**3. Current Clinical Applications of Oncolytic Adenoviral Therapies**

ONYX-015 (also referred to as ∆1520) was one of the first replication selective oncolytic adenoviruses tested in a clinical setting for the treatment of head and neck cancer [97,98]. ONYX-015 harbours an E1B55K deletion, which was originally thought to be essential for viral replication as it sequesters p53 and promotes cell cycle transition [99]. p53 is commonly lost or downregulated through mutations in multiple cancer cells [100], and it is therefore considered that ONYX-015 would replicate almost exclusively in cancer cells lacking p53. However, more recent

research suggests that this mutation is more likely to work through the loss of late viral RNA export, rather than through p53 status alone. The mechanism of action may be more complex than originally thought, likely due to multi-modal action of the p53 pathway [49,101,102]. Whilst ONYX-015 and a variant H101 (E1B55K and E3 deletion) demonstrated the safety of oncolytic adenoviruses, the efficacy was limited by attenuated viral replication and spread [96,103]. Subsequent generations of tumour selective oncolytic adenoviruses contain mutations in the E1A gene that functions through binding the retinoblastoma protein (pRb) [91]. Several oncolytic adeno-virotherapies have since entered clinical trials (Table 1) and have demonstrated safety and feasibility. However, delivery and efficacy must be improved if oncolytic adenovirus is to be used as an effective cancer therapy, especially as a systemically administered agent capable of effectively targeting tumours and metastases [59,60].

The vast majority of adenovirus research to date has focused on the species C, Ad5. Several oncolytic adenoviruses (OAds) have demonstrated limited efficacy in clinical trials as a result of poor viral persistence [104,105]. Although this is, in part, related to the early design of these viruses, it may also result from high levels of pre-existing immunity [106]. A substantial proportion of the population will have experienced an acute adenovirus infection, and many will have developed neutralising antibodies against the most common Ad serotypes [32]. Activation of anti-tumour immunity whilst dampening the innate host anti-viral immune response is essential to the success of OAds. An alternative approach, therefore, may be through the development of alternative, low seroprevalence adenoviral species, such as those from Species B or D, which tend to have naturally low levels of pre-existing immunity in the population [107]. Such serotypes may also exhibit naturally lower levels of off target uptake due to reduced interactions with components of the blood. Ad5 is known to bind to FX in serum which mediates sequestration by the liver and can impact virus delivery to the tumour [108]. FX binds the hexon protein on Ad5 capsid and can result in off target uptake [109]. It was observed that alternative species, for example species D Ad26, does not bind FX in the same manner as Ad5 [109]. This knowledge was used to identify key residues in FX binding through sequence alignment and has fed into the production of retargeted Ad vectors however the use of alternative species as an alternative to Ad5 has not been fully explored [71]. The use of novel oncolytics developed from rarely isolated serotypes from species D may represent an exciting and alluring possibility, where the diverse nature of this species represents a significant and largely untapped repository for investigation. Recent significant progress has been made in this regard by the Ehrhardt laboratory, who have investigated a larger spectrum of adenoviral vectors and begun to evaluate their potential for oncology applications [110,111].

Alternative adenoviral species offer many advantages over the commonly used Ad5-based therapies however, despite their increasing popularity as platforms for vaccine applications, they are poorly represented in the clinical oncology setting. Ad5/kn3 pseudotype has demonstrated some limited efficacy in ovarian cancer [112,113]. This targets via species B receptors which are not cancer specific and maintain the previously outlined issues associated with Ad5-based vectors.




Species B adenovirus have demonstrated the potential to play an important role in the field of adenoviral oncolytics. An alternative strategy to the rational development of novel Ad serotypes for oncology applications has been to develop a novel chimeric OV through a process of natural selection for recombinants with enhanced cell killing activity in cancer cells such as with enadenotucirev (EnAd, formally known as ColoAd1; PsiOxus Therapeutics Ltd., Abingdon, UK). Several adenovirus serotype recombinants were selected for on colon cancer cells (HT29) by this method of "directed evolution" (Figure 5) [114]. Despite the initial pool representing diverse serotypes from species B–F, the resultant chimera is a fully species B recombinant, derived from Ad11p and Ad3 [114]. This virus demonstrated potency and selectivity greater than ONYX-015 and Ad5 [115]. The tumour selectivity mechanism for EnAd is not fully understood however initial clinical trials have demonstrated durability and tolerability, not only for colorectal cancers but with other solid cancers [115,116]. EnAd is thought to act through a non-apoptotic mechanism termed ischemic cell death and possesses pro-inflammatory properties [117]. Species B therefore represents an exciting avenue for development of oncolytics, however the lack of intrinsic tumour specificity and high prevalence of species B receptors, CD46 and DSG-2, on healthy cells is an important consideration that may result in the depletion of virotherapy available for active targeting of tumours through off-target sequestration. Ablation of these native interactions or use of Ads that bind receptors with weak affinity may result in improved novel retargeted oncolytics. Species B adenovirus have demonstrated the potential to play an important role in the field of adenoviral oncolytics. An alternative strategy to the rational development of novel Ad serotypes for oncology applications has been to develop a novel chimeric OV through a process of natural selection for recombinants with enhanced cell killing activity in cancer cells such as with enadenotucirev (EnAd, formally known as ColoAd1; PsiOxus Therapeutics Ltd., Abingdon, UK). Several adenovirus serotype recombinants were selected for on colon cancer cells (HT29) by this method of "directed evolution" (Figure 5) [114]. Despite the initial pool representing diverse serotypes from species B–F, the resultant chimera is a fully species B recombinant, derived from Ad11p and Ad3 [114]. This virus demonstrated potency and selectivity greater than ONYX-015 and Ad5 [115]. The tumour selectivity mechanism for EnAd is not fully understood however initial clinical trials have demonstrated durability and tolerability, not only for colorectal cancers but with other solid cancers [115,116]. EnAd is thought to act through a non-apoptotic mechanism termed ischemic cell death and possesses proinflammatory properties [117]. Species B therefore represents an exciting avenue for development of oncolytics, however the lack of intrinsic tumour specificity and high prevalence of species B receptors, CD46 and DSG-2, on healthy cells is an important consideration that may result in the depletion of virotherapy available for active targeting of tumours through off-target sequestration. Ablation of these native interactions or use of Ads that bind receptors with weak affinity may result in improved novel retargeted oncolytics.

*Cancers* **2020**, *12*, x 10 of 24

**Figure 5.** Method of production for oncolytic virus, EnAd (PsiOxus Therapeutics Ltd., Abingdon, UK). Serial passages of an Adenoviral library in tumour cells lead to chimeric Ads development. Selection with healthy cells and blood factors removes those that bind off-target receptors, lead to Ad11p/Ad3 chimeric EnAd being selected for. Created with BioRender.com. **Figure 5.** Method of production for oncolytic virus, EnAd (PsiOxus Therapeutics Ltd., Abingdon, UK). Serial passages of an Adenoviral library in tumour cells lead to chimeric Ads development. Selection with healthy cells and blood factors removes those that bind off-target receptors, lead to Ad11p/Ad3 chimeric EnAd being selected for. Created with https://biorender.com.
