*5.1. OVs with Immune Checkpoint Inhibitors*

The idea behind this combinatorial approach is that these two therapeutic tools can improve each other by addressing one another's shortcomings. Oncolytic viruses present some limitations related mainly to antiviral immunity, which makes it challenging to exploit the bloodstream to reach distant metastatic sites [91]. Therefore, triggering a tumor-specific adaptive immune response is a fundamental feature as OVs cannot travel inside the body to reach other sites, while T cells that have been sensitized to tumor cells surely can, thus assuring an antitumor response even in different sites to that of the primary tumor [91]. From this perspective, ICIs help guarantee the correct activation of the immune system, targeting specific molecules expressed either on the tumor or on immune cells (CTLA-4, PD-1, PD-L1), while viral infections obtained using OVs makes the TME more immunogenic, creating a microenvironment in which ICIs are known to work much better [92].

This combinatorial approach has been explored in numerous clinical trials [91], among which was a phase II clinical trial with 198 stage IIIB–IV melanoma patients, which was organized to evaluate: (1) ipilimumab as a monotherapy and (2) ipilimumab combined with T-VEC [92]. The results showed that the objective response rate of the combination therapy was 39%, while ipilimumab alone had an objective response rate of 18% [91,92]. T-VEC has also been investigated in combination with other ICIs such as pembrolizumab, an anti-PD-1 antibody. In the phase IB portion of the clinical trial

Masterkey-265, T-VEC was administered to 21 patients with stage IIB and IV melanoma in combination with intravenous pembrolizumab. Among the criteria that have been evaluated, the safety profile of the combination was favorable, with no dose-limiting toxicities, and the objective response rate was 62%, while 33% of patients showed a complete response [92].

Multiple adenoviruses are undergoing clinical and preclinical testing in combination with ICIs, both in melanoma and other types of tumor.

The Hemminki group exploited a murine model of melanoma to establish the mechanism under the combination of the anti-PD-1 antibody with the oncolytic viruses encoding for TNFα and IL-2 [93]. What emerged from the combination therapy was a marked increase in intratumoral CD8+ T cells and a statistically significant tumor growth suppression, along with increased survival in animals. Researchers reported complete tumor regression after the course of the combinatory therapy. This preclinical research provides the rationale for a clinical trial where oncolytic adenovirus coding for TNFa and IL-2 (TILT-123) is used in melanoma patients receiving an anti-PD-1 antibody NCT04217473) [92,93].

Thomas et al. reported development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1. Researchers developed various oncolytic vectors expressing e.g., GMCSF, an anti-CTLA-4 antibody-like molecule. Anti-cancer assessment was performed in vivo and in nude mouse xenograft models (melanoma, lymphoma, gliosarcoma). The combination therapy with the virus expressing GALV-GP-R- and mGM-CSF and an anti-murine PD1 antibody showed improved anti-tumor effects compared to the control. The treatment of mice with derivatives of this virus coding for anti-mCTLA-4, mCD40L, m4-1BBL, or mOX40L showed enhanced anti-cancer efficacy in un-injected tumors (abscopal effect) [94].

Also, in our previous study we have investigated the anti-cancer potency of ONCOS-102 and pembrolizumab in the humanized melanoma mouse model. Humanized mice engrafted with A2058 melanoma cells showed significant tumor volume reduction after ONCOS-102 treatment. The combination of anti-PD1 with the virus further reduced tumor volume, while pembrolizumab alone did not show therapeutic benefit by itself [45]. Systemic abscopal was also observed when combining oncolytic adenovirus and checkpoint inhibitor in a humanized NOG mouse model of melanoma [44]. These data support the scientific rationale for the ongoing clinical study of combination therapy of ONCOS-102 and pembrolizumab for the treatment of melanoma (NCT03003676).

Currently, there are many oncolytic vectors are under development and investigation in melanoma: coxsackieviruses, HF-10, adenoviruses, reoviruses, echoviruses, and Newcastle disease viruses. Therefore, it is probable that oncolytic vectors will have long-term application in the treatment of advanced melanoma not only as a monotherapy but as a part of combinatory therapies. [95].

T-VEC is the first oncolytic vector approved for the melanoma treatment. Reported data have shown improved therapeutic responses to T-VEC in combination with immune checkpoint blockade in patients with melanoma without additive toxicity [96]. T-VEC combined with anti-PD-1 based immunotherapy for unresectable stage III-IV melanoma showed an overall response rate for on-target lesions of 90%, with 6 patients resulting in a complete response in injected lesions (NCT02263508) [97]. Also, the treatment with T-VEC in patients with advanced melanoma with disease progression following multiple previous systemic therapies (vemurafenib, metformin, ipilimumab, dabrafenib, trametinib, and pembrolizumab) showed signs of anti-cancer effect, and provides potential clinical and immunotherapeutic utility of T-VEC application [98].

CAVATAK, an oncolytic immunotherapy, is an oncolytic strain of Coxsackievirus A21 (CVA21). The virus infects ICAM-1 expressing tumor cells, resulting in cell lysis, and anti-tumor immune response. The Phase II CALM study investigated the efficacy and safety of CVA21 in patients with advanced melanoma (NCT01227551). The treatment with CAVATAK resulted in elevation of the immune CD8+ T cell infiltrates within the tumor (5 of 6 patients), and increased expression of PD-L1+ cells. It was also reported that the virus was able to reconstitute immune cell infiltrates in lesions resistant to immune-checkpoint blockade [99]. The combinatory therapy trials have been conducted where

CAVATAK was administered with ipilimumab (NCT02307149) or pembrolizumab (NCT02565992). The treatment with CAVATAK and anti-CTLA-4 has shown durable response with minimal toxicity. The preliminary ORR rate for the ITT population of 50.0% is higher than published rates for either agent used alone (CAVATAK: ~28% and ipilimumab: ~15–20%) in advanced melanoma patients [100]. Among the evaluable patients (intratumoral CAVATAK and systemic pembrolizumab in advanced melanoma patients), the ORR was 73% (8/11). The DCR (CR + PR + SD) was 91% (10/11). In patients with stage IVM1c disease, the ORR and the DCR is 100% (5/5). Combination therapy of the virus1 and anti-PD1 may present a new strategy for the treatment of patients with injectable advanced melanoma (CAPRA clinical trial) [101].

Another oncolytic adenovirus that has been investigated in combination with pembrolizumab is ONCOS-102 (AdV5/3-∆24-GM-CSF), which is now under clinical trial (NCT03003676) to investigate its safety and efficacy, supported by preclinical data showing increased CD8+ T cell infiltration in tumor mass upon viral administration [92]. The therapeutics efficacy and safety of the virus was previously tested in C1 study (NCT01598129). The treatment with the virus was safe and well tolerated at the tested doses. Therapy resulted in infiltration of CD8+ T cells to tumors and up-regulation of PD-L1, highlighting the potential of ONCOS-102 as an immunosensitizing agent for combinatory therapies with checkpoint inhibitors [102]. Therefore, providing a scientific rationale for the combinatory therapy with CPIs.

To date, approximately one third of all clinical trials concerning OVs have investigated a combinatorial approach with at least one ICI [91]. Therefore, it is expected that oncolytic viruses have the capability to promote a 'hotter' immune microenvironment which can improve the efficacy of ICI [103,104]. Oncolytic viruses can be thought of as matches—they can light up a fire inside the tumor and this fire will make the TME "hot" enough for ICIs to strike a blow. Many clinical and preclinical models of melanoma and other solid tumors have provided strong evidence that the infection of tumor cells with OVs can result in the creation of a pro-inflammatory tumor microenvironment, which in turn translates into a new influx of T cells that can be protected from inactivation by ICIs [104,105]. Furthermore, some adenoviruses administered in combination with ICIs have been reported to boost release the pool of tumor neoantigens which can be recognized by CD8+ T cells [106]; this is a particularly important finding, because OVs (both as monotherapy and in combination) have most difficulty affecting low mutational burden cancers, which typically have a very small number of TAAs [91].

Nowadays, one of the major challenges for researchers investigating this field is to assess not only which combinations are most effective, but the dosing regimens and schedules to adopt to maximize the synergy and minimize the side effects [91,92]. This is why further clinical trials results are so impatiently awaited. ICIs have contributed to revolutionize cancer treatment. Nevertheless, the best response rates to these agents do not exceed 35% to 40% [107]. Therefore, the goal of combining OVs with ICIs is to enhance clinical efficacy. Oncolytic vectors are used in order attract the immune cells into the lesion, prime anti-tumor immune responses by development of innate and adoptive anticancer immunity. In turn, CPI therapy will prevent inhibition of activates cancer specific T cells. It is expected that those two agents can result in synergistic or additive anti-cancer effect. Interestingly, it has been demonstrated that local OV injection can modulate tumor-specific CD8<sup>+</sup> T-cell responses rendering distant tumors susceptible to immune checkpoint inhibitor therapy [108]. Therefore, due to the preclinical success of this combination therapy, there is huge interests in clinical trials: results obtained from patients who have progressed after immune checkpoint inhibition (e.g., NCT 03003676) could shed the light on OV's role in overcoming resistance to immunotherapy. By elucidating the potential of the combination of OVs and checkpoint inhibitors, further development in treatment regimens employing these novel therapeutic agents could be beneficial for patients.

Apart from combinatorial strategies, another aspect concerning the use of ICIs is often investigated to reach some improvement—the response predictions with biomarkers. There are several biomarkers associated with the response of ICIs, some of which have been approved and are currently being exploited to predict the response rate in patients before treatment begins, while others are under further study to establish whether they have a strong correlation with the extent of patients' responses to ICIs. The most important predictive biomarker for anti-PD-1/PD-L1 antibodies is PD-L1 expression [41,82], which is evaluated by immunohistochemistry. PD-L1 expression by cancer cells is recognized as both a prognostic and predictive biomarker in patients with cutaneous melanoma. Approx. 35% of cutaneous melanomas express PD-L1, The PD-L1 immunohistochemistry (IHC) has been approved by FDA as a complement diagnostic to select patients with non-small-cell lung carcinoma (NSCLC) suitable for pembrolizumab therapy. Nevertheless, absence of PD-L1 does not necessarily translates into a poor response to anti-PD-1/PD-L1 inhibitors. Some patients with low PD-L1 expression exhibits clinical efficacy. However, further efforts are still needed to improve the clinical use of PD-L1 expression as biomarkers Apart from combinatorial strategies, another topic concerning the use of ICIs is often investigated to study prediction biomarkers. There are several biomarkers associated with the response of ICIs, some of which have been approved and are currently being exploited to predict the response rate in patients before treatment begins, while others are under further study to establish whether they have a strong correlation with the extent of patients' responses to ICIs. The most important predictive biomarker for anti-PD-1/PD-L1 antibodies is PD-L1 expression [41,109], which is evaluated by immunohistochemistry and is a prerequisite for treatment with drugs such as atezolizumab or pembrolizumab. However, this biomarker may not be enough to identify all of the patients who could benefit from this type of therapy, and this observation led scientists to begin further studies to find more appropriate predictive biomarkers. This biomarker's use is already well established in conventional chemotherapy regimens, but recent studies suggest that it could be exploited to predict the response to immunotherapy and, most importantly, that it could also help discriminate real disease progression from pseudo-progression in patients treated with immunotherapy, avoiding re-biopsy in these patients [109–111].
