**4. Engineering NDV to Modulate Innate and Adaptive Immune Responses**

With the development of reverse genetics, it has become possible to modify the NDV viral genome and introduce foreign sequences to potentially enhance oncolytic and immunostimulatory properties of these agents [82]. Several strategies to enhance innate and/or adaptive antitumor immunity by engineering NDV to express cytokines, antibodies, ligands, or tumor antigens have been explored, and a few are reviewed below (Figure 2). Given its ability to activate antigen-presenting cells, granulocyte–macrophage colony stimulating factor (GM-CSF) has been explored as a therapeutic transgene within the context of multiple oncolytic viruses, and T-VEC, an oncolytic herpes simplex virus expressing GM-CSF, was approved by the FDA for treatment of metastatic melanoma [74]. A recombinant strain based on the mesogenic NDV 73T strain currently in clinical development, MEDI5395, expressing human GM-CSF was recently shown to increase secretion of pro-inflammatory cytokines such as IFN-α, IL-6, IL-8, and TNF-α in PBMC samples from healthy volunteers, and stimulated PBMCs to exert antitumor effects in vitro [83]. In addition, infection of dendritic cells led to their maturation, and co-culture of dendritic cells with allogeneic T cells increased the levels of T cell effector cytokines IL-2 and IFN-γ [83]. In a separate study using NDV Hitchner B1 strains engineered to express either murine IL-2, IFN-γ, and GM-CSF in vivo, only NDV expressing IL-2 led to a significant increase in overall animal survival when compared to parental NDV [82]. Similar results were recently demonstrated with a lentogenic recombinant NDV strain expressing IL-24 [84].

*Cancers* **2020**, *12*, x 7 of 16

**Figure 2.** Strategies to enhance the NDV antitumor immune response by recombinant genetic engineering. Genetic engineering can be used to generate NDV strains with greater potential to stimulate antitumor immune response. First, NDV engineered to express cytokines such as GM-CSF or interleukins can increase recruitment of innate effector cells such as antigen-presenting cells (APCs). Second, NDV can be used as a therapeutic vaccine targeted to specific tumor antigens such as oncogenic viral antigens, frame shift mutations, or mutated self-antigens. Third, NDV can be engineered express single-chain variable fragments or full antitumor antibodies to induce antibodydependent cellular cytotoxicity by effector cells. **Figure 2.** Strategies to enhance the NDV antitumor immune response by recombinant genetic engineering. Genetic engineering can be used to generate NDV strains with greater potential to stimulate antitumor immune response. First, NDV engineered to express cytokines such as GM-CSF or interleukins can increase recruitment of innate effector cells such as antigen-presenting cells (APCs). Second, NDV can be used as a therapeutic vaccine targeted to specific tumor antigens such as oncogenic viral antigens, frame shift mutations, or mutated self-antigens. Third, NDV can be engineered express single-chain variable fragments or full antitumor antibodies to induce antibody-dependent cellular cytotoxicity by effector cells.

Optimal immune mechanisms for intratumoral targeting with oncolytic virus are unknown. Gene expression profiling of tumors after NDV injection revealed the upregulation of T cell costimulatory receptors ICOS, 4-1BB, GITR, OX40, CD27, and CD40, all of which are currently being evaluated as therapeutic targets in clinic using monoclonal antibodies [60]. Targeting of ICOS within the context of tumor microenvironment using engineered cellular vaccines expressing ICOS ligand (ICOSL) has in particular been previously demonstrated to improve systemic efficacy of CTLA-4 blockade through potentiation of cytotoxic T cell function [85]. Intratumoral administration of engineered NDV LaSota expressing ICOSL resulted in enhanced infiltration of CD8+ and CD4+ T cells, tumor growth delay of both injected and non-injected tumors, and prolonged survival, as compared to wild type NDV, and this effect that was further enhanced when combined with anti-CTLA-4 blockade [60]. These findings highlight that stimulation of both innate and adaptive immune response pathways within the context of intratumoral NDV therapy may be required for optimal activation of antitumor immune response. Recently, recombinant NDV LaSota strains expressing soluble single-chain variable fragments for anti-CD28, anti-PD1, and anti-PDL1 were generated, as Optimal immune mechanisms for intratumoral targeting with oncolytic virus are unknown. Gene expression profiling of tumors after NDV injection revealed the upregulation of T cell co-stimulatory receptors ICOS, 4-1BB, GITR, OX40, CD27, and CD40, all of which are currently being evaluated as therapeutic targets in clinic using monoclonal antibodies [60]. Targeting of ICOS within the context of tumor microenvironment using engineered cellular vaccines expressing ICOS ligand (ICOSL) has in particular been previously demonstrated to improve systemic efficacy of CTLA-4 blockade through potentiation of cytotoxic T cell function [85]. Intratumoral administration of engineered NDV LaSota expressing ICOSL resulted in enhanced infiltration of CD8+ and CD4+ T cells, tumor growth delay of both injected and non-injected tumors, and prolonged survival, as compared to wild type NDV, and this effect that was further enhanced when combined with anti-CTLA-4 blockade [60]. These findings highlight that stimulation of both innate and adaptive immune response pathways within the context of intratumoral NDV therapy may be required for optimal activation of antitumor immune response. Recently, recombinant NDV LaSota strains expressing soluble single-chain variable fragments for anti-CD28, anti-PD1, and anti-PDL1 were generated, as well as versions fused to IL-12 [86]. All of these strains showed improved tumor control and survival in a melanoma mouse model [86].

well as versions fused to IL-12 [86]. All of these strains showed improved tumor control and survival in a melanoma mouse model [86]. Engineering NDV to express a tumor-associated antigen represents another attractive strategy due to its potential to overcome immune tolerance within the context of NDV-induced inflammatory environment [59]. Such strategy was explored with NDV Hitchner B1 expressing an MHC class I restricted epitope of β-galactosidase (β-gal), a model antigen expressed by murine CT26 colorectal carcinoma cells [87]. Intratumoral therapy of CT26 tumor-bearing mice induced a β-gal-specific immune response and significant increase in the number of complete tumor regressions compared to parental NDV. This response was further boosted by co-administration of NDV expressing IL-2, with 90% tumor regression seen [87]. These findings warrant investigation of NDVs expressing other tumor-associated antigens, such as those caused by oncogenic viral antigens, frame shift mutations, and mutated self-antigens, but also highlight that combinatorial strategies using oncolytic viruses targeting different mechanisms (e.g., antigens and adaptive immunity) may be required to achieve Engineering NDV to express a tumor-associated antigen represents another attractive strategy due to its potential to overcome immune tolerance within the context of NDV-induced inflammatory environment [59]. Such strategy was explored with NDV Hitchner B1 expressing an MHC class I restricted epitope of β-galactosidase (β-gal), a model antigen expressed by murine CT26 colorectal carcinoma cells [87]. Intratumoral therapy of CT26 tumor-bearing mice induced a β-gal-specific immune response and significant increase in the number of complete tumor regressions compared to parental NDV. This response was further boosted by co-administration of NDV expressing IL-2, with 90% tumor regression seen [87]. These findings warrant investigation of NDVs expressing other tumor-associated antigens, such as those caused by oncogenic viral antigens, frame shift mutations, and mutated self-antigens, but also highlight that combinatorial strategies using oncolytic viruses targeting different mechanisms (e.g., antigens and adaptive immunity) may be required to achieve optimal anti-tumor response.

optimal anti-tumor response.
