Immunotherapeutic targets have emerged in the past decade as a promising addition to the oncology treatment arsenal for selected types of cancers ^[1][2][1,2]. With the rapid development of new immune anticancer drugs and viruses, accurate and reliable preclinical validation including assessment of tumor growth and response, evaluation of complications, drug resistance, and mechanistic effects became of utmost importance to expedited drug development [3]. Recent advances, including the recognition by the Nobel Prize in 2018 of Dr. James P. Allison and Dr. Tasuku Honjo for their discoveries in immune-oncology treatment, highlight the current research focus on the development of new immunotherapeutic drugs ^[3][4][3,4].

With the advent of new immunotherapeutic targets and new developments in oncolytic virus (OV) immunotherapeutic applications, problems with preclinical validation and safety have emerged [5]. Classically, simple preclinical models often use orthotopic or syngeneic tumors, either in immunocompromised or immunocompetent mice [6]. Although they are essential to initial target and mechanism determination as well as time and cost-effective, these models have demonstrated limited reproducibility in clinical trials [7].

Along those lines, multiple animal models have been developed in an attempt to suffice the need for a reliable preclinical assessment of these drugs. Humanized mice, which comprise immune-deficient mice engrafted with human hematopoietic cells and therefore with a human immune system, are the most promising models [8].

2. Oncologic Immunomodulation

The relationship between tumor tissues and the immunologic system has been increasingly explored with a new concept of immunologic modulation in the fight against cancer cells. The thought that tumors escape immune surveillance is not recent, but evolved significantly in the past decade with the development of immune checkpoint inhibitors (ICI), specifically the ones targeting the cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death 1 (PD1), that showed encouraging results in initial clinical trials ^[9][10][9,10]. Ipilimumab and tremelimumab emerged as therapeutic strategies to augment anti-tumor immunity against cancer [11]. Although initial studies demonstrated improved outcomes with a combination of ICI therapy, long-term follow-up showed no significant clinical difference of combined nivolumab and ipilimumab survival compared to single therapies [12]. Furthermore, ICI’s high cost and immune-related adverse effects became a concern ^[13][14][13,14]. Thus, there is growing interest in complementary agents with safer toxicity profile. CTLA4 intrinsic function is closely linked to T lymphocyte modulation and a major responsibility for immunologic tolerance [15]. CTLA4 is released from intracellular vesicles to the immunologic synapse following T cell receptor (TCR) activation [16]. CTLA4 attenuates T cells by competitive binding to ligands B7-1 and B7-2. CD28 interaction with ligands B7-1 and B7-2 is intrinsically positive through downstream signaling mediated by phosphoinositide 3-kinase (PIK3) and protein kinase B (AKT) ^[17][18][17,18]. Both co-stimulatory positive and negative interactions are similar in strength, allowing for modulation swift. CTLA4 acts primarily in sites of lymphocyte priming but may also be encountered in varying degrees in peripheric tissues where antigen-presenting cells and activated lymphocytes express B7 ligands [19]. In addition to intrinsic function, CTLA4 also modulates T lymphocyte activation extrinsic mechanisms through regulatory T cells (Tregs) modulation. Loss of extrinsic CTLA4 modulation is sufficient to induce aberrant T cell activation and autoimmunity. In this context, CTLA4 appears to modulate B7 ligand activation in antigen-presenting cells, possibly by a mechanism of trans-endocytosis [20]. CTLA4 modulation occurs by selective competitive attenuation of high strength TCR expression, allowing for medium-strength T cell activation [21]. The PD1-PD-ligand 1 (PD-L1) pathway has shown to significantly improve survival in preclinical and clinical trials and is currently in clinical use for many types of cancer. PD-L1 is expressed by many types of cells including tumor cells while PD-L2 is expressed mainly by normal dendritic cells [22]. Thus, the PD-1 mechanism primarily acts in the periphery. PD-1 signaling pathway is activated upon T and B cell activation and, different from CTLA4, appears to directly inhibit TCR activation through activation of the downstream signaling mediated by the tyrosine phosphatase SHP2 ^[23][24][23,24]. In addition, CD28 has also been demonstrated to confer a primary target for PD-1, indicating a common pathway with CTLA4. The PD-1—PD-L1 interaction attenuates T lymphocyte immune response, and in the setting of cancer, is a major contributor to impaired anti-cancer immune response [25].

3. Oncolytic Viruses and Immuno-Oncologic Modulation

OVs present a natural or genetically engineered tropism for tumor cells that can be further enhanced to increase both innate and adaptive immune responses. Over the years, OVs, particularly adenovirus, have been continuously modified to increase tumor selectivity and minimize toxicity. OV mechanism of action is thought to occur through three main mechanisms: the primary lysis of tumor cells caused by intracellular viral replication, gene modification delivery, and the secondary increase in antigen-presenting molecules leading to an increased adaptive immunologic response. One of the main advantages is that given the selective nature of OVs, these mechanisms may be used systemically to reach tumor metastatic tissue that was not directly inoculated. Viruses and other pathogens naturally stimulate stronger immune responses than over-expressed self-antigens normally encountered in solid tumors. OVs have evolved through the years to express multiple cell receptors and lack intrinsic replication capabilities. OVs mechanism rely on virus-mediated cytolysis promoting the release of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), which are cancer cell death sensors ^[26][65]. Tumor oncolysis and release of PAMPs and DAMPs, which are recognized by pattern recognition receptors (PRR), lead to DC activation and subsequent CD4 and CD8 T cell priming. With a growing body of evidence in immunologic modulation for cancer therapy, OVs followed a similar trend by genetically adding immunologic capabilities. Talimogene laherparepvec (T-VEC) is a herpes-simplex virus encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and was the first OV-based immunotherapy to reach a phase III trial. It is currently FDA approved for the treatment of selected patients with metastatic melanoma that are not candidates for surgical resection. Following intratumoral administration, T-VEC induces cell lysis followed by the release of tumor antigen and subsequent circulation of tumor-specific effective T-cells. Thus, although the administration is local, the treatment effect reaches systemic levels. GM-CSF controls both myeloid differentiation and the function of mature presenting antigen cells. This effect has been replicated with adenovirus (Ad5-D24-GMCSF), which demonstrated complete tumor regression and tumor-specific cytotoxic T lymphocyte response ^[27][66].

Oncolytic Viruses and Immunotherapeutic Drugs

Sipuleucel-T autologous cell-based immunotherapy vaccine FDA-approved for the treatment of hormone-refractory prostate cancer. Production is individualized by leukapheresis of the patient’s peripheral blood antigen-presenting cells followed by ex vivo antigen loading with prostate acid phosphatase (PAP) and GM-CSF enhancement ^[28][67]. Approval was based upon the IMPACT trial, a multicenter phase 3 clinical trial comparing sipuleucel-T or placebo in men with asymptomatic metastatic castration-resistant prostate cancer. In this population, sipuleucel-T showed modest improvement in overall survival (25.8 months in the sipuleucel-T group vs. 21.7 months in the placebo group) but no effect in time to disease progression ^[29][68]. Based on the effects of cancer vaccines such as Sipuleucel-T, several OVs armed with GM-CSF (HSV T-Vec, VV JX-594, Ad Ad5/3-D24-GMCSF, and CG0070 have entered clinical trials ^[30][69]. The combination of immunotherapy drugs has been attempted as an approach to increase tumor cell response. However, the combination of immunotherapy drugs carries concerns with increased adverse effects ^[31][70]. The concept of utilizing a combination of oncolytic virus and immunotherapy emerged to enhance immunomodulation, particularly to overcome immunosuppressive effects within the TME while maintaining a safety profile. Along those lines, a recent phase II clinical trial demonstrated the improved response of T-VEC combined with ipilimumab with decreased visceral lesion decreases in 52% of patients in the combination arm and 23% of patients in the ipilimumab arm. This researchtudy included selected patients bearing advanced melanoma. There was no significant increase in adverse events with the combination approach ^[32][71]. With the same concept of vaccine cancer therapy, researcheuthors recently reported on the use of cancer stem cells lysate-pulsed dendritic cell vaccine with induced tumor-specific humoral and cellular immunologic response ^[33][34][35][72,73,74]. Cancer stem cells are encountered in higher number in selected tumors subtypes and are known for their high differentiation capabilities, contributing to tumor heterogeneity and therapy resistance ^[36][75]. Although innovative, response with single cancer stem cell vaccine therapy was limited by the immunosuppressive tumor microenvironment that hampered immunologic response ^[37][76]. In a more recent article, researcheuthors explored the use of combination dual or triple therapy with PD-L1 or CTLA4 inhibitors, demonstrating increased T cell proliferation, improved tumor-specific CD8 + T cell response, and inhibition of tumor necrosis factor-beta (TNFβ), resulting in a dramatic tumor response in animal models ^[38][77]. Chimeric antigen receptor-modified T cell therapy (CAR-T) is an adoptive autologous T cell therapy strategy targeting cells or TME. CAR-T synthetically generates personalized effector T cells with a high affinity for tumor antigens independent of MHC ^[39][78]. CAR-T therapy demonstrated dramatic clinical response in B cell malignancies ^{[40][41][42][43]}[79,80,81,82]. However, CAR-T therapy in solid tumors is much less successful, likely secondary to immunosuppressive TME changes ^[44][45][46][83,84,85]. Li et al. evaluated the combination of CAR-T therapy and oncolytic adenovirus expressing TNFβ receptor II-Fc (rAd.sT) in a triple-negative breast cancer model ^[47][86]. This modified adenovirus targets and inhibits TNFβ signaling, decreasing immunosuppressive effects in the TME ^[48][87]. The researcheuthors constructed CAR-T cells targeting mesothelin, a protein that is normally expressed by various mesothelial tissues but also over-expressed by a wide range of cancers ^[49][88]. The researcheuthors demonstrated moderate mesothelin expression in the MDA-MB-231 cell line. The combination therapy elicited increased apoptosis and the researcheauthor detected a synergistic effect in the expression of IL-2 and IL-6 cytokines, important immunogenic cytokines. In a different work by Watanabe et al., an engineered oncolytic adenovirus expressing tumor necrosis-alpha (TNFα) and interleukin-2 (IL2) (Ad5/3-E2F-D24-TNFa-IRES-IL2 or Ad-mTNFa-mIL2) was administered to humanized mice bearing pancreatic adenocarcinoma xenografts. Pancreatic adenocarcinoma has a highly immunosuppressive TME. The researcheuthors demonstrated the increased intensity of T cell infiltration with sustained tumor regression after combined meso-CAR-T cells and oncolytic virus therapy. Pancreatic adenocarcinoma was resistant to meso-CAR-T cells alone, but co-administration with Ad-mTNFa-mIL2 elicited tumor regression. Moreover, Ad-mTNFa-mIL2 demonstrated induced increased M1 polarization of macrophages and DC maturation compared to control adenovirus, again indicating that oncolytic therapy enhances innate and adaptive immunity ^[50][89]. TMAs and OV interplay, however, is not completely clear and often contradictory in the literature. Although it has been reported that tumor inflammation inhibits viral replication through interferon release, some tumors have demonstrated a response to virus-based anti-tumor immune activation ^[51][52][90,91]. This relationship is, however, not linear and the type of macrophage polarization (M1 or M2) appears to vary by tumor and type of virus ^[53][92]. Recently, researcheuthors tested the anti-tumor effect of a recombinant Newcastle disease virus (MEDI5395) expressing GM-CSF and demonstrated enhanced immune-cell activation and pro-inflammatory cytokine release in vitro ^[54][93].