*1.2. Oncolytic Viruses*

One rejuvenated area of research for targeted therapy is oncolytic viruses (OVs). These cancer-killing viruses exploit the natural ability of a virus to infect, replicate and lyse cells. An additional benefit of OVs is that the lytic nature of cell killing induces immunogenic cell death (ICD), increasing recruitment of immune cells to the tumour site. This ability of OVs to enhance host anti-tumour immune responses through ICD has the potential to turn immunologically "cold" tumours "hot", thus sensitising otherwise refractory tumours to subsequent immunotherapies. This exciting potential of OVs to synergise with immunotherapies is the subject of significant ongoing clinical investigation, with compelling preliminary data obtained in several early phase clinical trials in brain tumours [8] and breast cancer [9], with other trials continuing in sarcoma, melanoma and breast cancer [10–12]. Efficacy may be further enhanced, with correspondingly reduced dose-limiting toxicities, through the development of rationally and effectively tumour targeted OVs suitable for intravenous applications.

The overarching aim in the development of OVs is to engineer viruses that can selectively target and/or replicate within cancer cells, leaving normal cells and tissues uninfected. The pro-immunogenic environment induced by OVs through the release of tumour antigens during cell lysis can be further enhanced by engineering the viral genome to over-express therapeutic transgenes, thus adding an extra layer to their therapeutic power. These multiple levels of activity are overviewed in Figure 1.

To limit "off target" uptake of OVs and subsequent killing of healthy cells, genetic engineering approaches have been undertaken. These have broadly focused on two approaches—firstly the active targeting of virions to tumour cells through the manipulation of viral capsid proteins to enhance uptake of virus into tumour cells and limit uptake via healthy cells. This has been largely achieved through the rational engineering of the viral genome and thus capsid to engineer tumour tropism via tumour associated antigens and receptors [13–15]. A second approach is to engineer in selectivity "post entry", such that viral replication is blocked in non-transformed cells, but subtle modifications within the viral early genes permit replication to proceed within malignantly transformed cells. Using combinations of these approaches, it is possible to achieve tightly controlled tumour cell killing, and thus creating a more e *Cancers*  ffective cancer treatment. **2020**, *12*, x 3 of 24

**Figure 1.** The mode of action of an oncolytic virus. Oncolytic viruses leave healthy cells undamaged, whilst leading to a range of effects in tumour cells which lead to lysis, further infection and an immunological response. Created with BioRender.com. **Figure 1.** The mode of action of an oncolytic virus. Oncolytic viruses leave healthy cells undamaged, whilst leading to a range of effects in tumour cells which lead to lysis, further infection and an immunological response. Created with https://biorender.com.

To limit "off target" uptake of OVs and subsequent killing of healthy cells, genetic engineering approaches have been undertaken. These have broadly focused on two approaches—firstly the active targeting of virions to tumour cells through the manipulation of viral capsid proteins to enhance uptake of virus into tumour cells and limit uptake via healthy cells. This has been largely achieved through the rational engineering of the viral genome and thus capsid to engineer tumour tropism via tumour associated antigens and receptors [13–15]. A second approach is to engineer in selectivity "post entry", such that viral replication is blocked in non-transformed cells, but subtle modifications within the viral early genes permit replication to proceed within malignantly transformed cells. Using combinations of these approaches, it is possible to achieve tightly controlled tumour cell killing, and thus creating a more effective cancer treatment. The first OV licensed in the western world by both the FDA and EMA is talimogene laherparepvec (T-Vec, Imlygic™), which exemplifies the early therapeutic potential of oncolytic viruses. T-Vec is a herpes simplex virus 1, with several genetic modifications. Deletions of the ICP34.5 gene reduces neurovirulence whilst also suppressing its ability to reactivate, which prevents it producing cold sores in the patient. A deletion in the ICP47 gene leads to enhanced antigen loading on MHC class I molecules. The MHC loading results in infected cells presenting tumour antigens, leading to enhanced immune recognition. These deletions improve the safety of this treatment as it limits the ability of the virus to completely evade immune responses. In addition, T-Vec is engineered to overexpress the cytokine GM-CSF (granulocyte-macrophage colony-stimulating factor) to further enhance immunogenicity through T-cell priming [16]. A truncated version of the US11 gene is also The first OV licensed in the western world by both the FDA and EMA is talimogene laherparepvec (T-Vec, Imlygic™), which exemplifies the early therapeutic potential of oncolytic viruses. T-Vec is a herpes simplex virus 1, with several genetic modifications. Deletions of the ICP34.5 gene reduces neurovirulence whilst also suppressing its ability to reactivate, which prevents it producing cold sores in the patient. A deletion in the ICP47 gene leads to enhanced antigen loading on MHC class I molecules. The MHC loading results in infected cells presenting tumour antigens, leading to enhanced immune recognition. These deletions improve the safety of this treatment as it limits the ability of the virus to completely evade immune responses. In addition, T-Vec is engineered to overexpress the cytokine GM-CSF (granulocyte-macrophage colony-stimulating factor) to further enhance immunogenicity through T-cell priming [16]. A truncated version of the US11 gene is also included to enhance the lytic activity of the virus by partial de-attenuation [17]. The virus takes advantage of disrupted anti-viral pathways in cancer cells to enable selective virus replication. One such pathway is the PKR (protein kinase R) pathway which is key in regulating cell proliferation. Normally, this pathway is activated by the dsRNA produced when the virus replicates, thus triggering protein synthesis inhibition. However, in cancer cells with aberrant regulation, the dsRNA production warning sign is ignored, allowing the virus to replicate, leading to viral propagation and cell lysis. Disruption in the PKR pathway has been found in approximately 70% of melanoma cells, and thus T-Vec is a good replication selective therapy for melanoma cancers [18,19].

included to enhance the lytic activity of the virus by partial de-attenuation [17]. The virus takes advantage of disrupted anti-viral pathways in cancer cells to enable selective virus replication. One such pathway is the PKR (protein kinase R) pathway which is key in regulating cell proliferation. Normally, this pathway is activated by the dsRNA produced when the virus replicates, thus triggering protein synthesis inhibition. However, in cancer cells with aberrant regulation, the dsRNA production warning sign is ignored, allowing the virus to replicate, leading to viral propagation and cell lysis. Disruption in the PKR pathway has been found in approximately 70% of melanoma cells, T-Vec was licensed for localised treatment of recurrent melanoma [20,21]. In phase III trials using T-Vec to treat unresectable late stage melanoma, durable response rates of 16% vs. 2% were shown, compared to control group, increasing the survival rates of these patients [22,23]. Moreover, recent studies have demonstrated that virotherapies such as T-Vec can be a powerful tool in adjuvant therapy and have been used to sensitise triple negative breast cancers to follow up treatment with immunotherapies [9,24].

#### and thus T-Vec is a good replication selective therapy for melanoma cancers [18,19]. T-Vec was licensed forlocalised treatment of recurrent melanoma [20,21]. In phase III trials using *1.3. Adenovirus as an Oncolytic Virotherapy*

T-Vec to treat unresectable late stage melanoma, durable response rates of 16% vs. 2% were shown, compared to control group, increasing the survival rates of these patients [22,23]. Moreover, recent studies have demonstrated that virotherapies such as T-Vec can be a powerful tool in adjuvant therapy and have been used to sensitise triple negative breast cancers to follow up treatment with immunotherapies [9,24]. Despite the success of T-Vec, the therapy is only licensed for local intra-tumoural applications. Therefore, its use for treatment of metastases, which kills the majority of cancer patients, is limited. Metastasis treatment would require blood system disseminated therapy to individually target each lesion through intravenous (IV) delivery, or utilising the abscopal effect from treatment of one or more

lesions, which could lead to a systemic anti-tumour immune response extending to the milieu of micro metastatic deposits in the body [25–27]. Therefore, significant research is ongoing into other viral vectors that may be better suited to IV delivery, including adenoviruses (Ads) [28], reovirus [29] and vaccinia virus [30]. Ads have been shown to be the most durable of these options and are the most studied clinically and experimentally. Ads also have the advantage of being naturally lytic, immunogenic and can be produced to high titres and purity, all important considerations when developing such therapeutics for widespread clinical application. Therefore, its use for treatment of metastases, which kills the majority of cancer patients, is limited. Metastasis treatment would require blood system disseminated therapy to individually target each lesion through intravenous (IV) delivery, or utilising the abscopal effect from treatment of one or more lesions, which could lead to a systemic anti-tumour immune response extending to the milieu of micro metastatic deposits in the body [25–27]. Therefore, significant research is ongoing into other viral vectors that may be better suited to IV delivery, including adenoviruses (Ads) [28], reovirus [29] and vaccinia virus [30]. Ads have been shown to be the most durable of these options and are the most studied clinically and experimentally. Ads also have the advantage of being naturally lytic,

immunogenic and can be produced to high titres and purity, all important considerations when

*Cancers* **2020**, *12*, x 4 of 24

Despite the success of T-Vec, the therapy is only licensed for local intra-tumoural applications.

#### 1.3.1. Adenovirus Cell Entry and Trafficking developing such therapeutics for widespread clinical application.

Human adenoviruses can be classified into seven species (termed A–G) comprised of over 100 serotypes [31]. Ads commonly infect the respiratory system however different serotypes can also infect the gastrointestinal, cardiac, neurological, ophthalmological and genitourinary tissues which can result in an array of clinical pathologies [32]. Ads bind to receptors on the membrane surface. Ad5 has been well described as recognising the coxsackievirus and adenovirus receptor (hCAR) [33]. Other receptors involved in adenoviral attachment are CD46 [34], desmoglein 2 (DSG2) [35] and sialic acid [36]. Primary receptor binding is dependent on the Ad serotype but generally species A, C, E and F interact with hCAR while species B and D utilise other receptors [37]. Species B is reported as using primarily CD46 and DSG2, and, although the individual receptors for many species D serotypes are unknown, it is thought there is sialic acid involvement [38,39]. Initial receptor attachment is followed by internalisation mediated by αvβ3/5 integrin binding through a conserved Arg-Gly-Asp (RGD) motif [40]. Upon cell entry, the adenovirus is partially disassembled via endosome acidification and the uncoated is released and transports viral DNA into the nucleus [41]. Viral replication takes place in two phases: early phase and late phase. An overview of the adenoviral replication cycle is illustrated in Figure 2. 1.3.1. Adenovirus Cell Entry and Trafficking Human adenoviruses can be classified into seven species (termed A–G) comprised of over 100 serotypes [31]. Ads commonly infect the respiratory system however different serotypes can also infect the gastrointestinal, cardiac, neurological, ophthalmological and genitourinary tissues which can result in an array of clinical pathologies [32]. Ads bind to receptors on the membrane surface. Ad5 has been well described as recognising the coxsackievirus and adenovirus receptor (hCAR) [33]. Other receptors involved in adenoviral attachment are CD46 [34], desmoglein 2 (DSG2) [35] and sialic acid [36]. Primary receptor binding is dependent on the Ad serotype but generally species A, C, E and F interact with hCAR while species B and D utilise other receptors [37]. Species B is reported as using primarily CD46 and DSG2, and, although the individual receptors for many species D serotypes are unknown, it is thought there is sialic acid involvement [38,39]. Initial receptor attachment is followed by internalisation mediated by αvβ3/5 integrin binding through a conserved Arg-Gly-Asp (RGD) motif [40]. Upon cell entry, the adenovirus is partially disassembled via endosome acidification and the uncoated is released and transports viral DNA into the nucleus [41]. Viral replication takes place in two phases: early phase and late phase. An overview of the adenoviral replication cycle is illustrated in Figure 2.

**Figure 2.** Adenovirus replication cycle. (**1**) Virus attachment to receptors on the host cell surface. (**2**) Internalisation of the virus by endocytosis. (**3**) Low pH results in endosomal acidification and partial disassembly of the virion. (**4**) Virion released from endosome and trafficked to the nuclear pore complex where it releases viral DNA into the nucleus. (**5**) Early phase: Transcription and subsequent translation of early genes to the regulatory early proteins. (**6**) Late phase: Transcription and subsequent translation of late genes to the late structural proteins. (**7**) Assembly of progeny virion. (**8**) Cell lysis resulting in release of mature virus. Created with https://biorender.com. Figure adapted from [41–44].
