**5. Immunovirotherapy**

While early efforts in engineering oncolytic MeV mainly focused on maximizing direct tumor cell killing, there has been a recent shift from mainly oncolytic to mainly immunotherapeutic treatment strategies, spurred by the developments in cancer immunotherapy which have revolutionized medical oncology.

MeV oncolysis per se has pleiotropic effects on the anti-tumor immune response and supports all phases of the "cancer immunity cycle" (Figure 4; reviewed in [99]). MeVinduced cell death is immunogenic [100], induces a distinctive immunopeptidome [101], and promotes cross-priming of antitumor T cell responses by conventional and plasmacytoid dendritic cells [19,102]. MeV oncolysis has also been reported to increase tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by myeloid and plasmacytoid DCs [103] as well as modulation of macrophages towards an antitumor phenotype [104]. Neutrophil activation also occurs, leading to secretion of IL-8, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and IFN-α, TRAIL

expression, and degranulation [105], which may be beneficial or not depending on the tumor model [106]. mor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and IFN-α, TRAIL expression, and degranulation [105], which may be beneficial or not depending on the tumor model [106].

While early efforts in engineering oncolytic MeV mainly focused on maximizing direct tumor cell killing, there has been a recent shift from mainly oncolytic to mainly immunotherapeutic treatment strategies, spurred by the developments in cancer immuno-

MeV oncolysis per se has pleiotropic effects on the anti-tumor immune response and supports all phases of the "cancer immunity cycle" (Figure 4; reviewed in [99]). MeV-induced cell death is immunogenic [100], induces a distinctive immunopeptidome [101], and promotes cross-priming of antitumor T cell responses by conventional and plasmacytoid dendritic cells [19,102]. MeV oncolysis has also been reported to increase tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by myeloid and plasmacytoid DCs [103] as well as modulation of macrophages towards an antitumor phenotype [104]. Neutrophil activation also occurs, leading to secretion of IL-8, tu-

*Cancers* **2021**, *13*, x 7 of 19

therapy which have revolutionized medical oncology.

**5. Immunovirotherapy** 

**Figure 4.** Measles virus as an oncolytic immunotherapy. Measles virus-mediated oncolysis has been shown to support different phases of the antitumor immune response: Oncolysis induces immunogenic cell death, which promotes dendritic cell activation, antigen presentation, and crosspriming of T cells. Measles virotherapy remodels the tumor microenvironment, thereby enhancing innate (macrophage repolarization and neutrophil degranulation) as well as adaptive antitumor immunity (T cell infiltration and CD8+ effector responses). **Figure 4.** Measles virus as an oncolytic immunotherapy. Measles virus-mediated oncolysis has been shown to support different phases of the antitumor immune response: Oncolysis induces immunogenic cell death, which promotes dendritic cell activation, antigen presentation, and crosspriming of T cells. Measles virotherapy remodels the tumor microenvironment, thereby enhancing innate (macrophage repolarization and neutrophil degranulation) as well as adaptive antitumor immunity (T cell infiltration and CD8+ effector responses).

These immunotherapeutic effects can be enhanced by insertion of immunomodulatory transgenes into the MeV genome (Table 1). Further, MeV can serve as a vector to deliver immunomodulators to the tumor site which can be highly toxic when administered systemically. The first immunomodulatory transgene reported in the context of many oncolytic viruses and also MeV was the granulocyte macrophage colony stimulating factor, GM-CSF [14]. In a lymphoma xenograft model, MV GM-CSF led to increased neutrophil infiltration, which correlated with tumor regression. Further immunomodulators have been shown to increase innate immune activation in the context of MeV oncolysis. A MeV vector encoding IFN-β was reported to induce immune infiltration and remodeling of the tumor microenvironment in mesothelioma xenografts [107]. MeV encoding the immunomodulatory neutrophil-activating protein (NAP) of *H. pylori* prolonged survival and induced a beneficial cytokine response in breast cancer xenograft pleural effusion and lung colonization models [108]. These immunotherapeutic effects can be enhanced by insertion of immunomodulatory transgenes into the MeV genome (Table 1). Further, MeV can serve as a vector to deliver immunomodulators to the tumor site which can be highly toxic when administered systemically. The first immunomodulatory transgene reported in the context of many oncolytic viruses and also MeV was the granulocyte macrophage colony stimulating factor, GM-CSF [14]. In a lymphoma xenograft model, MV GM-CSF led to increased neutrophil infiltration, which correlated with tumor regression. Further immunomodulators have been shown to increase innate immune activation in the context of MeV oncolysis. A MeV vector encoding IFN-β was reported to induce immune infiltration and remodeling of the tumor microenvironment in mesothelioma xenografts [107]. MeV encoding the immunomodulatory neutrophil-activating protein (NAP) of *H. pylori* prolonged survival and induced a beneficial cytokine response in breast cancer xenograft pleural effusion and lung colonization models [108].

Introduction of the first fully immunocompetent mouse model of MeV oncolysis, MC38cea [93], was the prerequisite to further study immunomodulatory MeV vectors and demonstrate induction of tumor-specific adaptive immune responses. This model consists of murine colorectal adenocarcinoma MC38, syngeneic to C57BL/6 mice and stably expressing the carcinoembryonic antigen (CEA), which are susceptible to CEA-targeted MeV [93]. In this model, treatment with MV GM-CSF led to prolonged survival compared to control MV. Forty percent of treated mice experienced complete tumor remission and were subsequently protected from tumor re-engraftment, indicating a tumor vaccination effect. Further, treatment with MV GM-CSF enhanced intratumoral T cell infiltration as well as tumor-specific T cell responses [109].

**Table 1.** Immunomodulatory oncolytic MeV. Overview of immunomodulatory transgenes that have been encoded in MeV, their anticipated immunological effects in the context of MeV oncolytic immunotherapy, and the outcome of the respective preclinical studies. GM-CSF: granulocyte–macrophage colony-stimulating factor; IFN: interferon; NAP: neutrophil activating protein; CTLA-4: cytotoxic T lymphocyte antigen-4; PD-L1: programmed cell death 1-ligand 1; Th: T helper cell; Teff: effector T cell; Treg: regulatory T cell; IL: interleukin; AICD: activation-induced cell death; BiTE: bispecific T cell engager; TAA: tumor-associated antigen; IFNAR: IFN-α receptor.


Italic: bacterial taxa.

To develop a second immunocompetent model of MeV oncolysis in C57BL/6 mice, B16 melanoma cells were transduced for stable expression of the CD20 surface antigen for treatment with CD20-targeted MeV. In this model, MeV vectors encoding immune checkpoint antibodies against cytotoxic T lymphocyte-associated-4 (CTLA-4) and programmed cell death-ligand 1 (PD-L1) prolonged survival compared to MeV encoding the antibody constant region only [110]. Combination with systemically administered antibodies against CTLA-4, PD-1, and PD-L1 has also demonstrated the therapeutic value

of this approach [110,116]. In the MC38cea model, systematic comparison of transgenes targeted at different phases of the cancer immunity cycle—GM-CSF, IFN-γ induced protein 10 (IP-10), membrane-bound CD80, anti-CTLA-4, IL-12, and anti-PD-L1 identified the latter two as the most potent [111]. MeV encoding IL-12 induced complete tumor remissions in 90% of treated mice, which were mediated by CD8+ effector T cell responses. Oncolytic MeV vectors encoding an IL-15 superagonist mediated T and NK cell activation, but were less effective than MeV encoding IL-12 [112]. Bispecific T cell engagers (BiTEs) simultaneously bind CD3 on T cells and a tumor surface antigen, thereby redirecting T cells to tumor cells to mediate antitumor T cell cytotoxicity. Oncolytic measles viruses encoding BiTEs were shown to promote T cell infiltration and activation in syngeneic and patient-derived tumor models [113].

For induction of T cell responses against specific antigens, MeV can also be employed as a heterologous, highly immunogenic vaccine vector (reviewed in [117]). This strategy has been used to develop vaccines against a range of pathogens, including emerging SARS CoV-2 [118]. This strategy has been adopted in oncolytic immunotherapy by encoding tumor-associated antigens in the MeV vector. MeV vectors encoding ovalbumin (OVA) as model antigen or the tumor antigen claudin-6 either in native form or in association with lentivirus-like particles were shown to induce antigen-specific humoral and cellular immune responses in IFN-α receptor (IFNAR)-deficient, CD46-transgenic mice and prolong survival in B16-derived tumor models [114]. Employing OVA and the melanoma antigen tyrosinase-related protein-2 (TRP-2), MeV vectors encoding the full-length antigens or their respective immunodominant CD8+ epitope or epitope variants were generated. The epitope variants are either secreted or targeted to the proteasome. Using these MeV vectors, activation and dendritic cell-mediated priming of cognate T cells was demonstrated ex vivo [115].

As another modality of immunovirotherapy, combination of oncolytic MeV with adoptive transfer of antitumor immune effector cells such as NK cells [119] or CD8+ NKG2D+ cells [120] has been reported.

Importantly, antitumor immune activation by MeV oncolysis has not only been demonstrated in preclinical models. Clinical data also suggest augmentation of antitumor immunity by oncolytic measles virotherapy. In cutaneous T cell lymphoma, a shift towards a Th1-biased T cell population in lymphoma lesions was noted after treatment [121]. In four ovarian cancer patients treated with MV-NIS, IFN-γ and IL-4 responses against the tumor antigens FRα and IGF binding protein 2 (IGFBP2) were detected by ELISPOT [122]. Increases in IFN-γ ELISPOT counts against cancer testis antigens were also observed in the majority of tested multiple myeloma patients treated with MV-NIS [123]. The myeloma patient with an exceptional response to MV-NIS had a high mutational load and high baseline T cell responses against several tumor antigens, which remained stable after virotherapy.

Of note, the clinical trials published thus far tested oncolytic MeV not encoding any additional immunotherapeutic payloads. Perhaps the fraction of patients showing immunological responses and overall therapeutic efficacy can be increased with novel immunomodulatory oncolytic MeV.
