Systemic Delivery of mLIGHT-Armed Myxoma Virus Is Therapeutic for Later-Stage Syngeneic Murine Lung Metastatic Osteosarcoma

Cancers that metastasize to the lungs represent a major challenge in both basic and clinical cancer research. Oncolytic viruses are newly emerging options but successful delivery and choice of appropriate therapeutic armings are two critical issues. Using an immunocompetent murine K7M2-luc lung metastases model, the efficacy of MYXV armed with murine LIGHT (TNFSF14/CD258) expressed under virus-specific early/late promoter was tested in an advanced later-stage disease K7M2-luc model. Results in this model show that mLIGHT-armed MYXV, delivered systemically using ex vivo pre-loaded PBMCs as carrier cells, reduced tumor burden and increased median survival time. In vitro, when comparing direct infection of K7M2-luc cancer cells with free MYXV vs. PBMC-loaded virus, vMyx-mLIGHT/PBMCs also demonstrated greater cytotoxic capacity against the K7M2 cancer cell targets. In vivo, systemically delivered vMyx-mLIGHT/PBMCs increased viral reporter transgene expression levels both in the periphery and in lung tumors compared to unarmed MYXV, in a tumor- and transgene-dependent fashion. We conclude that vMyx-mLIGHT, especially when delivered using PBMC carrier cells, represents a new potential therapeutic strategy for solid cancers that metastasize to the lung.

The Use of Oncolytic Viruses in the Treatment of Multiple Myeloma

Multiple myeloma accounts for 1% of all new cancers worldwide. It is the second most common haematological malignancy and has a low five-year survival rate (53.2%). Myeloma remains an incurable disease and is caused by the growth of malignant plasma cells in the bone marrow. Current anti-myeloma therapies (conventional chemotherapies, immunomodulatory drugs i.e., thalidomide and its’ analogues, proteasome inhibitors, monoclonal antibodies, and radiotherapy) initially substantially debulk tumour burden, but after a period of remission ‘plateau phase’ disease invariably relapses due to tumour recrudescence from foci of minimal residual disease (MRD) and accumulating drug resistance. Therefore, there is a compelling clinical need for the development of novel treatment regimens to target MRD and effectively eliminate all remaining tumour cells. This review will discuss the potential use of oncolytic virus (OV) therapies in the treatment of myeloma. Specifically, it will focus on preclinical studies using DNA viruses (adenovirus (Ad), vaccinia virus (VV), myxoma virus (MYXV), and herpes simplex virus (HSV)), RNA viruses (reovirus (reo), coxsackie virus, measles virus (MV) and bovine viral diarrhoea virus (BVDV), and vesicular stomatitis virus (VSV)), and on four types of viruses (VV, reo, MV-NIS and VSV-IFNβ-NIS) that have been assessed clinically in a small number of myeloma patients.

A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

947 Recombinant myxoma virus MC509-N1 demonstrates antitumor efficacy as monotherapy and in combination with immune checkpoint inhibitors

BackgroundThere are several obstacles to effective cancer immunotherapy including the heterogenic immune profile and the state of the tumor microenvironment. Oncolytic virotherapy provides an opportunity to overcome some of these limitations through high viral replication and the expression of therapeutic transgenes (TGs) within the tumor tissue. Myxoma virus (MYXV) belongs to the family of Poxviridae and represents a potent oncolytic virus and a safe platform as this virus is non-pathogenic in any hosts apart from lagomorphs. Importantly, MYXV has a high capacity of encoding for multiple TG payloads. Here we engineered MC509-N1, a novel double-encoding transgenes (TG1 and TG2) oncolytic MYXV designed for intravenous (IV) injection. The therapeutic TG1 acts to modify and remodel the immune state of the tumor microenvironment, and TG2 allows for prolonged self-evasion from the host immune defense.MethodsTransgenes expression upon infection was detected by ELISA and by flow cytometry. To determine anticancer efficacy, syngeneic B16F10 melanoma or MC38 colorectal cancer-bearing C57BL/6 mice were injected with MC509-N1 intratumorally or IV with or without immune checkpoint inhibitor (ICI). Tumor growth and survival was monitored after treatment and the immune profile within the tumor microenvironment was analyzed by flow cytometry. Mice cured of their tumors from the original treatment were rechallenged with primary tumor cells to examine anticancer immunity.ResultsCells upon infection with MC509-N1 were found to express both transgenes at high levels and stimulate downstream mechanisms. Importantly, the engineering of both transgenes did not affect MC509-N1 infectivity and productivity as compared to wild-type MYXV. Intratumoral injections of MC509-N1 effectively suppressed tumor growth and improved overall survival of both syngeneic cancer models. Furthermore, MC509-N1 therapy effectively modulated the immune profile within the tumor microenvironment, especially the ratio between tumor infiltrated CD8+ cytotoxic T cells and CD4+FoxP3+ T regulatory cells. In addition, IV injections of MC509-N1 showed improved inhibition of tumor growth compared to wild type MYXV. The combination therapy of MC509-N1 with the ICI anti-PD-L1 further promoted inhibition of tumor growth as demonstrated by higher rate of complete regression and improved survival rate. Furthermore, rechallenge experiments revealed that this combination regimen established specific anticancer immune memory and protected from cancer recurrence.ConclusionsOur results demonstrate that the novel engineered MC509-N1 exhibits potent anticancer efficacy, adequately modulates the immune state of the tumor microenvironment, and acts synergistically to eliminate cancer in combination with ICI.

742 Multi-armed myxoma virus induces potent anti-tumor responses in vitro and in vivo

BackgroundMyxoma virus (MYXV) has been shown to selectively infect cancer cells in humans in vitro and inhibit tumor growth in mice. The genome of MYXV is large and amenable to engineering for expression of multiple transgenes. We armed MYXV with mouse or human IL-12 and human decorin. IL-12 is an immune modulator. Cellular responses to decorin include tumor cell intrinsic signaling effects, tumor matrix remodeling, and inhibition of the TGF-beta pathway. We hypothesized that MYXV armed with decorin and IL-12 would be an effective anti-tumor therapy. The current work describes the oncolytic activity and transgene expression, following exposure to armed MYXV in human cancer cell lines in vitro and efficacy in in vivo in murine models, as single agents and in combination with immune checkpoint inhibition.MethodsCytotoxicity was measured by a cell viability assay. ELISAs were used to detect transgene expression, Caspase-3 activation, and TGF-beta induced SMAD phosphorylation. Mouse tumor models were treated with vehicle control or the indicated virus.ResultsMYXV carrying payloads of decorin and mouse IL-12 (vMYX-mIL-12/Dec) or human IL-12 (vMYX-hIL-12/Dec) were tested. Human tumor cell lines infected with vMYX-hIL-12/Dec in vitro showed independent effects when levels of transgene expression and cytotoxicity were compared, suggesting that oncolytic activity and transgene expression differentially contribute to MYXV activity. Virus-free supernatants derived from infected cells suggested a decorin specific response in caspase-3 activation, and inhibition of TGF-beta signaling. Human IL-12 is not active on mouse immune cells giving the opportunity to evaluate the role of decorin in tumor regression. B16-F10 murine melanoma mice treated with vMYX-mIL-12/Dec showed a robust response while vMYX-hIL-12/Dec showed an intermediate anti-tumor response suggesting decorin has cancer inhibitory activity and synergized with IL-12. We tested anti-PD-1 and vMYX-mIL-12/Dec in the colon adenocarcinoma model MC38. We observed that the combination for multi-armed MYXV with an immune checkpoint inhibitor showed dramatically reduced tumor growth and improved survival.ConclusionsOur data demonstrates that MYXV with IL-12 and decorin payloads have cytotoxic activity in vitro and inhibit tumor growth in vivo. Cellular responses to decorin in vitro included inhibition of processes intrinsic to tumor progression. In mouse tumor models decorin played a role in inhibiting tumor progression and synergized with IL-12 implying the combination has immune-modulatory activity. Interestingly, MYXV with IL-12 and decorin payloads significantly synergized with anti-PD-1 in preventing tumor growth, suggesting a potentially new approach towards anti-cancer therapy.Ethics ApprovalAll studies and procedures involving animals were carried out under the institutional guidelines of Translational Drug Development Institutional Animal Care and Use Committee

744 Multi-armed myxoma virus has therapeutic potential for treatment of multiple myeloma

BackgroundDespite improvements with new therapeutics, multiple myeloma (MM) patients still relapse and become refractory. Myxoma virus (MYXV) selectively replicates in human tumor cells and stimulates the immune system. MYXV selectively kills human patient MM cells and spares normal progenitors. MYXV also eradicates growth of a disseminated mouse MM in vivo. MYXV is a large, double stranded DNA poxvirus, and has a genome size amenable to insertion of multiple transgenes. We generated MYXV carrying IL-12 and decorin. IL-12 is an immune modulator that activates T- and NK-cells. Cellular responses to decorin include tumor cell intrinsic signaling effects, tumor matrix modulation, and inhibition of the TGF-beta pathway. This represents a promising therapeutic option for MM patients that do not respond well to immunotherapy. The current work suggests MYXV armed with IL-12 and decorin could be an effective anti-MM therapy.MethodsCytotoxicity assays were performed using a cell viability assay. Transgene expression levels were analyzed by microscopy, flow cytometry, and ELISA.ResultsHuman MM cell line U266 infected with MYXV (vMYX-hIL-12/Dec) carrying human IL-12, decorin, and green fluorescent protein (GFP) produced transgenes in a dose and time responsive manner. A panel of human MM cell lines was infected with vMYX-hIL-12/Dec and transgene expression in supernatant, cell killing EC50, and GFP levels were evaluated. Sensitive and resistant human MM cell lines were identified. The comparison of replication, cell killing capacity, and transgene expression highlighted the independent importance of these mechanisms in overall activity.ConclusionsThe current work describes the oncolytic activity and transgene expression following exposure to vMYX-hIL-12/Dec in human MM cell lines in vitro. Our initial studies suggest there is significant value in pursuing vMYX-hIL-12/Dec and other armed MYXV as a new approach towards MM therapy.

Viral Disease in Lagomorphs: A Molecular Perspective

Our understanding of molecular biology of the viruses that infect lagomorphs is largely limited to the leporipoxvirus myxoma virus (MYXV) and the lagoviruses rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) that infect the European rabbit (Oryctolagus cuniculus) and the European brown hare (Lepus europaeus) respectively. Thanks to the great effort of historic surveillance studies and careful sample archiving, the molecular evolution of these viruses is being resolved. Although historically considered viruses that cause species specific diseases recent reports show that several lagomorphs may now face the threat of these maladies. The driving factors behind these changes has not been determined and the effect of these species jumps on lagomorph populations has yet to be seen. Lagomorphs are also affected by several other lesser studied viral diseases. In addition, recent metagenomic studies have led to the identification of novel lagomorph viruses the importance of these to lagomorph health remains to be fully determined. In this chapter we summarize molecular aspects of viruses that infect lagomorphs, paying particular attention to recent interspecies infections.