Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased tumor-cell specificity, and are currently under investigation in clinical trials including a phase I study for glioblastoma multiforme (GBM). Recent preclinical studies have shown that the cellular tropism of several viruses can be controlled by inserting microRNA-target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. Since neuron-specific microRNA-7 is downregulated in gliomas but highly expressed in normal brain tissue, we engineered a microRNA-sensitive virus containing target sites for microRNA-7 in the 3'-untranslated region of the viral fusion gene. In presence of microRNA-7 this modification inhibits translation of envelope proteins, restricts viral spread, and progeny production. Even though highly attenuated in presence of microRNA-7, this virus retained full efficacy against glioblastoma xenografts. Furthermore, microRNA-mediated inhibition protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Importantly, endogenous microRNA-7 expression in primary human brain resections tightly restricted replication and spread of microRNA-sensitive virus. This is proof-of-concept that tropism restriction by tissue-specific microRNAs can be adapted to oncolytic MV to regulate viral replication and gene expression to maximize tumor specificity without compromising oncolytic efficacy.

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased antitumor activity, and are currently under investigation in clinical phase 1 trials. Approaches with other viral vectors have shown that insertion of immunomodulatory transgenes enhances the therapeutic potency. In this study, we engineered MV for expression of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). For the first time, therapeutic efficacy and adaptive immune response in the context of MV oncolysis could be evaluated in the previously established immunocompetent murine colon adenocarcinoma model MC38cea. MC38cea cells express the human carcinoembryonic antigen (CEA), allowing for infection with retargeted MV. Intratumoral application of MV-GMCSF significantly delayed tumor progression and prolonged median overall survival compared with control virus-treated mice. Importantly, more than one-third of mice treated with MV-GMCSF showed complete tumor remission and rejected successive tumor reengraftment, demonstrating robust long-term protection. An enhanced cell-mediated tumor-specific immune response could be detected by lactate dehydrogenase assay and interferon-γ enzyme-linked immunospot assay. Furthermore, MV-GMCSF treatment correlated with increased abundance of tumor-infiltrating CD3(+) lymphocytes analyzed by quantitative microscopy of tumor sections. These findings underline the potential of oncolytic, GM-CSF-expressing MV as an effective therapeutic cancer vaccine actively recruiting adaptive immune responses for enhanced therapeutic impact and tumor elimination. Thus, the treatment benefit of this combined immunovirotherapy approach has direct implications for future clinical trials.

Precise oncotropism is required for successful systemic administration of next-generation oncolytic measles viruses (MVs). We have previously established a system for efficient post-entry targeting by insertion of synthetic microRNA target sites (miRTS) into the MV genome, thereby repressing replication in the presence of cognate microRNAs. Thus, differential expression of microRNAs, as frequently observed in normal compared with malignant tissues, can be exploited to increase vector specificity and safety. Here we report the combination of miRTS for different microRNAs in a single vector to detarget pivotal organs at risk during systemic administration (liver, brain, gastrointestinal tract). Accordingly, miRTS for miR-122, miR-7 and miR-148a that are enriched in these tissues were inserted to create multi-tissue-detargeted MV (MV-EGFP(mtd)). Replication of MV-EGFP(mtd) is repressed in cell lines as well as in non-transformed primary human hepatocytes and liver slices expressing cognate microRNAs. Oncolytic potency of MV-EGFP(mtd) is retained in a model of pancreatic cancer in vitro and in vivo. This work is a proof-of-concept that favorable expression profiles of multiple microRNAs can be exploited concomitantly to reshape the tropism of MV without compromising oncolytic efficacy. This strategy can be adapted to different vectors and cancer entities for safe and efficient high-dose systemic administration in clinical trials.

No curative therapy is currently available for locally advanced or metastatic pancreatic cancer. Therefore, new therapeutic approaches must be considered. Measles virus (MV) vaccine strains have shown promising oncolytic activity against a variety of tumor entities. For specific therapy of pancreatic cancer, we generated a fully retargeted MV that enters cells exclusively through the prostate stem cell antigen (PSCA). Besides a high-membrane frequency on prostate cancer cells, this antigen is expressed on pancreatic adenocarcinoma, but not on non-neoplastic tissue. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cell lines, and we could show specific infection of pancreatic adenocarcinoma cell lines with both high- and low-level PSCA expression. Furthermore, we generated a fully retargeted and armed MV-PNP-anti-PSCA to express the prodrug convertase purine nucleoside phosphorylase (PNP). PNP, which activates the prodrug fludarabine effectively, enhanced the oncolytic efficacy of the virus on infected and bystander cells. Beneficial therapeutic effects were shown in a pancreatic cancer xenograft model. Moreover, in the treatment of gemcitabine-resistant pancreatic adenocarcinoma cells, no cross-resistance to both MV oncolysis and activated prodrug was detected.

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Virotherapy is a unique modality for the treatment of cancer with oncolytic viruses (OVs) that selectively infect and lyse tumor cells, spread within tumors, and activate anti-tumor immunity. Various viruses are being developed as OVs preclinically and clinically, several of them engineered to encode therapeutic proteins for tumor-targeted gene therapy. Scientists and clinicians in German academia have made significant contributions to OV research and development, which are highlighted in this review paper. Innovative strategies for "shielding," entry or postentry targeting, and "arming" of OVs have been established, focusing on adenovirus, measles virus, parvovirus, and vaccinia virus platforms. Thereby, new-generation virotherapeutics have been derived. Moreover, immunotherapeutic properties of OVs and combination therapies with pharmacotherapy, radiotherapy, and especially immunotherapy have been investigated and optimized. German investigators are increasingly assessing their OV innovations in investigator-initiated and sponsored clinical trials. As a prototype, parvovirus has been tested as an OV from preclinical proof-of-concept up to first-in-human clinical studies. The approval of the first OV in the Western world, T-VEC (Imlygic), has further spurred the involvement of investigators in Germany in international multicenter studies. With the encouraging developments in funding, commercialization, and regulatory procedures, more German engineering will be translated into OV clinical trials in the near future.

First-line treatment of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC) is based on platinum, 5-fluorouracil (5-FU) and the monoclonal antiEGFR antibody cetuximab. However, in most cases this chemoimmunotherapy does not cure the disease, and more than 50% of HNSCC patients are dying because of local recurrence of the tumors. In the majority of cases, HNSCC overexpress the epidermal growth factor receptor (EGFR), and its presence is associated with a poor outcome. In this study, we engineered an EGFR-targeted oncolytic measles virus (MV), armed with the bifunctional enzyme cytosine deaminase/ uracil phosphoribosyltransferase (CD/UPRT). CD/UPRT converts 5-fluorocytosine (5-FC) into the chemotherapeutic 5-FU, a mainstay of HNSCC chemotherapy. This virus efficiently replicates in and lyses primary HNSCC cells in vitro. Arming with CD/UPRT mediates efficient prodrug activation with high bystander killing of non-infected tumor cells. In mice bearing primary HNSCC xenografts, intratumoral administration of MV-antiEGFR resulted in statistically significant tumor growth delay and prolongation of survival. Importantly, combination with 5-FC is superior to virus-only treatment leading to significant tumor growth inhibition. Thus, chemovirotherapy with EGFR-targeted and CD/UPRT-armed MV is highly efficacious in preclinical settings with direct translational implications for a planned Phase I clinical trial of MV for locoregional treatment of HNSCC.

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review—as part of the special edition on “State-of-the-Art Viral Vector Gene Therapy in Germany”—the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.