Virotherapy Research in Germany: From Engineering to Translation

Adenovirus platform

Adenoviruses (Ads) are particularly suitable as OVs, because they possess potent lytic activity, their structure and replication cycle are well described, and ample opportunities for virus engineering are established. Over the last two decades, several groups in Germany have focused on different aspects of developing oncolytic Ads (OAds) for cancer therapy, including transcriptional regulation of viral replication, targeting viral cell entry and virus shielding, immunological aspects, therapeutic gene expression, as well as multimodal therapies (see also Table 1).

The group of Florian Kühnel and Stefan Kubicka at Hannover Medical School initially focused on developing conditionally replicating OAds that specifically respond to pan-cancer-associated molecular alterations such as telomerase activation and/or p53 dysfunction. ^31,32 The group achieved tumor-selective Ad replication using the telomerase promoter for transcriptional control of E1A, resulting in the OAd hTert-Ad. Because of the frequent telomerase activation in human cancer this concept is broadly applicable, and hTert-Ad has been used in several follow-up studies investigating the use of OAds as a central component of tumor immunotherapies. ^32,41 Furthermore, the group developed several OAds for selective replication in tumor cells with defective p53. ^{31 ,36z} These viruses use p53-dependent transrepression of E1A, p53-dependent expression of antiviral-microRNA networks, and a meganuclease-based “self-destruction” mechanism that was introduced to facilitate selective autodigestion of the viral genome in tumor cells.

Previous work of the group of Dirk M. Nettelbeck (German Cancer Research Center, Heidelberg) explored and optimized transcriptional targeting of Ad replication to tumor cells by expression of one or two essential Ad genes—or mutants thereof—using engineered cellular tyrosinase enhancers and promoters. ^33z

The group of Per Sonne Holm at the Technical University Munich, Klinikum rechts der Isar, in cooperation with Ulrike Naumann at the University of Tübingen, is focusing on treatment of glioblastoma and bladder cancer using YB-1-dependent OAds alone or in combination with cytostatic drugs and radiation. YB-1 is a multifunctional cellular (onco-)protein that plays important roles in tumor cell proliferation, progression, and drug resistance. Thus, YB-1 has emerged as a promising molecular target for the development of new virotherapy strategies. ⁷³ The important role of YB-1 in Ad replication was shown by the Holm group and has been exploited for the development of tumor-restricted, YB-1-dependent OAds.^30z,61,73 The Naumann group has established subcutaneous and orthotopic, both xenograft and syngeneic GBM (glioblastoma) mouse models and treatment modalities using intratumoral single injection as well as infusion techniques for the assessment of virotherapeutic approaches in vivo. ³⁰

In vivo, the therapeutic effect of the lead YB-1-dependent OAd XVir-N-31 alone or in combination with chemotherapy or radiotherapy ⁶¹ was investigated, among others, in an orthotopic GBM mouse model using a primary, chemotherapy-resistant GBM stem cell line. Compared with wild-type Ad, the genetic regions E1A and E1B of XVir-N-31 are modified to prevent expression of the proteins E1A13S and E1B19K, respectively, in order to ensure YB-1 dependence and to enhance oncolysis. Furthermore, an RGD motif is introduced into the fiber knob domain to modify the capsid of the virus. Intratumoral administration of XVir-N-31 significantly prolonged the median survival of tumor-bearing mice. ³⁰ On the basis of this research program, the Munich group, together with the spin-off company XVir Therapeutics GmbH, at present is preparing a phase I/II trial with XVir-N-31 for the treatment of recurrent glioblastoma (see below).

A further research focus in Germany has been to engineer the Ad capsid for targeted entry into cancer cells. In contrast to similar strategies well established for measles viruses, using genetic fusion of antibody fragments to viral glycoproteins (see Measles vaccine virus platform), entry targeting of Ad is hampered by the rigid structure of the Ad capsid. Furthermore, biosynthesis of the Ad capsid in the host cell nucleus represents an obstacle to the genetic insertion of affinity molecules that require glycosylation and/or disulfide bonds.

As an alternative to direct modification of capsid proteins, the Kühnel group generated bispecific adapter proteins to facilitate molecular retargeting of OAds toward polysialic acid, a cell surface antigen abundantly expressed on neuroendocrine tumors. Molecular retargeting can be used to enhance initial tumor infection to levels needed to stimulate anti-tumoral immune responses, even after systemic vector administration. ¹⁶

Florian Kreppel's group at the University Witten/Herdecke is working on improved delivery of OAds, with a special focus on systemic delivery through the bloodstream. Delivery through blood is mandatory to reach hardly accessible tumors or disseminated disease. Basic research, however, has revealed a multitude of interactions of various Ad types with noncellular and cellular blood components. Most of these interactions trigger premature sequestration and neutralization of viral particles as well as toxicity early after intravenous delivery. ^{74 –77} Therefore, the best characterized Ad types cannot be successfully delivered intravenously to the patients despite a great medical need.

The Kreppel group has developed a combination of genetic and chemical viral capsid modification technologies. ^17,78 Using this “geneti-chemical” modification technology, the viral capsids can be modified precisely with shielding molecules, targeting moieties or a combination thereof. Importantly, the choice of molecules that can be coupled to shield the viral capsid from unwanted virus–host interactions and target it to cancer tissue is not restricted by the substance class: proteins up to 150 kDa in size, (nonnatural) peptides, carbohydrates, lipids, small molecules, and synthetic or natural polymers can be chemically attached to the virus surface with high efficiency and specificity in a way that maintains the virus's oncolytic potency. Using mouse models and human blood, the group demonstrated that unwanted vector–host interactions such as neutralization by antibodies and complement or binding to blood coagulation factors can be completely prevented or significantly dampened. In fact, geneti-chemically modified Ad5-based viral particles exhibited strongly improved pharmacokinetics after intravenous delivery. ¹³ On the basis of their patented findings and with funding from the German Federal Ministry of Education and Research (BMBF) GO-Bio program and BMWi's EXIST Transfer of Research program, the Witten group is currently founding the spin-off company Ad-O-Lytics. ^* Ongoing work includes analysis of the viruses in various mouse tumor models and toxicity studies. The goal is to bring geneti-chemically modified OAds into the clinic.

For targeting of Ad cell entry to tumor cells by genetic engineering of the viral capsid, facilitating tropism modification also of progeny viruses, the Nettelbeck group inserted peptide ligands into an Ad capsid “blinded” for the native Ad receptor. To ablate native tropism, the shaft and knob domains of the cell-binding fiber protein of Ad serotype 5 were replaced by the corresponding domains of the short fiber of Ad serotype 41. ⁷⁹ Peptides binding to the emerging tumor target EphA2 could be genetically inserted into various positions of the chimeric fiber, resulting in peptide-mediated and cell type-specific Ad cell entry. ¹⁸ Peptide-mediated Ad cell entry was demonstrated for EphA2-overexpressing melanoma and pancreatic cancer cell lines in vitro and in vivo and for melanoma biopsies.

To target OAds to both tumor cells and tumor stroma of pancreatic cancer, the group of Stefan Kochanek at Ulm University genetically inserted a transforming growth factor (TGF)-β-binding peptide into the capsid protein hexon, resulting in enhanced cytotoxicity for both neoplastic and stellate cells or for cocultures. ¹⁹

In parallel to efforts on entry targeting and stringent replication control, the Hannover group also turned toward the function of OAds as an immunogenic stimulus. Besides their cytolytic function, OVs elicit innate and, even more important, adaptive tumor-directed immune responses due to effective cross-presentation of tumor antigens in virus-infected tumors. The group investigated the role of tumor selectivity of viral replication in anti-viral/anti-tumor immune balance. ^{31 ,32z} The studies strongly support the efforts for stringent regulation of OAds in order to optimize the immune balance, to reduce hepatotoxicity, and to improve therapeutic outcome.

At present, the Hannover group focuses on optimizing the immunogenic potency of OAds and their integration into systemic and/or personalized immunotherapeutic concepts. The heterologous expression of Flt3 ligand (Flt3L) and macrophage inflammatory protein (MIP)-1α was used to amplify the in situ vaccination properties of oncolytic virotherapy, and tumor-directed DC vaccines were used to shift the immune balance in favor of anti-tumor immunity.^32z These vaccines were effective only when applied during oncolytic tumor inflammation, suggesting that OVs are excellent tools to abrogate systemic tumor tolerance and resistance to tumor-directed vaccination. ICIs have shown great clinical success, but the vast majority of patients with tumors do not respond to therapy. In this regard, a highly relevant finding of the Hannover group is that intratumoral virotherapy combined with inhibition of the programmed death-ligand 1 (PD-1)/programmed death-ligand 1 (PD-L1) checkpoint leads to a significant spreading of CD8⁺ T cell responses against neoepitopes, associated with an improved therapeutic outcome. ⁴¹ Because these observations demonstrate that viral oncolysis is a promising trigger to overcome systemic tumor resistance to ICI, corresponding viro-immunotherapeutic concepts will be further propagated in Hannover toward clinical studies.

Ads also allow for the insertion of transgenes to improve viral oncolysis or to complement additional modes of action (“arming” of OVs). To this end, the Nettelbeck group developed strategies for therapeutic gene insertion at various positions of the Ad genome and exploited various genetic mechanisms for coexpression of inserted transgenes with viral genes.^33z These strategies allow for modulation of transgene expression both in terms of expression levels and timing during the Ad replication cycle. As such, a specific expression strategy was required to develop an armed OAd for the genetic delivery of a small antibody fusion protein, an immunoRNase (antibody virotherapy). The immunoRNase consisted of an epidermal growth factor receptor (EGFR)-binding antibody fragment fused to a cell death-triggering RNase. Insertion of the immunoRNase gene at a specific locus in the OAd genome and coexpression with a late viral gene, using a splice acceptor site, enabled both efficient OAd replication and immunoRNase expression. ⁵⁰ Thereby, oncolysis was combined with the expression of a targeted therapeutic biomolecule in tumor cells for the killing of (not infected) bystander cells in vitro and in vivo.

The development of clinically applicable armed OVs would benefit or even require strategies to switch transgene expression on and off. For example, it has been reported that the therapeutic benefit of OV-encoded immunostimulatory cytokines or chemokines depends on the timing of their expression during the virotherapy regimen, that is, delayed expression tended to preferentially support anti-tumor rather than anti-viral immune activation. ⁸⁰ The Nettelbeck group established inducible regulation of transgene expression by inserting short designer RNA sequences, aptazymes, into the transgene. These mediate transgene regulation at the mRNA level via activation or inactivation of intrinsic RNase activity (ribozyme part of aptazyme) by binding of small-molecule ligands (aptamer part of aptazyme). ⁴⁹ In cooperation with the Ungerechts group in Heidelberg, proof-of-principle for the feasibility of aptazymes to also control replication of DNA viruses (OAds) and RNA viruses (oncolytic measles vaccine viruses) via regulation of essential viral genes was demonstrated. ⁷² These results reveal the potential of aptazymes as safety switches that address safety concerns for (future generations of efficiency-enhanced) OVs for which antiviral compounds are not available. Aptazymes can be customized as ON or OFF switches and for regulation by various small molecules. Once advanced aptazymes featuring effective regulation in human cells and triggered by ligands applicable in patients are available, transgene or virus control by aptazymes might find applications in various OVs and virotherapeutic modalities.

The Berlin groups of Jürgen Eberle (Charité) and Henry Fechner (Technical University) develop armed OAds that trigger apoptosis for effective tumor cell killing, with a focus on the treatment of malignant melanoma. Deficiency in apoptosis programs critically contributes to melanoma therapy resistance, ^81,82 and targeting apoptosis pathways thus represents a promising strategy. The intrinsic, mitochondrial apoptosis pathway, which is of particular importance for apoptosis regulation in melanoma cells, is critically controlled by the family of pro- and antiapoptotic Bcl-2 proteins. Thus, Bcl-2 proteins appear to be suitable targets for melanoma therapy. ⁸³ On the other hand, the Eberle group has shown that melanoma cells can also be efficiently targeted by activating the extrinsic apoptosis pathway, using the death ligand TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand). However, additional strategies are needed for full enhancement of the extrinsic pathway by TRAIL and to prevent TRAIL resistance. ⁸¹

As proof-of-principle, the Berlin groups developed OAds, transcriptionally targeted to melanoma using a tyrosinase promoter, armed with doxycycline-inducible expression of CD95/Fas ligand. Virus treatment resulted in selective, inducible apoptosis and cell lysis in melanoma cells. ⁸⁴ Because CD95L may be hepatotoxic in vivo, a subsequent study replaced this molecule by TRAIL, which is supposed to selectively induce apoptosis in tumor cells. TRAIL-encoding OAds also mediated strong and selective melanoma cell killing by viral replication and by induction of apoptosis in cell culture. In xenograft mouse models, the virus resulted in significantly reduced tumor size. ³⁴ However, a particular limitation of this approach is inducible TRAIL resistance, which is strongly related to inhibition of mitochondrial apoptosis pathways and dysregulation of Bcl-2 proteins. ⁸¹ These results suggest that the efficiency of death ligand-encoding OAds can be further improved by simultaneous inhibition of antiapoptotic Bcl-2 proteins. To this end, new OAds expressing TRAIL together with (artificial) microRNAs directed against proapoptotic Bcl-2 proteins are currently under development. Furthermore, the viruses will be equipped with additional features concerning their safe application. For instance, microRNA target sites will be inserted to further restrict replication and transgene expression to melanoma cells. For increased efficiency, a telomerase promoter will be used to direct E1A expression and viral replication also to dedifferentiated, tyrosinase-negative melanoma cells. Finally, an Ad5/3 chimeric fiber shall be employed to also infect Ad5-resistant melanoma cells.

Further groups around Martina Breidenbach (University Hospital Düsseldorf) and Christine Spitzweg (Ludwig-Maximilian-University Munich) developed transcriptionally targeted OAds for the treatment of various malignancies. They explored OVs in combination with chemotherapy and arming of OAds with sodium iodide symporter for imaging of infected tumors or combined viro-radiotherapy, respectively. ^35,59

Arenavirus platform

The group of Karl Sebastian Lang at the University Hospital Essen studies arenaviruses (ArVs), which are round, pleiomorphic, and coated, with a diameter of 60 to 300 nm, and that contain two single-stranded RNA segments. Their rapid replication in susceptible tissues causes minor direct tissue destruction. Rather, it is the immune response against the infected cells that can cause severe tissue damage and disease symptoms. ⁸⁵ ArVs can infect humans, and disease outcome depends on the specific strain. ArVs inducing severe human disease include Lassa virus and Junin virus, which are responsible for the African and Argentinian hemorrhagic fever, respectively. ⁸⁵ In contrast, infections with the laboratory ArV strain lymphocytic choriomeningitis virus (LCMV, strain WE) and the strain Candid#1, a vaccine virus to protect against Argentinian hemorrhagic fever, are usually asymptomatic in humans or cause nonspecific symptoms such as fever and malaise. ⁸⁶ Because LCMV-WE induces a strong innate and adaptive immune response, recombinant LCMV is considered as a vaccine virus with wide applicability. ⁸⁷

The Lang group has analyzed whether the ArV-induced inflammatory response contributes to anti-tumor immunity. ⁴² The group demonstrated in murine and human cancer models that the ArVs LCMV and Candid#1 preferentially replicate in tumor cells. Viral replication leads to prolonged local immune activation, resulting in rapid regression of localized and metastatic cancers and long-term tumor control. In the models studied, LCMV-induced anti-tumor immunity was independent of the adaptive immune system, but depended on inflammatory monocytes, which showed the capacity to produce high levels of type I interferon within the tumor. High local IFN-I levels suppressed proangiogenic factors and tumor angiogenesis, engendered hypoxia, and caused apoptosis in established tumors. This clearly shows that independent of direct cytotoxic effects by ArVs, their immune activation capacity can strongly modulate tumor growth.

These data indicate that the most important hallmark for tumor therapy with ArVs is the strong and prolonged replication capacity in tumor cells, which then induces immune activation within the tumor. Ideally, future ArV-derived OVs should be designed to replicate only in tumor cells and a few antigen-presenting cells, as this most likely would induce the strongest immune response within the tumor. The Lang group's major goal for the future is to test whether Candid#1, if given to tumor patients, is indeed therapeutically active and can complement ICIs.

Measles vaccine virus platform

Measles vaccine virus (MV) tumor selectivity is assumed to result from sensitivity toward the interferon system, ⁵² which often is inactive in cancer cells, as well as from the overexpression of the MV entry receptors CD46 or nectin-4. ⁸⁸ Numerous preclinical approaches in Germany have endeavored to generate MV vectors with maximum tumor specificity and therapeutic efficacy. Three of the research groups analyzing oncolytic MV in Germany, namely the Buchholz, Mühlebach, and Ungerechts laboratories, are headed by principal investigators, who started their postdoctoral research activities in—and continue to collaborate with—the group of Roberto Cattaneo at the Department of Molecular Medicine, Mayo Clinic, Rochester, MN, which is spearheading the efforts concerning preclinical and clinical development of oncolytic MV.

The glycoproteins of MV can be genetically engineered to use any cell surface receptor of choice for cell entry, thereby achieving enhanced tumor specificity. ²⁷ Essentially, this is accomplished through ablating natural receptor usage by introducing selected point mutations in the viral H gene and by adding a targeting domain (including ligands, a single-chain variable fragment [scFv], or designed ankyrin repeat proteins [DARPins]) that provides specificity for the selected cell surface protein. A series of different MVs has been generated in Germany by this approach, each targeting a particular tumor-specific surface protein as entry receptor. The group of Guy Ungerechts at the National Center for Tumor Diseases in Heidelberg demonstrated tumor-specific targeting to pancreatic cancer (anti-PSCA [prostate stem cell antigen] ²¹ ), head and neck squamous cell carcinoma (anti-EGFR ²² ), and melanoma cells (in cooperation with the Nettelbeck group; anti-HMWMAA ²³ ). The groups of Christian Buchholz und Michael Mühlebach at the Paul-Ehrlich-Institut in Langen have shown that DARPins can be used as alternative targeting domains for MV hemagglutinin ⁸⁹ and therefore are useful also for oncolytic MV. ²⁴ These synthetically selected protein domains are only approximately half the molecular size of scFvs, exhibit high protein stability, and a lateral binding surface. Therefore, different DARPin units can be linked to generate multispecific targeting domains where the single modules do not block each other. Indeed, DARPin-displaying MVs targeting the tumor markers HER2/neu, EGFR, or EpCAM (epithelial cell adhesion molecule) have been successfully generated, and DARPin-displaying MV targeting HER2/neu revealed higher stability and better anti-tumor efficacy both in vitro and in vivo compared with their scFv-displaying counterpart. ²⁴ Moreover, DARPin domains allowed the generation of bispecific MVs simultaneously targeting HER2/neu or EpCAM ^24,28 that were extraordinarily efficacious in mixed tumor cell cultures in vitro as well as in orthotopic models of ovarian carcinoma. ²⁸ Interestingly, the engineered MV glycoproteins can also target lentiviral vectors to cell types of interest. ²⁷

Among the tumor markers targeted, those expressed on cancer stem cells (CSCs) are particularly attractive. CSCs are thought to differentiate into tumor cells, thereby forming the tumor mass, and to confer resistance against conventional therapies such as radio- and chemotherapy. ⁹⁰ Various transmembrane proteins are under discussion as potential markers for CSCs, including CD133 (prominin-1) and EpCAM. The Langen MV groups revealed that display of an scFv derived from the hybridoma 141.7 renders MV-CD133 highly selective for CD133-positive tumor cells and, interestingly, significantly more active than nontargeted MV in various mouse tumor models. ⁹¹ To become applicable to a clinical situation, in which CSCs are present in tumor tissues as single dispersed cells, the oncolytic activity of MV-CD133 must be further enhanced. The well-established safety of recombinant MVs as a vaccine platform including the insertion of genes from severe pathogens such as, for example, MERS-CoV (Middle East respiratory syndrome coronavirus), provides a wide scope for this undertaking ⁹² by including tumor antigens as immunotherapeutic payload. ^† Accordingly, initial attempts of the Langen MV groups included exchange of the measles vaccine strain P gene for a wild-type variant to more potently counteract innate antiviral responses of infected cells. ²⁵ In a glioblastoma model, a virus with retained CD46 receptor and additional CD133 targeting showed the most promising results. ²⁵ Interestingly, in some modes oncolytic activity was further enhanced by transferring the CD133-targeted envelope to vesicular stomatitis virus (VSV). However, the resulting VSV-CD133 vector induced neurotoxicity when administered intracerebrally. This finding points out further testing of VSV-CD133 for extracerebral applications, for example, in hepatocellular carcinoma. ²⁵ In this context of vector safety, the Heidelberg MV group integrated microRNA technology for increased safety and tumor specificity at the postentry level by introducing target sequences of selected microRNAs into the viral genome. ³⁷ Detargeting multiple normal tissues at risk for off-target toxicity was achieved by the combination of various microRNA target sequences within a single MV. ³⁸ These modifications do not compromise anti-tumor efficacy. Furthermore, in cooperation with the Nettelbeck group, proof-of-principle was reported for aptazyme regulation of MV infection as a safety switch (see Adenovirus platform ⁷² ). Such approaches may be of special importance in high-dose systemic virotherapy applications.

To control for viral replication and to achieve bystander killing, integration of various suicide genes including CD/UPRT (encoding the bifunctional enzyme cytosine deaminase/uracil phosphoribosyltransferase), locally converting the nontoxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil and PNP (encoding the purine nucleoside phosphorylase, locally converting the active metabolite of fludarabine phosphate), into the MV genome was pursued by the Ungerechts group in Heidelberg and the group of Ulrich Lauer at the University Hospital Tübingen. ^{21 –23,52 –56, ‡}

The Tübingen group, in cooperation with the former Neubert group in Martinsried, generated a Schwarz strain-identical MV vector backbone encoding CD/UPRT (MV-SCD). MV-SCD exerts strong oncolytic effect against human hepatocellular carcinoma (HCC) cell lines both in vitro and in vivo, which is accompanied by an apoptosis-like cell death. However, MV-SCD-induced cell death was shown to be independent of intact apoptosis. ⁵⁷ This apoptosis independence of MV-SCD raises hopes for the treatment of patients whose tumor cells harbor defects in this cell death mechanism. In further work, efficient MV replication after therapy-induced senescence (TIS) in various cell types could be demonstrated. ⁶³

The Tübingen MV group, in cooperation with the Langen MV groups, assessed both the safety and pharmacokinetics of MV-SCD after intrahepatic injection in two animal models, IFNAR(tm)-CD46(Ge) transgenic mice and rhesus macaques. ⁵⁸ In these models single direct intrahepatic injections of MV-SCD did not cause any overt symptoms. The transient presence of infectious viral RNA was detected in several organs. However, no shedding of viral particles was detected. Histopathology and blood parameters including liver enzymes were normal. Both mice and macaques raised a humoral immune response shortly after virus administration. These findings provide preclinical safety data for possible future clinical applications of intrahepatic MV-SCD injection in combination with systemic 5-FC prodrug administration.

Regarding clinical translation and personalized therapy, the Tübingen group identified human precision cut liver tumor slices (PCLS) as comprehensive predictive test systems for the oncolytic effectiveness of MV and also other virotherapeutics. ^93,94

The Heidelberg MV group is focusing on the development of oncolytic MV vectors specifically engineered toward targeted immunomodulation. Vector-mediated modulation of the immune system is currently under broad investigation to reinforce efficacy of viral monotherapy, to break immune tolerance and therapy resistance. A potent cytokine encoded by many OVs, including T-VEC and Pexa-Vec, is GM-CSF. Also in the context of MV oncolysis, this proved to be an effective strategy. ⁴⁷ A genuine MV-GM-CSF vector was found to significantly improve survival in a fully immunocompetent mouse model. This was accompanied by T cell infiltration at the tumor invasive margin and a tumor-specific, protective immune response. ⁴⁷ To overcome limitations of immune checkpoint-modulating antibodies, that is, resistance of T cell-neglected tumors and side effects of systemic administration, MV vectors encoding CTLA-4 (cytotoxic T lymphocyte-associated protein 4) and PD-1/PD-L1 blocking antibodies were designed. ⁴⁸ These vectors were shown to be safe and effective in a B16 mouse model and to increase the ratio of intratumoral effector to regulatory T cells. ⁴⁸ A systematic screening for the most effective immunomodulatory MV was performed in the MC38cea model of MV oncolysis. ⁴³ This study identified MV encoding interleukin (IL)-12 as a vector with superior efficacy, achieving 90% complete tumor remissions. Therapeutic activity was associated with a helper T cell type 1 (Th1)-directed immune response and dependent on CD8⁺ T cells. ⁴³ Current developments by the Heidelberg MV group include the generation of MV-TAA and MV-BiTE vector families. MV-TAAs are defined as OVs encoding tumor-associated antigens (TAAs), thereby suitable to prime a TAA-specific immune response and activate TAA-specific T cells, which then direct OV-induced immunity against target antigens of choice. ^§ In contrast, MV-BiTEs encode bispecific T cell engagers that crosslink T cells to tumor cells via two single-chain antibody fragments. ⁹⁵ MV-BiTE treatment recruits T cells to immunologically “cold” tumors and subsequent oncolysis further enhances the anti-tumor T cell response. ^**

To circumvent neutralizing antibodies that could potentially hamper MV therapy, the Ungerechts group has developed Tupaia paramyxovirus, an MV relative, as an alternative virus platform. ^14,15

A general theme in cancer immunotherapy trials comprises combination approaches. Thus, aside from encoding immunomodulators within the MV genome, rational combination strategies for MV are explored. Most prominently, radiotherapy is employed to sensitize the tumor microenvironment for OV therapy. ⁹⁶ The Heidelberg group explores whether radiotherapy induces abscopal effects that synergize with MV. ^††

The Tübingen MV group evaluated a novel combination of epigenetic and virotherapy, consisting of the oral histone deacetylase inhibitor resminostat and MV-SCD for the treatment of pancreatic ductal adenocarcinoma. ^67,68 Notably, epi-virotherapy significantly reduced tumor cell mass, indicating synergistic effects of resminostat and MeV-SCD. The epigenetic modifier resminostat did not impair MV replication or activate antiviral signaling reported to be involved in resistance to OVs. Likewise, MV infection did not alter resminostat pharmacodynamics in pancreatic cancer cells.

Parvovirus platform

With a particle diameter of about 20 nm and a genomic DNA size of about 5 × 10³ nucleotides, protoparvoviruses (PVs) are the smallest OVs presently under clinical investigation. The research program of the groups of Jean Rommelaere at the German Cancer Research Center (DKFZ) in Heidelberg and Antonio Marchini, now heading the Laboratory of Oncolytic Virus Immuno-Therapeutics (LOVIT), a joint research unit of the DKFZ and the Luxembourg Institute of Health (LIH), focuses on the therapeutic applications of rodent PVs, in particular H-1PV, for which the natural host is the rat. The group of Jean Rommelaere pioneered the development of oncolytic PVs, with the first paper published in 1982 ⁹⁷ and the first-in-human clinical virotherapy study initiated in Germany in 2011 at the University Hospital Heidelberg (see below). The groups' interest in PVs is based on the genuine oncoselectivity of PV, that is, the preference of PV lytic infection for cells that have engaged the malignant transformation process. ⁹⁸ Importantly, a large number of human cancer cells have proven to be targets for H-1PV infection under conditions in which corresponding normal cells are resistant. PV oncoselectivity was found to result from the stimulation of postentry step(s) of the viral life cycle in tumor cells. It was traced back, at least in part, to cancer-associated alterations of cellular signaling pathways that lead to both overproduction and posttranslational activation of viral replication and cytotoxic proteins, as illustrated by the demonstration of the phosphorylation-dependent functionalization of the viral protein NS1 through the PKC/PDK1/PKB kinase cascade. NS1 exerts pleiotropic control over viral DNA amplification, gene expression, and cytopathic effects. ⁹⁸ These features are often correlated, although the induction of cell death does not necessarily require a productive virus infection, as in the case of human glioblastoma cells, which are efficiently killed by H-1PV while being poor virus producers. ³⁹ Interestingly, it was possible to restore productivity through the selection of H-1PV fitness mutants that harbor a small distinct deletion within the NS gene. ^39,40 PVs can kill cancer cells through various apoptotic and nonapoptotic processes, depending on the type and physiological state of target cells. This multiplicity of cytocidal activities allows PVs to circumvent resistance mechanisms developed by cancer cells, as exemplified by the capacity of H-1PV to kill apoptosis-refractory human glioblastoma cells through a lysosomal process. ⁹⁸

An increasingly recognized hallmark of oncolytic virotherapy is that it acts as immunotherapy by activating anticancer immune reactions that take over from the initial direct oncolytic effect of the virus to complete tumor destruction. In this context, the mode of action of PVs is congruent with this notion: while exerting a direct effect on some immune cells through their abortive infection, PVs mostly modulate host immune reactions in an indirect way by inducing an immunogenic type of tumor cell death. Various cellular factors were identified in viral oncolysates whose display or release leads to the activation of cells of both the native and adaptive arms of the immune system. This immune contribution to PV anti-tumor potency was demonstrated in (co-)culture systems and various animal cancer models. ⁹⁹ H-1PV has proven to suppress rat tumors, in particular glioblastoma and pancreatic carcinoma, in immunocompetent animals. ¹⁰⁰ Tumor suppression was found to rely at least in part on the induction of a tumor-specific T cell response, as evidenced by adoptive transfer and immunodepletion experiments. ^39,44 When used in high enough doses, the virus also achieved the eradication of a variety of human tumor xenografts (glioblastoma, lymphoma, various carcinomas) in immunocompromised animals. ^51,100 Yet, the efficiency of human pancreatic carcinoma xenograft suppression was enhanced through the reconstitution of these animals with viral oncolysate-primed autologous dendritic cells (DCs) and T cells, showing the virus–immune cell cooperation in achieving tumor cell killing. More recently, the dual (direct and immune-mediated) activity of H-1PV was further documented by analyses of tumors from patients with resected glioblastoma, that had been subjected to intratumoral or intravenous H-1PV administration. ¹⁰¹

However, the failure of H-1PV as monotherapy to achieve tumor eradication in patients with glioblastoma shows the need for optimization. This has prompted the Heidelberg group to set up an R&D program aiming to design and evaluate a second generation of replication-competent oncolytic PVs and combination strategies. Proof-of-concept studies have demonstrated that the oncolytic and immunostimulating activities of H-1PV can both be enhanced by arming the virus with specific shRNA expression cassettes and CpG motifs, respectively. ^46,51 While insertion of these short regulatory elements does not interfere with efficient virus multiplication, the limited packaging capacity of oncolytic PVs precludes the production of replication-competent recombinants harboring transgenes larger than a few hundred nucleotides. To overcome this limitation, an engineered version of the H-1PV genome has been cloned into a replication-defective adenoviral (Ad) vector. Ad particles containing the chimeric Ad/PV genome can be produced and serve as a shuttle to deliver the PV genome into the nucleus of cancer cells, where fully infectious oncolytic PV particles are then generated. ²⁹ These Ad/PV chimeras represent multipurpose tools for both cancer gene therapy and oncolytic virotherapy. Indeed, it is possible to insert therapeutic transgenes into the Ad backbone in order to complement the oncosuppressive activity of the PV component. The possibility of producing chimeric Ad/PVs at high titers under GMP conditions should accelerate the efforts for clinical translation of this approach.

Moving these innovative anticancer agents from the bench into the clinic is one of the major goals of the binational research unit LOVIT at DKFZ and LIH, headed by Antonio Marchini. Another area of active investigation concerns the identification of drugs that are able to synergize with H-1PV in killing cancer cells. Besides H-1PV compatibility with first-line radio- and chemotherapy,^64z,66 strong synergistic effects were demonstrated by combining H-1PV with sublethal doses of epigenetic modulators such as the histone deacetylase (HDAC) inhibitor valproic acid (VPA). ^69,70 VPA acts at least in part by increasing the level of acetylation of the PV replicative and oncotoxic protein NS1. Further cis- and trans-combinations of H-1PV with anticancer compounds (e.g., ICIs) represent a priority of the PV research program. ⁷⁰ Further strategies pursued by the Rommelaere and Marchini groups to improve PV-based virotherapy are listed in Table 1.

Vaccinia virus platform

Advances in DNA recombinant technology enabling purposeful manipulation of the viral backbone, coupled with the ever-increasing knowledge gains in the fields of molecular virology and cancer cell biology, have aided the development of safe and efficacious tumor-targeted oncolytic vaccinia viruses (VACVs). ^102,103 These VACV vector families are currently at the forefront of the most promising novel anticancer agents.

The group of Ulrich M. Lauer at University Hospital Tübingen focused on the preclinical and clinical development of Lister strain-derived oncolytic VACV GLV-1h68, which was constructed previously by inserting three expression cassettes encoding β-glucuronidase, β-galactosidase, and green fluorescent protein (GFP) at different gene loci of the virus. ¹⁰⁴ As a result, GLV-1h68 was found to be further attenuated when compared with wild-type VACV. In addition, expression of diagnostic markers also enabled close monitoring of virus infection, spread, and oncolysis, thus providing real-time insight into the success of tumor treatment, both in preclinical as well as in clinical studies (see also the section “Clinical Virotherapy Trial Activities in Germany,” below).

In detail, the Lauer VACV group contributed to the analysis of GLV-1h68 in HCC. ¹⁰⁵ GLV-1h68 efficiently colonized, replicated in, and lysed HCC cells in culture. In HCC xenografts, a single intravenous administration of GLV-1h68 led to a significant delay in tumor progression compared with untreated controls. Furthermore, GLC-1h68-mediated oncolysis induced inflammation and cytokine release along with dense infiltration of antigen-presenting cells in tumors. These data indicate that GLV-1h68 may be an effective virotherapeutic against HCC in a clinical setting.

In a further study, the Lauer VACV group contributed to proof-of-concept that GLV-1h68 can be employed to detect and reduce circulating metastatic tumor cells (CMTCs). CMTCs are important targets for cancer therapy and also a surrogate marker for patients' prognosis and treatment response. ⁶⁰ Infections with GLV-1h68 detected live CMTCs in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer. In xenograft models, early GLV-1h68 treatment prevented the formation of CMTCs and in mice with advanced tumors, GLV-1h68 reduced the number of CMTCs. Importantly, in a patient with advanced gastric cancer, a single intraperitoneal administration of GL-ONC1 (i.e., GMP-grade GLV-1h68) led to a significant reduction of tumor cells in ascites. ^‡‡ These results identify GLV-1h68 and potentially also other VACV vectors as highly instrumental theranostic tools for both quantitative detection and therapeutic reduction or even elimination of CMTCs.

A combination of virotherapy with established chemotherapy regimens (so-called “chemovirotherapy”) is an attractive modality to enhance anti-tumor efficacy. In this regard, the Lauer VACV group has investigated the addition of GLV-1h68 to a dual chemotherapy (nab-paclitaxel plus gemcitabine) in human pancreatic adenocarcinoma cell lines. ⁶⁵ Chemovirotherapy was superior to monotherapies provided that sustained productive viral replication was maintained during chemotherapy. This study identified important determinants of efficacy that must be considered when designing chemovirotherapy protocols.

Early monocenter trials in Germany

Multicenter clinical trials performed in Germany

German institutions are participating in several multicenter trials of T-VEC (talimogene laherparepvec; Imlygic), granted marketing approval for intratumoral therapy of nonresectable metastatic melanoma. ¹⁰ Because T-VEC appears to substantially enhance clinical responses to ICI therapy, three T-VEC multicenter trials performed in Germany are in combination with ICI, such as ipilimumab (NCT01740297) and pembrolizumab (NCT02263508), both focusing on advanced melanomas, as well as T-VEC in combination with pembrolizumab (NCT02626000), addressing advanced head and neck tumors. The two other T-VEC trials with participation from Germany, not employing ICIs, focus on the correlation between the overall response rate (ORR) and baseline intramelanoma CD8⁺ cell densities (NCT02366195), or constitute participation of a single German study center in an expanded access protocol of T-VEC for the treatment of European subjects with melanoma (NCT02297529). In total, T-VEC is being tested at 19 German study sites (Fig. 1).

Two other multicenter trials with participation of German institutions test Pexa-Vec (pexastimogene devacirepvec), a VACV-based oncolytic immunotherapy designed to preferentially replicate in and destroy tumor cells while stimulating anti-tumor immunity by expressing human granulocyte-macrophage colony-stimulating factor (hGM-CSF). ¹¹¹ In total, Pexa-Vec is tested at 10 German study sites (Fig. 1). Herein, the international multicenter randomized TRAVERSE phase IIb study (NCT01387555), in which patients with HCC who failed first-line treatment with sorafenib either received intratumoral applications of Pexa-Vec plus best supportive care (BSC) versus BSC alone, could not show a statistically significant survival benefit for virus-treated patients, but again established clinically meaningful immunological responses in some patients. As the study results are awaiting final publication, the PHOCUS phase III trial is ongoing (NCT02562755), comparing intratumoral applications of Pexa-Vec followed by oral application of sorafenib versus sorafenib alone in the first-line treatment of advanced HCC. As has been the experience with other immunotherapies, results suggest that less advanced patients in better physical condition may be more likely to benefit from oncolytic immunotherapy. Accordingly, current development of Pexa-Vec has been placed purposefully as a first-line therapy for HCC.