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As a clinical setting in which local live biological therapy is already well established, non-muscle invasive bladder cancer (NMIBC) presents intriguing opportunities for oncolytic virotherapy. Coxsackievirus A21 (CVA21) is a novel intercellular adhesion molecule-1 (ICAM-1)-targeted immunotherapeutic virus. This study investigated CVA21-induced cytotoxicity in a panel of human bladder cancer cell lines, revealing a range of sensitivities largely correlating with expression of the viral receptor ICAM-1. CVA21 in combination with low doses of mitomycin-C enhanced CVA21 viral replication and oncolysis by increasing surface expression levels of ICAM-1. This was further confirmed using 300-μm precision slices of NMIBC where levels of virus protein expression and induction of apoptosis were enhanced with prior exposure to mitomycin-C. Given the importance of the immunogenicity of dying cancer cells for triggering tumor-specific responses and long-term therapeutic success, the ability of CVA21 to induce immunogenic cell death was investigated. CVA21 induced immunogenic apoptosis in bladder cancer cell lines, as evidenced by expression of the immunogenic cell death (ICD) determinant calreticulin, and HMGB-1 release and the ability to reject MB49 tumors in syngeneic mice after vaccination with MB49 cells undergoing CVA21 induced ICD. Such CVA21 immunotherapy could offer a potentially less toxic, more effective option for the treatment of bladder cancer. Keywords: bladder cancer, coxsackievirus A21, intercellular adhesion molecule-1

// Subhransu S. Sahoo 1 , Min Yuan Quah 2 , Sarah Nielsen 3 , Joshua Atkins 4 , Gough G. Au 2 , Murray J. Cairns 4 , Pravin Nahar 5 , Janine M. Lombard 6 and Pradeep S. Tanwar 1 1 Gynaecology Oncology Group, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia 2 The Picornaviral Research Unit, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia 3 Hunter Cancer Biobank, University of Newcastle, Callaghan, New South Wales, Australia 4 Discipline of Pharmacy and Experimental Pharmacology, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia 5 Department of Maternity and Gynaecology, John Hunter Hospital, New Lambton Heights, New South Wales, Australia 6 Department of Medical Oncology, Calvary Mater Newcastle, Waratah, New South Wales, Australia Correspondence to: Pradeep S. Tanwar, email: // Keywords : microenvironment, ECM, endometrial cancer, TGF-β signalling, metastasis Received : May 05, 2017 Accepted : May 07, 2017 Published : May 22, 2017 Abstract Although aggressive invasion and distant metastases are an important cause of morbidity and mortality in patients with endometrial cancer (EC), the requisite events determining this propensity are currently unknown. Using organotypic three-dimensional culture of endometrial cancer cell lines, we demonstrated anti-correlated TGF-β signalling gene expression patterns that arise among extracellular matrix (ECM)-attached cells. TGF-β pathway seemed to be active in EC cells forming non-glandular colonies in 3D-matrix but weaker in glandular colonies. Functionally we found that out of several ECM proteins, fibronectin relatively promotes Smad phosphorylation suggesting a potential role in regulating TGF-β signalling in non-glandular colonies. Importantly, alteration of TGF-β pathway induced EMT and MET in both type of colonies through slug protein. The results exemplify a crucial role of TGF-β pathway during EC metastasis in human patients and inhibition of the pathway in a murine model impaired tumour cell invasion and metastasis depicting an attractive target for therapeutic intervention of malignant tumour progression. These findings provide key insights into the role of ECM-derived TGF-β signalling to promote endometrial cancer metastasis and offer an avenue for therapeutic targeting of microenvironment derived signals along with tumour cells.

Purpose: The CANON [CAVATAK in NON–muscle-invasive bladder cancer (NMIBC)] study evaluated a novel ICAM-1–targeted immunotherapeutic-coxsackievirus A21 as a novel oncolytic agent against bladder cancer. Patients and Methods: Fifteen patients enrolled in this “window of opportunity” phase I study, exposing primary bladder cancers to CAVATAK prior to surgery. The first 9 patients received intravesical administration of monotherapy CAVATAK; in the second stage, 6 patients received CAVATAK with a subtherapeutic dose of mitomycin C, known to enhance expression of ICAM-1 on bladder cancer cells. The primary endpoint was to determine patient safety and maximum tolerated dose (MTD). Secondary endpoints were evidence of viral replication, induction of inflammatory cytokines, antitumor activity, and viral-induced changes in resected tissue. Results: Clinical activity of CAVATAK was demonstrated by induction of tumor inflammation and hemorrhage following either single or multiple administrations of CAVATAK in multiple patients, and a complete resolution of tumor in 1 patient. Whether used alone or in combination with mitomycin C, CAVATAK caused marked inflammatory changes within NMIBC tissue biopsies by upregulating IFN-inducible genes, including both immune checkpoint inhibitory genes (PD-L1 and LAG3) and Th1-associated chemokines, as well as the induction of the innate activator RIG-I, compared with bladder cancer tissue from untreated patients. No significant toxicities were reported in any patient, from either virus or combination therapy. Conclusions: The acceptable safety profile of CAVATAK, proof of viral targeting, replication, and tumor cell death together with the virus-mediated increases in “immunological heat” within the tumor microenvironment all indicate that CAVATAK may be potentially considered as a novel therapeutic for NMIBC.

Summary Oncolytic viruses are attractive biological agents for the control of human malignancy. This study assessed the capacity of Coxsackievirus A21 (CVA21) to target and destroy multiple myeloma (MM) and precursor aberrant plasma cells in vitro using established MM cell lines and 15 patient bone marrow (BM) biopsies [n = 10 MM and five monoclonal gammopathy of undetermined significance (MGUS)]. Cell surface analysis revealed that all tumour cells lines expressed high levels of intercellular adhesion molecule-1 (ICAM-1) and decay-accelerating factor (DAF), the receptor molecules to which CVA21 can bind, leading to subsequent cell-entry and infection. MM cell lines were remarkably susceptible to CVA21 lytic infection, producing 100–1000-fold increases in viral progeny within 24 h. In contrast, normal peripheral blood cells were refractile to CVA21 infection. Furthermore, challenge of patient BM biopsies with CVA21 for 48 h resulted in specific purging of up to 98·7% of CD138+ plasma cells, with no significant decrease in progenitor cell function. Data generated in this study suggests that CVA21 virotherapy may have potential applications as a systemic anti-tumour agent for MM, or in the ex vivo purging of malignant plasma cells prior to autologous stem cell transplantation.

Purpose: The incidence of malignant melanoma continues to increase worldwide; however, treatment of metastatic melanoma remains unsatisfactory, and there is an urgent need for development of effective targeted therapeutics. A potential biological target on the surface of malignant melanoma cells is the up-regulated expression of intercellular adhesion molecule (ICAM)-1 and decay-accelerating factor (DAF), relative to surrounding benign tissue. Coxsackievirus A21 (a common cold virus) targets and destroys susceptible cells via specific viral capsid interactions with surface-expressed virus receptors comprising ICAM-1 and DAF. Experimental Design: The oncolytic capacity of a genetically unmodified wild-type common cold-producing human enterovirus (Coxsackievirus A21, CAV21) was assessed against in vitro cultures and in vivo xenografts of malignant human melanoma cells. Results: In vitro studies established that human melanoma cells endogenously express elevated levels of ICAM-1/DAF and were highly susceptible to rapid viral oncolysis by CAV21 infection, whereas ICAM-1/DAF-expressing peripheral blood lymphocytes were refractile to infection. In vivo studies revealed that the tumor burden of nonobese diabetic severe combined immunodeficient mice bearing multiple s.c. melanoma xenografts was rapidly reduced by oncolysis mediated by a single administration of CAV21. The antitumor activity of CAV21 was characterized by highly efficient systemic spread of progeny CAV21, with oncolysis of tumors also occurring at sites distant to the primary site of viral administration. Conclusions: Overall, the findings presented herein demonstrate an important proof of principle using administration of replication-competent CAV21 as a potential biological oncolytic agent in the control of human metastatic melanoma.

Background: Coxsackievirus A21 (CAVATAKTM) is a bio-selected oncolytic immunotherapy virus. Following intravenous (i.v) infusion, CAVATAK preferential infects ICAM-1 expressing tumor cell, resulting in tumor cell lysis and a systemic immune-mediated anti-tumor response. A Phase I/II trial of i.v delivered CAVATAK (NCT01227551) in advanced cancer patients displayed signs of antitumor activity in some lesions. Blockade of programmed death protein-1 (PD-1) and or CTLA-4 in many advanced cancer patients has resulted in substantial tumor responses via a mechanism involving reversal of tumor induced T cell suppression and loosening the host “immunological handbrake”. We investigated host immune activity in a B16-ICAM-1 melanoma immune competent mouse following a combination of intravenous delivered CAVATAK and PD-1 or CTLA-4 blockade. Methods: Preclinical studies in C57BL mice were conducted to assess the antitumor activity of CAVATAK and anti-mouse PD-1 (mPD-1) mAb or anti-CTLA-4 (mCTLA-4) mAb in a B16-ICAM-1 melanoma immune competent mouse model. CAVATAK was administered i.v, while anti mPD-1 or mCTLA-4 mAbs were delivered via the intraperitoneal route. Following treatment of the primary cutaneous B16-ICAM-1 tumor with 4 cycles of CAVATAK injections and 4 cycles of anti-PD-1 or anti-CTLA-4 mAbs, mice were then re-challenged with an additional intradermal administration of B16 cells. Sequential serum samples were taken to monitor viral loads, inflammatory cytokines and anti-viral neutralising antibodies. Results: Significant single agent antitumor activities against the primary B16-ICAM-1 tumors were observed in mice treated with either CAVATAK, anti-PD-1 or anti-CTLA-4 mAbs relative to saline controls. Interestingly, the depth of the anti-tumor activity appeared to be greater in treatments involving anti-CTLA-4 blockade. Surprisingly, levels of serum anti-CVA21 neutralising antibody were enhanced in both the anti-CTLA-4/ CVA21 and anti-CTLA-4/ anti-PD-1/CVA21 groups relative to mice treated with CVA21 alone of with anti-PD-1/CVA21. Despite the presence of higher levels of serum anti-CVA21 neutralising antibody, no reductions in anti-tumor activity from the combination therapy were observed in mice within the anti-CTLA-4 groups. Conclusion: The significant anti-tumor activity mediated by the combination of CAVATAK and checkpoint inhibitor antibodies (anti-PD-1 and anti-CTLA-4) observed in the presented murine melanoma model supports clinical evaluation of such an immunotherapeutic combination treatment regime. Enhanced anti-CVA21 immune responses following immune-checkpoint blockade confirms the loosening of the host “immunological handbrake” and a general heightening of systemic immune surveillance. Citation Format: Min Yuan Quah, Yvonne Wong, Robert Andtbacka, Gough Au, Darren R. Shafren. Elevated immune activity following an anticancer combination therapy of a novel oncolytic immunotherapeutic agent, CAVATAK (Coxsackievirus A21), and immune checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2341.

e16016Background: CAVATAK is a novel, bio-selectedICAM-1 targeted immunotherapeutic Coxsackievirus A21 (CVA21). Surface ICAM-1 is up-regulated on NMIBC. CVA21 displays potent oncolytic activity aga...

The dissemination of malignant gastric cells to the peritoneum occurs frequently, usually as an early event in disease, and results in poor patient prognosis. Surgery and chemotherapy offer limited therapeutic success. The low-pathogenic human enterovirus, Echovirus 1 (EV1), is an oncolytic virus that selectively targets and destroys malignant prostate and ovarian cancer xenografts in vivo. Lytic EV1 infection requires the cell surface expression of alpha(2)beta(1), an integrin involved in the dissemination of gastric cancer cells to the peritoneum. Herein, we evaluated the capacity of EV1 for anti-neoplastic cell action in gastric peritoneal carcinomatosis. Flow cytometric analysis demonstrated that alpha(2)beta(1) was abundantly surface expressed on a panel of gastric cancer cell lines, rendering the majority of lines highly susceptible to in vitro lytic EV1 infection and supportive of efficient viral progeny production. A bioluminescent MKN-45-Luc SCID mouse model of peritoneal dissemination was developed to allow real-time non-invasive monitoring of peritoneal tumor burden. Employing this mouse model, we demonstrated a therapeutic dose-response for escalating oncolytic EV1 doses. Taken together, these results emphasize the exciting potential for EV1 as a single or adjunct therapy for the control of the peritoneal dissemination of gastric cancer.

BACKGROUND. Oncolytic virotherapy offers a unique treatment modality for prostate cancer, especially stages that are resistant to current therapies, with the additional benefit of preferentially targeting tumor cells amongst an environment of healthy tissue. Herein, the low pathogenic enteroviruses; Coxsackievirus A21 (CVA21), as well as a bio-selected variant of Coxsackievirus A21 (CVA21-DAFv) and Echovirus 1 (EV1) are evaluated as novel oncolytic agents against human prostate cancer. METHODS. The surface expression of viral receptors required for enterovirus cell attachment/entry, including intercellular adhesion molecule-1 (ICAM-1), decay-accelerating factor (DAF) and integrin alpha2beta1 on a number of human prostate cancer lines was assessed by flow cytometry. Susceptibility to viral oncolysis was determined via in vitro cell lysis assays performedoncellmonolayerscultured inmicrotiterplates.Theinvivooncolytic efficacyofthe enteroviruses was assessed using xenograft models in immune compromized SCID-mice following systemic challenge. RESULTS. ThemajorityofprostatecancerlinestestedexpressedsurfaceICAM-1and/orDAF, or alpha2beta1, facilitating significant degrees of oncolysis following in vitro viral challenge. Systemic delivery of each of the three viruses induced reduction of xenograft tumor burdens in vivo, and a therapeutic dose-response was demonstrated for escalating doses of EV1 in the LNCaP animal model. CONCLUSION. Enteroviruses CVA21, CVA21-DAFv, and EV1 are potentially potent oncolytic agents against human prostate cancer. Prostate 68: 577–587, 2008. # 2008 Wiley-Liss, Inc.

Cultured melanoma cell lines despite exhibiting similar in vitro morphology, display significant phenotypic and growth rate differences when propagated as in vivo xenografts. Previously we have shown that Coxsackievirus A21 (CVA21) lytically infects in vitro cultures of malignant melanoma cells and is efficient at reducing the tumor burden of mice bearing slow-growing SK-Mel-28 melanoma xenografts. The oncolytic activity of CVA21 against in vivo melanoma xenografts, which possess rapid growth rates and more extensive vascular structure than SK-Mel-28 xenografts warrants further investigation. In the present study we evaluated the oncolytic action of CVA21 against rapidly growing melanoma xenografts (ME4405) which exhibit a highly vascular phenotype. Flow cytometric analysis indicated that in vitro cultures of ME4405 cells expressed comparable levels of the CVA21 cellular receptors, ICAM-1 (intercellular adhesion molecules-1) and DAF (decay accelerating factor) to SK-Mel-28 cells. Despite similar levels of CVA21 receptor expression, SK-Mel-28 cells appear to be more susceptible to viral lysis than ME4405 cells, even though the kinetics of virus replication in both lines was comparable. Intratumoral, intraperitoneal or intravenous administration of CVA21 were equally effective in reducing the tumor volume of ME4405 xenografts in immunodeficient mice, and provides further evidence for the use of CVA21 as a novel oncolytic agent against varying phenotypes of malignant melanoma.

Aim: Coxsackievirus A21 (CAVATAKTM) is a bio-selected oncolytic immunotherapy virus. Following intratumoral (i.t) injection, CAVATAK preferential infects ICAM-1 expressing tumor cells, resulting in tumor cell lysis and a systemic immune-mediated anti-tumor response. A Phase II trial of i.t delivered CAVATAK in advanced melanoma patients has highlighted antitumor activity in both injected and distant non-injected lesions. Blockade of programmed death receptor-1 (PD-1) in patients with metastatic melanoma has resulted in substantial tumor responses via a mechanism involving reversal of tumor induced T cell suppression. We hypothesized that combination of CAVATAK and PD-1 blockade may enhance antitumor responses, potentially leading to improved clinical activity. Methods: Preclinical studies in C57BL mice were conducted to assess the antitumor activity of CAVATAK and anti-mouse PD-1 (mPD-1) mAb (2A3-IgG2a) in a B16-ICAM-1 melanoma immune competent mouse model. B16-ICAM-1 cells are murine melanoma B16 cells stably transfected to express human ICAM-1 allowing CAVATAK binding and cell infection. CAVATAK (∼108TCID50) was administered i.t, while anti mPD-1 mAb (12.5 mg/kg) was delivered via the intraperitoneal route. Following treatment of the primary cutaneous B16-ICAM-1 tumor with 8 cycles of CAVATAK injections and 4 cycles of anti-PD-1mAb, mice were then challenged with an additional subcutaneous administration of B16 cells. Results: Significant single agent antitumor activities against the primary B16-ICAM-1 tumor were observed in mice treated with either CAVATAK or anti-PD-1 mAb relative to saline controls. Furthermore, combination of CAVATAK and anti-PD-1 mAb mediated significantly greater antitumor activity and offered greater survival benefit when compared to use of either agent alone. Of particular interest was the finding that a combination of CAVATAK and anti-PD-1 mAb was able to noticeably delay the onset of palpable tumor development following B16 cell challenge when compared to all other single agent treatment regimes. Conclusions: The significant anti-tumor activity mediated by the combination of CAVATAK and anti-PD-1 mAb blockade observed in the presented murine melanoma model supports clinical evaluation of such an immunotherapeutic combination treatment regime in patients with advanced melanoma. Disclosure: D. Shafren: Author is CSO of Viralytics and holds stock; M. Quah and Y. Wong: Receives research support from Viralytics; R.H. Andtbacka: has received travel support from Viralytics; G. Au: Receives research support from Viralytics and holds stock

Meeting abstracts Coxsackievirus A21 (CAVATAKTM) is a bio-selected oncolytic immunotherapy virus. Following intravenous (i.v) administration, CAVATAK can preferentially infect ICAM-1 expressing tumor cell, resulting in tumor cell lysis and generate a potential systemic immune-mediated anti-tumor

Meeting abstracts As a clinical setting in which local live biological therapy is already well established, nonmuscle invasive bladder cancer (NMIBC) presents intriguing opportunities for oncolytic virotherapy. Coxsackievirus A21 (CVA21) is a novel intercellular adhesion molecule-1 (ICAM-1)

Coxsackievirus A21 (CAVATAK™) is a bio-selected oncolytic immunotherapy virus. Following intratumoral (i.t) injection, CAVATAK selectively infects ICAM-1-expressing tumor cells, resulting in tumor cell lysis and a systemic immune-mediated anti-tumor response. A Phase II trial of i.t delivered CAVATAK (NCT01227551) in advanced melanoma patients has highlighted antitumor activity in both injected and distant non-injected lesions. Such responses have occurred at times when no circulating infectious CAVATAK was detected in patient serum and in an environment of high levels of anti-CAVATAK neutralizing antibodies. In further support of the generation of CAVATAK-mediated immune anti-tumor activity is the identification of a possible novel serum cytokine signature of elevated levels of IL-8 and IFN-γ in treated patients associated with tumor inflammation and systemic tumor response. Blockade of programmed death-1 (PD-1) in patients with metastatic melanoma has resulted in substantial tumor responses via a mechanism involving reversal of tumor-induced T cell suppression. We hypothesized that a combination of CAVATAK and PD-1 blockade may enhance anti-tumor responses, potentially leading to improved clinical activity. Preclinical studies in C57BL mice were conducted to assess the anti-tumor activity of CAVATAK and anti-mouse PD-1 (mPD-1) mAb in a B16-ICAM-1 melanoma immune competent mouse model. B16-ICAM-1 cells are murine melanoma B16 cells stably transfected to express human ICAM-1 allowing CAVATAK binding and cell infection. CAVATAK was administered i.t, while anti mPD-1 mAb was delivered intraperitoneally. Following treatment of the primary cutaneous B16-ICAM-1 tumor with 8 cycles of CAVATAK injections and 4 cycles of anti-PD-1mAb, mice were challenged with additional subcutaneous administration of B16 cells. Significant single agent anti-tumor activities against the primary B16-ICAM-1 tumor were observed in mice treated with either CAVATAK or anti-PD-1 mAb relative to saline controls. Combination of CAVATAK and anti-PD-1 mAb mediated significantly greater anti-tumor activity and offered greater survival benefit when compared to use of either agent alone. Of particular interest was the finding that a combination of CAVATAK and anti-PD-1 mAb was able to noticeably delay the onset of palpable tumor development following B16 cell challenge when compared to all other single agent treatment regimes. The significant anti-tumor activity mediated by the combination of CAVATAK and the checkpoint inhibitor antibody (anti-PD-1) observed in the presented murine melanoma model supports clinical evaluation of such an immunotherapeutic combination treatment regimen in patients with advanced melanoma.