*5.7. Arming with T Cell Engager Proteins*

A promising strategy to redirect T cells to cancer cells are bispecific T-cell engagers (BiTEs) [121]. BiTEs are composed of a tumor ligand and a single-chain antibody fragment, which facilitates binding to CD3. BiTE-mediated clustering of tumor cells and T cells leads to T-cell activation and antitumor cytotoxicity thereby circumventing T-cell receptor (TCR)-mediated antigen recognition. However, the side effects of BiTEs can be considerable and therapeutic success in solid tumors has been rather limited. OVs armed with BiTEs can warrant high BiTE levels in tumor tissue and thus optimizes the ratio of on-target/off-tumor toxicity. Furthermore, viral tumor inflammation promotes intratumoral T-cell infiltration and thus provides an appropriate T-cell pool for BiTE-mediated T-cell retargeting. Fajardo et al. engineered an oAd to express an EGFR-BiTE (ICOVIR-15K-cBiTE) [122]. After expression in cancer cells, the BiTEs activated T cells in PBMCs in vitro. In murine xenograft models, ICOVIR-15K-cBiTE supported tumor infiltration and persistence of adoptively transferred T cells. ICOVIR-15K-cBiTE was also employed to overcome the limits of a CAR T-cell therapy because the EGFR-BiTE was able to redirect T cells against tumor cells that had lost the recognition antigen of the CAR [123]. Freedman et al. constructed a variant of enadenotucirev (EnAd) expressing a BiTE which targets epithelial cell adhesion molecule (EpCAM) [124]. The authors showed that crosslinking of EpCAM-expressing target cells and PBMC-derived T cells activated both CD8 and CD4 T cells. Furthermore, T cells in ascites fluid from cancer patients were activated by the virus-encoded BiTE and EpCAM-positive tumor cells were successfully depleted.

The use of BiTEs targeting components of the tumor microenvironment is a promising approach to reverse tumor immunosuppression. Freedman et al. modified EnAd to express a fibroblast activation protein (FAP)-targeting BiTE to redirect T cells to cancer-associated fibroblasts (CAFs) [125]. Treatment of biopsies of ascites or solid prostate cancer tissue samples with FAP-BiTE- expressing variant of EnAd was capable of activating tumor-infiltrating PD1<sup>+</sup> T cells to kill CAFs. This in turn interfered with CAF-associated immunosuppression and resulted in an upregulation of proinflammatory cytokines, increased the presentation of tumor antigen, and finally led to improved T cell function. A comparable strategy has been pursued by generating the oAd ICO15K-FBiTE [126]. In a xenograft model, the expression of FBiTE led to an increased intratumoral T-cell accumulation and decreased the intratumor levels of FAP.

Tumor-associated macrophages (TAMs), and particularly the M2-polarized subset, contribute to immunosuppression. To deplete cancer-promoting TAMs and to reverse immunosuppression, Scott et al. recently developed EnAd-variants equipped with bi- and trivalent T-cell engagers targeting CD206 or folate receptor β on M2-like macrophages [127]. By detecting selective T-cell cytotoxicity against M2-TAMs in cancer patient biopsies, they could demonstrate that these BiTEs allow selective depletion of tumor-promoting TAMs whilst sparing those with potential antitumor features.
