Enhanced oncolysis mediated by Coxsackievirus A21 in combination with doxorubicin hydrochloride.

Virotherapy is an emerging strategy for the treatment of cancer that utilizes both replication-competent and genetically modified viruses to selectively kill tumor cells. We have previously shown that Coxsackievirus A21 (CVA21), a common-cold producing enterovirus, is an effective oncolytic agent against human melanoma, prostate, and breast cancer xenografts in vivo. CVA21 specifically targets and lytically infects susceptible cells expressing the CVA21 cellular receptors, intercellular adhesion molecule-1 (ICAM-1) and decay-accelerating factor (DAF). Herein, the efficacy of CVA21 administered in combination with doxorubicin hydrochloride as a new therapeutic regimen for cancer was investigated. Flow cytometric analysis demonstrated that the human breast, colorectal, and pancreatic cancer cell lines examined expressed moderate levels of surface ICAM-1 and DAF, whilst a normal breast cell line expressed only minimal levels. When CVA21 was combined with doxorubicin hydrochloride, synergistically enhanced cell death was observed when CVA21 was administered both simultaneously or 24 h prior to doxorubicin hydrochloride exposure. Doxorubicin hydrochloride had no effect on CVA21 replication. Through the use of an orthotopic (MDA-MB-231-luc) xenograft SCID mouse model of human breast cancer we showed that a single intravenous injection of CVA21 in combination with an intraperitoneal injection of doxorubicin hydrochloride resulted in significantly greater tumor reduction compared to either agent alone. Overall, these findings highlight the exciting potential of CVA21, administered in combination with doxorubicin hydrochloride, as a new therapeutic regimen for cancer.

Oncolysis of malignant human melanoma tumors by Coxsackieviruses A13, A15 and A18.

Many RNA viruses are displaying great promise in the field of oncolytic virotherapy. Previously, we reported that the picornavirus Coxsackievirus A21 (CVA21) possessed potent oncolytic activity against cultured malignant melanoma cells and melanoma xenografts in mice. In the present study, we demonstrate that three additional Group A Coxsackieviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15) and Coxsackievirus A18 (CVA18), also have similar oncolytic activity against malignant melanoma. Each of the viruses grew quickly to high titers in cancer cells expressing ICAM-1 and intratumoral injection of preformed subcutaneous SK-Mel-28 xenografts in mice with CVA13, CVA15 and CVA18 resulted in significant tumor volume reduction.As preexisting immunity could potentially hinder oncolytic virotherapy, sera from stage IV melanoma patients and normal controls were tested for levels of protective antibody against the panel of oncolytic Coxsackieviruses. Serum neutralization assays revealed that 3 of 21 subjects possessed low levels of anti-CVA21 antibodies, while protective antibodies for CVA13, CVA15 and CVA18 were not detected in any sample. Serum from individuals who were seropositive for CVA21 failed to exhibit cross-neutralization of CVA13, CVA15 and CVA18. From these studies it can be concluded that the administration of CVA13, CVA15 or CVA18 could be employed as a potential multivalent oncolytic therapy against malignant melanoma.

Regional administration of oncolytic Echovirus 1 as a novel therapy for the peritoneal dissemination of gastric cancer.

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.

Systemic targeting of metastatic human breast tumor xenografts by Coxsackievirus A21.

Breast cancer is the most commonly diagnosed malignancy in women worldwide. Metastatic development is associated with poor prognosis and current therapies provide only limited success. Virotherapy is an emerging strategy for the treatment of cancer that utilizes both replication-competent and genetically modified viruses to selectively kill tumor cells. We have previously shown that Coxsackievirus A21 (CVA21), a wild-type common-cold producing enterovirus, is an effective oncolytic agent against human melanoma xenografts in vivo. CVA21 specifically targets and lytically infects susceptible cells expressing the CVA21 cellular receptors, intercellular adhesion molecule-1 (ICAM-1) and/or decay-accelerating factor (DAF). Herein, the efficacy of CVA21 as a therapeutic agent against human breast cancer was investigated both in vitro and in vivo. Flow cytometric analysis revealed that the human breast cancer cell lines examined expressed significantly elevated levels of surface ICAM-1 and DAF compared to normal breast cell lines, and that all cancerous lines were more susceptible to lytic infection by CVA21 than the normal cells. Through the use of subcutaneous (T47D cells) and orthotopic (MDA-MB-231-luc cells) xenograft SCID mouse models it was demonstrated that a single intravenous injection of CVA21 produced significant regression of pre-established tumors in vivo, as well as targeting and elimination of metastases in the orthotopic model. Taken together, these findings highlight the exciting potential of CVA21 as a therapeutic agent against both primary and metastatic human breast cancer.

Potent oncolytic activity of human enteroviruses against human prostate 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 alpha(2)beta(1) 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 performed on cell monolayers cultured in micro titer plates. The in vivo oncolytic efficacy of the enteroviruses was assessed using xenograft models in immune compromised SCID-mice following systemic challenge. RESULTS: The majority of prostate cancer lines tested expressed surface ICAM-1 and/or DAF, or alpha(2)beta(1), 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.

Oncolysis of vascular malignant human melanoma tumors by Coxsackievirus A21.

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.

Oncolysis of human ovarian cancers by echovirus type 1.

A small number of enteroviruses possess the capacity to induce rapid and marked lytic infections in cells of various human malignancies. During screening of representative human enteroviruses for their oncolytic capacity, we observed that echovirus type 1 (EV1) displayed a high level of tropism for human ovarian cancer cells. EV1 is an enterovirus which largely causes asymptomatic infections in humans and whose tissue tropism is primarily regulated via interactions with the I domain of the alpha subunit of cell surface-expressed integrin alpha2beta1. We evaluated the capacity of wild-type EV1 to act as an oncolytic agent of ovarian cancers propagated as cell monolayers, multicellular spheroids or xenografts in SCID mice. EV1 infection of in vitro propagated ovarian cell lines expressing high levels of integrin alpha2beta1 was assessed for specific viral attachment, antibody blockade, induction of cytopathic effect and production of progeny virions. EV1 lytically infected all 8 human ovarian cancer cell lines tested (2008, DOV13, JAM, OVCA-429, OVCAR-3, OVHS-1, OAW-42 and IGROV-1) but not the immortalized normal ovarian surface epithelial cell line (HOSE) or human PBMCs. EV1 challenge was equally effective in the oncolysis of human ovarian cancer cells whether in monolayer or spheroidal environments. The therapeutic efficacy of EV1 was demonstrated by rapid reduction of tumor burden by a single viral intratumoral injection in SCID mice bearing multiple preformed s.c. xenografts. Using an in vivo i.p. human ovarian cancer xenograft model, administration of EV1 was further shown to significantly inhibit the formation and burden of ascites tumors. These findings demonstrate an important proof of principle for employing wild-type EV1 as a potential oncolytic agent in the control of human ovarian cancers.

Novel role for decay-accelerating factor in coxsackievirus A21-mediated cell infectivity.

Decay-accelerating factor (DAF) is involved in the cell membrane attachment of many human enteroviruses. Presently, further specific active roles of DAF in mediating productive cell infection and in the pathogenesis of natural enterovirus infection are poorly understood. In an attempt to more fully understand the role of DAF in lytic cell infection we examined the specific interactions of the prototype strain of coxsackievirus A21 (CVA21) with surface-expressed DAF. Investigations into discrete DAF-CVA21 interactions focused on viral binding; viral particle elution with respect to the parameters of time, temperature, and pH; and subsequent cell infection. Radiolabeled-virus binding assays revealed that peak elution of CVA21 from DAF occurred within 15 min of initial attachment and that the DAF-eluted virus increased in a linear fashion with respect to temperature and pH. CVA21 eluted from endogenous surface-expressed DAF was highly infectious, in contrast to CVA21 eluted from intercellular adhesion molecule 1 (ICAM-1), which retained little to no infectivity. Using an adenovirus transduction system, we demonstrate that CVA21 can remain infectious for up to 24 h after DAF binding and is capable of initiating a multicycle lytic infection upon delayed ICAM-1 surface expression. Taken together, the data suggest that a major role of DAF in cell infection by the prototype strain of CVA21 is to provide membrane concentration of infectious virions, effectively increasing viral interactions with endogenous or induced ICAM-1.

Systemic therapy of malignant human melanoma tumors by a common cold-producing enterovirus, coxsackievirus a21.

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.

Enterovirus capsid interactions with decay-accelerating factor mediate lytic cell infection.

The cellular receptor usage of numerous human enteroviruses can differ significantly between low-cell-culture-passaged clinical isolates and highly laboratory-passaged prototype strains. The prototype strain of coxsackievirus A21 (CVA21) displays a dual-receptor specificity as determined with a receptor complex consisting of decay-accelerating factor (DAF) and intercellular adhesion molecule 1 (ICAM-1). In this study, the cellular receptor interactions of low-cell-passage CVA21 clinical isolates with respect to their interactions with cell surface-expressed DAF and ICAM-1 were compared to those of the CVA21 prototype (Kuykendall) strain. Dual-receptor usage of DAF and ICAM-1 by CVA21 clinical isolates was confirmed by cell transfection and radiolabeled binding assays. The cellular attachment of clinical and prototype CVA21 strains to cells that coexpressed DAF and ICAM-1 was not additive compared to the viral binding to cells expressing one or other receptor. In fact, the binding data suggest there is an inhibition of CVA21 cellular attachment in environments where high-level coexpression of both DAF and ICAM-1 occurs. Antibody cross-linking of DAF rendered cells susceptible to lytic infection by the CVA21 clinical isolates. In a novel finding, three clinical isolates could, to various degrees, infect and lyse DAF-expressing cells in the absence of DAF-antibody cross-linking and ICAM-1 expression. Sequence analysis of the P1 region of clinical and prototype virus genomes identified a number of coding changes that may contribute to the observed enhanced DAF usage phenotype of the clinical CVA21 isolates. None of the amino acid changes was located in the previously postulated ICAM-1 footprint, a receptor-binding environment that was conserved on the capsid surface of all CVA21 clinical isolates. Taken together, the data suggest that community-circulating strains of CVA21 can infect target cells expressing either ICAM-1 or DAF alone and that such interactions extend tissue tropism and impact directly on viral pathogenesis.

Cellular receptor interactions of C-cluster human group A coxsackieviruses.

The cellular receptor complex of coxsackievirus A21 (CVA21), a C-cluster human enterovirus, is formed by the subtle interaction of individual cellular receptors, decay accelerating factor (DAF) and intercellular adhesion molecule-1 (ICAM-1). In this receptor complex, DAF functions in the membrane sequestration of the virus, while the role of ICAM-1 is as the functional cellular internalization receptor. However, despite the elucidation of the CVA21-cell receptor interactions, there have been few definite investigations into cellular receptor usage of other coxsackie A viruses (CVAs) belonging to the C-cluster. In the present study, radiolabelled virus-binding assays demonstrated that CVA13, -15, -18 and -20, a subset of the human enterovirus C-cluster, bind directly to surface-expressed ICAM-1, but not to surface-expressed DAF. Furthermore, lytic infection of ICAM-1-expressing rhabdomyosarcoma (RD) cells by this C-cluster subset of viruses was inhibited by specific ICAM-1 monoclonal antibody blockade, except for that of CVA20. Despite possessing ICAM-1-binding capabilities, CVA20 employed an as yet unidentified internalization receptor for cell entry and subsequent productive lytic infection of ICAM-1-negative RD cells. In a further example of C-cluster cellular receptor heterogeneity, CVA13 exhibited significant binding to the surface of CHO cells expressing neither DAF nor ICAM-1. Despite a common receptor usage of ICAM-1 by this subset of C-cluster CVAs, the amino acid residues postulated to represent the ICAM-1-receptor footprint were not conserved.