**1. Introduction**

Interleukin 12 (IL-12) is a powerful master regulator of both innate and adaptive anti-tumor immune responses. As a heterodimeric cytokine, it produces multifaceted anti-tumor e ffects [1,2], including stimulation of growth and cytotoxic activity of natural killer (NK) cells and T cells (both CD4+ and CD8+) [1,3–5], induction of di fferentiation of CD4+ T cells towards Th1 phenotype [6,7], increased production of IFN-γ from NK and T cells [1,8,9], and inhibition of tumor angiogenesis [1,10]. Despite encouraging success in preclinical studies [4], the early stages of IL-12 clinical trials did not meet expectations. Severe adverse events were first reported on 15 out of 17 patients in a phase II clinical trial following intravenous IL-12 administration, and the trial was immediately terminated by the FDA following two cases of death [11,12]. Although success was observed in cutaneous T cell lymphoma variants [13,14], AIDS-related Kaposi sarcoma [15] and non–Hodgkin's lymphoma [16], severity of side e ffects outweighed e ffectiveness of IL-12 based therapies in the vast majority of oncology clinical trials [17]. In an e ffort to optimize e fficacy and enhance the safety profile, alternative approaches are being studied to localize IL-12 expression at the tumor microenvironment.

*Cells* **2020**, *9*, 400

Recent studies show that systemic toxicity of IL-12 is limited when expressed by oncolytic viruses (OVs) locally in the tumors [18–20] and in the brains of non-human primates [21]. OVs are a distinct class of anti-cancer agents with unique mechanisms of action: (i) selectively replicating in and killing cancer cells (i.e., oncolysis) without harming healthy cells or tissues [22–24], and (ii) exposing viral/tumor antigens, which promote a cascade of anti-tumor innate and adaptive immune responses (i.e., in situ vaccine e ffects) [25,26]. The OV-induced vaccine e ffects can be further enhanced through viral expression of anti-tumor cytokines such as IL-12 [18–20], as illustrated in Figure 1. Cancer immunotherapy involving OVs is an emerging and increasingly examined therapeutic approach for the treatment of cancer [27,28]. Among OVs, oncolytic herpes simplex virus (OHSV) is the furthest along in the clinic and approved by the FDA for the treatment of advanced melanoma [29]. To utilize the therapeutic potential of IL-12, there are several OHSVs encoding IL-12 which have been genetically engineered and tested in various cancers (Table 1). In addition to OHSVs, several di fferent OVs such as adenoviruses, measles virus, maraba virus, Newcastle disease virus, Semliki forest virus, vesicular stomatitis virus and Sindbis virus are also being engineered to express IL-12 (Table 2). Our literature research found that 25 di fferent types of OV-IL12s are either being or have recently been explored (see Tables 1 and 2). Despite this rapidly rising interest, there are no reviews on IL-12 expressing viruses that exploit their potential e fficacy and safety to translate into human subjects. This review presents the most current data on this topic and provides a basic understanding of OV-IL12 as a promising treatment approach in cancer immunotherapy, which ultimately could support continued research in the future. More specifically, in this review, we will discuss safety, tumor-specificity, and anti-tumor/anti-vascular e ffects of OHSV-IL12 as monotherapy or combination therapy. In addition to OHSV-IL12 viruses, we will also review other IL-12 expressing OVs and their application in cancer therapy.

**Figure 1.** Graphic presentation of mechanism of action of oncolytic virus encoding IL-12. ( **A**) Infection of tumor with oncolytic virus encoding IL-12 (OV-IL12). (**B**) OV-IL12 replicates in and kills cancer cells (i.e., oncolysis) and releases IL-12 in the tumor microenvironment. ( **C**) Neoantigens from lysed cancer cells activate and recruit dendritic cells (DCs) into the tumor microenvironment. DCs process neoantigens, travel to nearest lymphoid organs, and present the antigen to T cells (CD4+ and CD8+ T cells). (**D**,**E**) T cells migrate to the site of infection (referred as tumor-infiltrating T cells or TILs), di fferentiate into Th1 cells, produce anti-tumor cytokines and kill cancer cells. (**F**) IL-12-induced production of IFN-γ and interferon inducible protein 10 (IP-10) produces anti-angiogenetic e ffect through reduction of tumoral vascular endothelial growth factor (VEGF) and CD31+ tumor endothelial cells.



*Cells* **2020**, *9*, 400

