*3.2. Improving Antitumor Efficacy*

Genetic modifications of OVs to increase the expression of cytokines, chemokines, costimulatory molecules, tumor extracellular matrix (ECM)-degrading enzymes, and antiangiogenic molecules can enhance their antitumor effects (Figure 1). Granulocytemacrophage colony-stimulating factor (GM-CSF) gene-bearing OVs such as T-VEC, Pexa-Vec, and CG0070 recruit antigen-presenting cells (APCs) and CTLs, resulting in a better TAA presentation with minimal antiviral response induction [6]. GMOVs expressing proinflammatory cytokines showed enhanced antitumor efficacy. Despite the considerable antitumor response, IL-2-secreting OVs cause systemic toxicity. The design of VACV expressing membranous IL-2 rather than secretory form increases local antitumor response with significantly reduced toxicity [138]. The use of IL-12, IL-15, IL-18, TNF-α, IL-24, and IFN-γ genes in OVs also enhances antitumor effects with much lower toxicity than IL-2 [6,139–141]. Interestingly, the application of the non-secretory form of these cytokines causes local effects rather than systemic AEs [142]. Expression of specific chemokines such

as CCL5, CCL19, CCL20, CCL21 by engineered OVs (mainly VACV) increases the infiltration of naïve and memory T lymphocytes and DCs into the TME [143–146]. Simultaneously, employment of one or multiple costimulatory ligands, including CD40L, 4-1BBL, OX40L, and B7-1 in OVs such as LOAd703 (the combination of CD40L and 4-1BBL) increases antigen presentation and T cell priming [6,26,96]. Besides, insertion of TLR ligands such as CpG-rich regions in the OVs genome stimulates TLRs and further activates innate and acquired immunity [138].

Another way to enhance the immune responses in the TME is the elimination of immunosuppressive cells. GMOVs that express the hydroxyprostaglandin dehydrogenase (HPGD) enzyme inactivate PGE2 and reduce the presence of MDSCs in the TME [147]. Soluble CXCR4 expressed by GMOVs binds to CXCL12 secreted by tumor cells as a decoy receptor and inhibits the effects of CXCL12 on angiogenesis, metastasis, and recruitment of MDSCs [148].

Although OVT can release TAAs through various mechanisms, the expression of TAAs by GMOVs or coating the TAA-derived peptides on the surface of OVs increases T cell response and improves OVT. A large number of TAAs and peptides have been studied so far [26]. The advantage of peptide coating over peptide expression is the convenience, speed, lower cost, and the possibility of personalization for each patient in the peptide coating method [26].

OVs can be engineered to express proapoptotic proteins such as TNF-related apoptosisinducing ligand (TRAIL) and apoptin that can induce specific apoptosis in tumor cells [149,150]. Insertion of the oncogene suppressor small interfering RNAs (siRNAs) in OVs could also suppress oncogene expression and inhibit tumor growth [151,152].

The host antiviral response ensures that OVs disappear after a while and prevents the AEs of their long presence. However, the host antiviral response might cause rapid clearance of OVs before fulfilling their antitumor activity [153]. Expression of IFN-I antagonists by OVs or some non-pathogenic bacteria reduces the innate immune response against OV and delays their clearance [154]. Also, the use of stem cells, polymers, and liposomes as OV carriers reduces the immunogenicity of OVs, shields them from neutralizing antibodies (nAbs), and improves their transmission to the TME, which is listed in Table 2. An interesting way to optimize cytokine production with minimal antiviral responses is to insert inducible promoters or regulatory genes so that the cytokine expression is exogenously induced after sufficient replication of OVs in tumor cells [155].

**Figure 1.** Recent approaches in oncolytic virotherapy. Expression of TAA-receptors and scFvs, recombination of specific domains and motifs, using tumor-specific promoters, and application of miRNA-TS could enhance the tumor targeting. GMOVs expressed inflammatory cytokines, enzymes, chemokine receptors, costimulatory molecules, and proapoptotic proteins achieve high antitumor potency. ECM and vasculature degradation by enzymes and molecules result in a higher spread of OVs. Administration routs are a critical factor in achieving better results with lower adverse effects. Besides, deleting virulence genes and recombination of OVs together could diminish the concerns of adverse events. However, several biosafety concerns still remained unmet. The combination of OVT with other immunotherapy, such as ICIs, TIL therapy, CART cell therapy, DC vaccines, mAbs, BiTEs, and metabolic inhibitors could potentiate the immunotherapy against tumors. OV. Oncolytic virus; OVT. OV therapy; TAA. Tumor-associated antigen; scFv. Single-chain variable fragment; Her-2. Human epidermal growth factor receptor 2; EpCAM. Epithelial cell adhesion molecule; CEA. Carcinoembryonic antigen; AdV. Adenovirus; RGD. Arginine-glycine-aspartate; GP-160. Glycoprotein-160; miRNA-TS. microRNA targeting sequence; PSA. Prostate-specific antigen; hTERT. Human telomerase reverse transcriptase; DC. Dendritic cell; CD. Cytosine deaminase; 5-FC. 5-fuorocytosine; 5-FU. 5-fluorouracil; HPGD. Hydroxyprostaglandin dehydrogenase; PGE2. Prostagalndin-E2; MDSC. Myeloid-derived suppressor cell; IFN. Interferon; TRAIL. TNF-related apoptosis-inducing ligand; siRNA. Small interfering RNA; TLR-9. Toll-like receptor-9; ICI. Immune checkpoint inhibitor; BiTE. Bispecific T cell engager; mAb. Monoclonal antibody; TIL. Tumor0infiltraring lymphocyte; CART cell. Chimeric-antigen receptor T cell; TME. Tumor microenvironment; ECM. Extracellular matrix; VEGF. Vascular-endothelial growth factor. Another way to enhance the immune responses in the TME is the elimination of im-**Figure 1.** Recent approaches in oncolytic virotherapy. Expression of TAA-receptors and scFvs, recombination of specific domains and motifs, using tumor-specific promoters, and application of miRNA-TS could enhance the tumor targeting. GMOVs expressed inflammatory cytokines, enzymes, chemokine receptors, costimulatory molecules, and proapoptotic proteins achieve high antitumor potency. ECM and vasculature degradation by enzymes and molecules result in a higher spread of OVs. Administration routs are a critical factor in achieving better results with lower adverse effects. Besides, deleting virulence genes and recombination of OVs together could diminish the concerns of adverse events. However, several biosafety concerns still remained unmet. The combination of OVT with other immunotherapy, such as ICIs, TIL therapy, CART cell therapy, DC vaccines, mAbs, BiTEs, and metabolic inhibitors could potentiate the immunotherapy against tumors. OV. Oncolytic virus; OVT. OV therapy; TAA. Tumor-associated antigen; scFv. Single-chain variable fragment; Her-2. Human epidermal growth factor receptor 2; EpCAM. Epithelial cell adhesion molecule; CEA. Carcinoembryonic antigen; AdV. Adenovirus; RGD. Arginine-glycine-aspartate; GP-160. Glycoprotein-160; miRNA-TS. microRNA targeting sequence; PSA. Prostate-specific antigen; hTERT. Human telomerase reverse transcriptase; DC. Dendritic cell; CD. Cytosine deaminase; 5-FC. 5-fuorocytosine; 5-FU. 5-fluorouracil; HPGD. Hydroxyprostaglandin dehydrogenase; PGE2. Prostagalndin-E2; MDSC. Myeloid-derived suppressor cell; IFN. Interferon; TRAIL. TNF-related apoptosis-inducing ligand; siRNA. Small interfering RNA; TLR-9. Toll-like receptor-9; ICI. Immune checkpoint inhibitor; BiTE. Bispecific T cell engager; mAb. Monoclonal antibody; TIL. Tumor0infiltraring lymphocyte; CART cell. Chimeric-antigen receptor T cell; TME. Tumor microenvironment; ECM. Extracellular matrix; VEGF. Vascular-endothelial growth factor.

munosuppressive cells. GMOVs that express the hydroxyprostaglandin dehydrogenase








OV. Oncolytic virus; OVT. OV therapy; ECM. Extracellular matrix; BiTE. Bispecific T cell engager; VEGF. Vascular-endothelial growth factor; bFGF. basic fibroblast GF; PI3K. Phosphoinositide 3-kinase; TAF. Tumor-associated fibroblast; Lox. Lipoxygenase.

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