the plasma is due to tumor specific replication. Tissue viral genome distribution differences could also be detected in C57BL/6 mice expressing huCD46, the viral tropism of ICVB-1042. Conclusions: ICVB-1042 viral genomes can be detected using ddPCR. This assay can be used to assess viral replication in vitro and PK and distribution in vivo. Use of ddPCR to detect ICVB-1042 in patient samples will be key to the characterization of clinical efficacy.

Chimeric Oncolytic Adenovirus Armed Chemokine Rantes for Treatment of Breast Cancer.

The immunosuppressive state in the tumor microenvironment (TME) of breast cancer makes it difficult to treat with immunotherapy. Oncolytic viruses not only lyse tumor cells but also reshape the TME. Therefore, they can play a multi-mechanism synergistic effect with immunotherapy. In this study, an oncolytic adenovirus Ad5F11bSP-Rantes was constructed and used as a vector to express the chemokine Rantes. The objective of this study was to test the dual mechanisms of the oncolytic effect mediated by virus replication and the enhanced anticancer immune response mediated by Rantes chemotaxis of immune cells. It was found that Ad5F11bSP-Rantes has strong infectivity and effective killing activity against breast cancer cells. In the established triple negative breast cancer (TNBC) xenograft model in NCG mice whose immune system was humanized with human peripheral blood mononuclear cells (PBMCs), Ad5F11bSP-Rantes achieved 88.33% tumor inhibition rate. Rantes expression was high in mouse blood, a large number of CD3+ lymphocytes infiltrated in tumor tissues and E-cadherin was up-regulated in cancer cells, suggesting that Ad5F11bSP-Rantes altered the TME and induced a reversal of cancer cell epithelial–mesenchymal transition (EMT). In conclusion, oncolytic adenovirus can exert the oncolytic effect and the chemotactic effect of immune cells and realize the synergy of multiple anticancer effects. This strategy creates a candidate treatment for the optimization of breast cancer, especially TNBC, combination therapy.

Triple-serotype chimeric oncolytic adenovirus exerts multiple synergistic mechanisms against solid tumors

BackgroundOncolytic virotherapy has become an important branch of cancer immunotherapy. This study investigated the efficacy of an oncolytic adenovirus (OAV), OncoViron, with synergistic mechanisms in the treatment of multiple solid...

1083P A pilot study of engineered adenovirus ONCOS-102 in combination with pembrolizumab (pembro) in checkpoint inhibitor refractory advanced or unresectable melanoma

Patients (pts) with advanced melanoma resistant to immune checkpoint inhibitors (ICI) may benefit from oncolytic viruses. ONCOS-102 (Ad5/3-D24-GM-CSF), a chimeric oncolytic adenovirus expressing human granulocyte-macrophage colony stimulating factor, has demonstrated innate and adaptive immune responses in solid tumors. This study assessed the safety, clinical and immune responses of sequential (Part 1) or combined (Part 2) ONCOS-102 and pembro for pts with advanced melanoma after prior PD-1 blockade.

Systemic Delivery of Oncolytic Adenovirus to Tumors Using Tumor-Infiltrating Lymphocytes as Carriers.

Immunotherapy with tumor-infiltrating lymphocytes (TIL) or oncolytic adenoviruses, have shown promising results in cancer treatment, when used as separate therapies. When used in combination, the antitumor effect is synergistically potentiated due oncolytic adenovirus infection and its immune stimulating effects on T cells. Indeed, studies in hamsters have shown a 100% complete response rate when animals were treated with oncolytic adenovirus coding for TNFa and IL-2 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) and TIL therapy. In humans, one caveat with oncolytic virus therapy is that intratumoral injection has been traditionally preferred over systemic administration, for achieving sufficient virus concentrations in tumors, especially when neutralizing antibodies emerge. We have previously shown that 5/3 chimeric oncolytic adenovirus can bind to human lymphocytes for avoidance of neutralization. In this study, we hypothesized that incubation of oncolytic adenovirus (TILT-123) with TILs prior to systemic injection would allow delivery of virus to tumors. This approach would deliver both components in one self-amplifying product. TILs would help deliver TILT-123, whose replication will recruit more TILs and increase their cytotoxicity. In vitro, TILT-123 was seen binding efficiently to lymphocytes, supporting the idea of dual administration. We show in vivo in different models that virus could be delivered to tumors with TILs as carriers.

External Beam Radiation Therapy and Enadenotucirev: Inhibition of the DDR and Mechanisms of Radiation-Mediated Virus Increase.

Ionising radiation causes cell death through the induction of DNA damage, particularly double-stranded DNA (dsDNA) breaks. Evidence suggests that adenoviruses inhibit proteins involved in the DNA damage response (DDR) to prevent recognition of double-stranded viral DNA genomes as cellular dsDNA breaks. We hypothesise that combining adenovirus treatment with radiotherapy has the potential for enhancing tumour-specific cytotoxicity through inhibition of the DDR and augmentation of virus production. We show that EnAd, an Ad3/Ad11p chimeric oncolytic adenovirus currently being trialled in colorectal and other cancers, targets the DDR pathway at a number of junctures. Infection is associated with a decrease in irradiation-induced 53BP1 and Rad51 foci formation, and in total DNA ligase IV levels. We also demonstrate a radiation-associated increase in EnAd production in vitro and in a pilot in vivo experiment. Given the current limitations of in vitro techniques in assessing for synergy between these treatments, we adapted the plaque assay to allow monitoring of viral plaque size and growth and utilised the xCELLigence cell adhesion assay to measure cytotoxicity. Our study provides further evidence on the interaction between adenovirus and radiation in vitro and in vivo and suggests these have at least an additive, and possibly a synergistic, impact on cytotoxicity.

Antitumor effect of hypoxia response element chimeric tumor specific promoter regulated oncolytic adenovirus on renal cell carcinoma

Objective To investigate the effect of chimeric promoter composed of hypoxia response element (HRE) and tumor specific cell proliferation-associated nuclear antigen (Ki-67) promoter on the replication ability of oncolytic adenovirus and its cytotoxic activity under hypoxic conditions. Methods The HRE sequence, as an enhancer, was inserted upstream of the Ki-67 promoter and, at the same time carried the reporter gene enhanced green fluorescent protein (EGFP) to construct oncolytic adenovirus Ad-HRE-Ki-67-EGFP, and the titer of adenoviruses was measured by the tissue culture infective dose (TCID50). Normal renal epithelial cells HK-2 and renal cancer cells OSRC-2 and ACHN were infected with two kinds of viruses under normoxic (21% O2) or hypoxic (1% O2) conditions respectively for 24 h, and the virus tumor-targeting was detected by fluorescence microscopy and flow cytometry. Western blotting was performed to detect the expression of hypoxia-related protein hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and the expression of E1A protein. Cell counting kit-8 (CCK-8) assay was used to detect the cytotoxity of virus on cells. Results The recombinant oncolytic virus Ad-HRE-Ki67-EGFP was successfully constructed. Under normoxic conditions, the efficiency of ACHN and OSRC-2 infection by chimeric HRE oncolytic adenovirus in renal cancer cells was (47.5±2.6)% and (35.2±3.4)%, while that in hypoxic conditions was (86.4±3.4)% and (73.5±2.3)%, indicating that the infection efficiency was higher under hypoxic conditions (ACHN: t=15.760; OSRC-2: t=16.190; P<0.01); Normal renal tubular epithelial cells HK-2 were infected with Ad-Ki-67-EGFP or Ad-HRE-Ki-67-EGFP under normoxic conditions. The infection efficiency was (22.4±3.1)% and (22.9±2.2)% respectively, compared with that of ACHN and OSRC-2 cells [ACHN: (68.4±3.3)% and (72.1±2.9)%; OSRC-2: (56.1±3.1)% and (61.4±2.3)%], the difference was statistically significant (Ad-Ki-67-EGFP: t=17.650, 13.390; Ad-HRE-Ki-67-EGFP: t=23.680, 21.430, P<0.01), indicating that the two adenoviruses have a targeting effect on renal cancer cells. Western blotting data showed that the expression of HIF-1α, VEGF and p53 protein were increased under hypoxic conditions and was time-dependent, indicating that hypoxia can regulate the transcription and translation of HIF-1α, VEGF and p53 proteins. Under hypoxic conditions, the expression level of E1A protein in OSRC-2 and ACHN infected with Ad-HRE-Ki67-EGFP was significantly higher than that after Ad-Ki67-EGFP infection. It indicated that HRE can enhance the replication ability of virus under hypoxic conditions. CCK-8 results showed that when the multiple infection (MOI) values were 20, 100, the survival rate of the Ad-HRE-Ki-67-EGFP-normoxia virus group in ACHN and OSRC-2 cells was (73.4±2.0)%, (56.4±1.5)% and (79.9±1.8)%, (61.3±2.7)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group was (63.1±2.0)%, (31.6±2.1)% and (68.1±2.6)%, (35.8±3.1)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group showed a stronger proliferation inhibitory effect [MOI (20): t=6.328, 6.441; MOI (100): t=16.410, 10.790, P<0.01]. Conclusion Under hypoxic conditions, chimeric HRE sequence recombinant oncolytic adenovirus has stronger replication ability and cell killing effect in renal carcinoma cells. Key words: Renal cell carcinoma; Oncolytic adenovirus; Hypoxia response element; Hypoxia

Interleukin-24 enhancing antitumor activity of chimeric oncolytic adenovirus for treating acute promyelocytic leukemia cell.

Background:Acute promyelocytic leukaemia (APL) is a clonal disease arising by hematopoietic stem cell (HSC), which characterized by inappropriate proliferation/differentiation or survival of immature myeloid progenitors. Oncolytic adenoviruses have been under widespread investigation as anticancer agents. Recently, our data suggested that tumor cells were cured by AdCN205-IL-24, an adenovirus serotype 5-based conditionally replicating adenovirus expressing IL-24 after infection.Methods:In this study, we created a novel fiber chimeric oncolytic adenovirus AdCN306-IL-24 that has Ad11 tropism and approved CAR (coxsackie adenovirus receptor, CAR)-independent cell entry, which could allow development of selective cytopathic effects (CPE) in APL cells in vitro.Results:Formidable cytotoxic effect was specifically implemented in APL cells after infection with AdCN306-IL-24. The expression of IL-24 was up-regulated upon treated with accepted tumors. And the vector also induced superior cytolytic effects activity in APL cells by activation of programmed cell death.Conclusions:Taken together, our data suggested that chimeric oncolytic adenovirus AdCN306-IL-24 could express IL-24 gene, representing a potential therapeutics for acute promyelocytic leukemia.

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.

Synergistic anti-tumor efficacy of immunogenic adenovirus ONCOS-102 (Ad5/3-D24-GM-CSF) and standard of care chemotherapy in preclinical mesothelioma model.

Malignant mesothelioma (MM) is a rare cancer type caused mainly by asbestos exposure. The median overall survival time of a mesothelioma cancer patient is less than 1-year from diagnosis. Currently there are no curative treatment modalities for malignant mesothelioma, however treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase life expectancy. Pemetrexed-Cisplatin is the only standard of care (SoC) chemotherapy for malignant mesothelioma, but the median PFS/OS (progression-free survival/overall survival) from the initiation of treatment is only up to 12 months. Therefore, new treatment strategies against malignant mesothelioma are in high demand. ONCOS-102 is a dual targeting, chimeric oncolytic adenovirus, coding for human GM-CSF. The safety and immune activating properties of ONCOS-102 have already been assessed in phase 1 study (NCT01598129). In this preclinical study, we evaluated the antineoplastic activity of combination treatment with SoC chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and ONCOS-102 in xenograft BALB/c model of human malignant mesothelioma. We demonstrated that ONCOS-102 is able to induce immunogenic cell death of human mesothelioma cell lines in vitro and showed anti-tumor activity in the treatment of refractory H226 malignant pleural mesothelioma (MPM) xenograft model. While chemotherapy alone showed no anti-tumor activity in the mesothelioma mouse model, ONCOS-102 was able to slow down tumor growth. Interestingly, a synergistic anti-tumor effect was seen when ONCOS-102 was combined with chemotherapy regimens. These findings give a rationale for the clinical testing of ONCOS-102 in combination with first-line chemotherapy in patients suffering from malignant mesothelioma.

661. Synergistic Anti-Tumor Efficacy of Immunogenic Adenovirus ONCOS-102 and Standard of Care Chemotherapy in Preclinical Mesothelioma Model

Malignant mesothelioma is a rare cancer type with no effective treatment therapies so far. Most of the mesothelioma cases are caused due to asbestos exposure. The median survival time of mesothelioma cancer patient is short, around one year from diagnosis and conventional treatments such as surgery, chemotherapy and radiotherapy can help only to improve mesothelioma prognosis and finally increase patient's life expectancy. Unfortunately, currently there is no cure for mesothelioma, and new treatment strategies against mesothelioma are in high demand. ONCOS-102 is a double targeted, chimeric oncolytic adenovirus, coding for human GM-CSF. Its potency as an antitumor agent has been reported in pre-clinical and clinical studies. In this study we evaluated the anticancer activity of combined therapy: first-line chemotherapy (Pemetrexed, Cisplatin, Carboplatin) with ONCOS-102 in a human malignant mesothelioma xenograft model in BALB/c mice. ONCOS-102 was able to kill all human mesothelioma cell lines in vitro and exhibited anti-tumor activity in treatment refractory H226 mesothelioma xenograft model. Current standard of care chemotherapy regimens for malignant mesothelioma presented no anti-tumor efficacy in tested mesothelioma model. Importantly, a synergistic anti-tumor effect was shown when ONCOS-102 was combined with chemotherapy regimens. Our findings support the clinical use of ONCOS-102 with first-line chemotherapy against malignant mesothelioma.

Abstract B51: Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM-CSF: Results in vitro, in rodents and in humans

Abstract Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as ipilimumab or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models in combination with low-dose cyclophosphamide, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. Four out of nine melanoma patients treated in ATAP were evaluable for possible therapy benefit with modified Response Evaluation Criteria In Solid Tumors (RECIST): one patient had minor response, two had stable disease, and one had progressive disease. Treatments appeared safe and well-tolerated. Two patients were alive at 559 and 1501 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma. Citation Format: Simona Bramante, Johanna K. Kaufmann, Ville Veckman, Dirk M. Nettelbeck, Otto Hemminki, Ilkka Liikanen, Lotta Vassilev, Vincenzo Cerullo, Minna Oksanen, Raita Heiskanen, Timo Joensuu, Anna Kanerva, Sari Pesonen, Markus Vähä-Koskela, Anniina Koski, Akseli Hemminki. Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM-CSF: Results in vitro, in rodents and in humans. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B51.

Abstract B80: Patients treated with oncolytic adenovirus Ad5/3-hTERT-E2F-Δ24-GMCSF induce immunological antitumor responses

Abstract Background: Gene therapy with oncolytic viruses is a promising novel treatment modality for cancer due to their selective replication and lytic effect on cancer cells. Although the clinical safety profiles of individual agents are encouraging, the efficacy of oncolytic viruses as monotherapy has been limited. Arming the virus with an immune stimulatory molecule is an appealing strategy and may increase overall antitumor potency. Granulocyte-macrophage colony-stimulating factor (GMCSF) is one of the most potent inducers of antitumor immunity. GMCSF is associated with recruitment and maturation of antigen presenting cells (APCs), mainly dendritic cells, as well as activating the innate immunity by recruiting natural killer cells and neutrophils. Local GMCSF expression could ensure sufficient local concentration for induction of an anti-tumor immune response circumventing the problems with toxic systemic exposure and side effects. Results: 5/3 chimeric oncolytic adenovirus selective for the p16-Rb pathway engineered to contain a tumor specific E2F promoter and carrying GMCSF (Ad5/3-hTERT-E2F-Δ24-GMCSF aka. CGTG-602) was constructed. Preclinical studies in vivo showed promising efficacy in immune competent Syrian hamster model. Encouraged by these results, 13 patients were treated with Ad5/3-hTERT-E2F-Δ24-GMCSF in Advance Therapy Access Program. Treatments were well-tolerated and overall signs of antitumor efficacy were seen in 9/12 patients (75%) when evaluated by PET-CT or tumor markers. Patient samples were analyzed for virus circulation in blood, neutralizing antibody titers, tumor- and adenovirus-specific PBMCs and antibodies against selected tumor associated antigens. Microarray analysis and immunohistochemistry were performed on available tumor biopsies and indicated accumulation of immunological cells, especially T-cells, to tumors after treatment. RNA expression analyses of tumors indicated immunological activation and metabolic changes secondary to virus replication. Conclusions: These findings demonstrate that Ad5/3-hTERT-E2F-Δ24-GMCSF was safe and able to show promising signs of efficacy in preclinical studies and in patients. Clinical trials are needed to confirm these preliminary findings in larger patient cohorts. Citation Format: Otto Hemminki, Suvi Parviainen, Akseli Hemminki. Patients treated with oncolytic adenovirus Ad5/3-hTERT-E2F-Δ24-GMCSF induce immunological antitumor responses. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B80.

109. Replicating Oncolytic Chimeric Adenovirus Expressing shRNA Improves Inhibition of Human Colorectal Carcinoma (CRC) Growth

RNAi combined with oncolytic viral vector therapy may provide a new approach to control malignant disease since allele specific shRNA to a retrogene that modulates stemness will inhibit growth of human CRC in vivo. Our original delivery vector was nonreplicating Lentiviral vectors (LV) but now oncolytic adenovirus is the vector. We have created a conditionally replicating chimeric oncolytic adenovirus (CRAd) expressing a shRNA to inhibit growth of established tumors in preclinical models. shRNA is directed to NANOGP8, a retrogene that differs from parental NANOG primarily in a single nucleotide variant: c.759G>C, p.Gln253His. Oncolytic adenoviruses were generated with a 5/3 fiber with either a control (Ad5/3-wtE1a-shNEG (Ad5/3shNEG)) or an anti-NANOGP8 shRNA (Ad5/3-wtE1a-shNp8 (Ad5/3shNp8)). LV shNEG or LV shNp8 were added 24 hr after creation of 3-D organoid cultures of human CRC cells (Clone A, CX-1 or LS174T) in serum-free medium. LV transduced 45 – 90% of cells in culture and shNp8 inhibited organoids by ~50% compared to either shNEG or untreated CRC organoids. In addition, LV injected into 7 -9 mm diameter CX-1 tumors in NOD/SCID mice reduced tumor growth in vivo by 40% for 11 days, but then tumors grew at an accelerated rate. NMF cluster analysis reveals that LVshNp8 affects a gene expression profile that is distinct from that of either LVshNEG or untreated controls. In contrast, Ad5/3shNp8 inhibited organoid growth by more than 70% and controlled growth of CX-1 and LS174T in vivo. Infection of cells in vitro was over 90%. Thus, shNp8 alters gene expression and replicating oncolytic virus with shNp8 has a more persistent inhibitory effect than that achieved with non-replicating LV-delivered shRNA.

Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM-CSF: Results in vitro, in rodents and in humans.

Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune-checkpoint-inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well-tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma.

Immunological effects of a tumor necrosis factor alpha-armed oncolytic adenovirus.

For long it has been recognized that tumor necrosis factor alpha (TNFa) has anticancer characteristics, and its use as a cancer therapeutic was proposed already in the 1980s. However, its systemic toxicity has limited its usability. Oncolytic viruses, selectively cancer-killing viruses, have shown great potency, and one of their most useful aspects is their ability to produce high amounts of transgene products locally, resulting in high local versus systemic concentrations. Therefore, the overall magnitude of tumor cell killing results from the combination of oncolysis, transgene-mediated direct effect such as TNFa-mediated apoptosis, and, perhaps most significantly, from activation of the host immune system against the tumor. We generated a novel chimeric oncolytic adenovirus expressing human TNFa, Ad5/3-D24-hTNFa, whose efficacy and immunogenicity were tested in vitro and in vivo. The hTNFa-expressing adenovirus showed increased cancer-eradicating potency, which was shown to be because of elevated apoptosis and necrosis rates and induction of various immune responses. Interestingly, we saw increase in immunogenic cell death markers in Ad5/3-d24-hTNFa-treated cells. Moreover, tumors treated with Ad5/3-D24-hTNFa displayed enhanced presence of OVA-specific cytotoxic T cells. We thus can conclude that tumor eradication and antitumor immune responses mediated by Ad5/3-d24-hTNFa offer a new potential drug candidate for cancer therapy.

"Arming" the chimeric oncolytic adenovirus enadenotucirev to deliver checkpoint inhibitors and other therapeutics directly to tumours

Enadenotucirev (EnAd; formerly called ColoAd1) is a potent, chimeric Ad11p/Ad3 adenovirus active against a range of epithelial cancer cells, with a shorter time-to-lysis than either wild type Ad11p, Ad3 or Ad5. In normal cells, EnAd is attenuated and shows little or no activity by either cytotoxicity or by qPCR. In vivo, EnAd shows efficacy in a range of xenograft human tumour models following intra-tumoural, intravenous and intra-peritoneal injection [1] and is currently being evaluated clinically for treatment of several different epithelial cancers. Data from an ongoing clinical mechanism of action study have shown that i.v. dosed EnAd infects and replicates in tumour cells, producing significant amounts of viral protein (hexon), indicating that transgene encoded proteins will also be made in significant amounts by tumours following i.v. delivery of an armed EnAd virus. To develop 'armed' variants for delivery of therapeutic agents that enhance EnAd's anti-tumour activity, we have developed a novel, efficient cloning system for rapid generation of modified viruses that can be dosed systemically to deliver immunomodulatory antibodies into tumours. We chose to first encode anti-VEGF antibodies since, unlike immunomodulators, they could be readily evaluated in vivo in immunodeficient mouse human tumour xenograft models. We have successfully produced EnAd variants encoding full-length (NG-135) and ScFv (NG-76) forms of anti-human VEGF antibodies which have similar virus activity profiles to EnAd in cancer cell lines in vitro (virus replication, gene expression and oncolytic action), but also express and release the respective anti-VEGF antibody forms into the culture supernatant. Using either HCT-116 or DLD human colon carcinoma xenograft models we have shown that the virus infection profile following intra-tumoural injection is similar to the parental EnAd virus (virus replication and hexon gene expression). Anti-VEGF antibody expression by these tumours could be detected in the tissue as both mRNA and functional antibody. Antibodies were detectable early (within 3 days of infection) and expression was sustained over several weeks. Furthermore, low levels of anti-VEGF antibody were detectable in the blood. Production and evaluation of viruses similarly expressing checkpoint inhibitor antibodies is now in progress, together with evaluation of anti-VEGF armed oncolytic viruses for their impact on the growth and microenvironment of tumour xenografts.