*5.1. Arming with Transgenes to Amplify Tumor Lysis*

Once the limitations of using first-generation oncolytic viruses as monotherapy became apparent, transgenes were introduced into existing oAd platforms with the intention to amplify tumor lysis and viral spread. When expressed in cells, the herpes simplex virus thymidine kinase (HSV-tk) gene converts a non-toxic prodrug ganciclovir (GCV) into a toxic agent, which is also distributed to neighboring cells to cause bystander cytotoxicity. Application of a E1B-55k-deleted oAd expressing HSV-tk and GCV improved survival of human colon carcinoma xenografts in mice [62]. Furthermore, oAds expressing HSV-tk and cytosine deaminase have been generated for the treatment of prostate cancer [63]. These oAds have also been used to deliver further cytotoxic or immunostimulatory payloads such as adenovirus death protein (ADP) or interleukin 12 (IL-12), respectively [64,65]. Ad5-yCD/mutTKSR39rep-mIL12, which expresses murine IL-12, improved local and metastatic tumor control in a preclinical prostate adenocarcinoma model accompanied by only mild local inflammation. The corresponding oAd expressing human IL-12 is being investigated in clinical trials.

The tumor necrosis factor- (TNF)-related apoptosis-inducing ligand (TRAIL) has been used in oAds with serotype 5/35 chimeric fibers. In vitro, Ad5/35.IR-E1A/TRAIL showed efficient virus spread and induction of apoptosis. Systemic administration eliminated preestablished liver metastasis in mice [66]. Fernández-Ulibarri et al. developed an oAd expressing a soluble RNase onconase fused to a tumor ligand (ONCEGFR). Upon internalization, the molecule induces tumor cell death through RNA degradation [67].

A critical aspect of arming with cytotoxic transgenes is the non-virus mediated cell killing, which may affect the productivity of viral infection [68]. It is also unclear how non-viral cell death affects the induction of tumor-directed immune responses.

Some monoclonal antibodies (mAb) against growth factor receptors are approved anticancer drugs. When systemically applied, these immunotherapies cannot exploit their full potential because of poor tumor penetration and side effects through normal tissue exposure. Taking advantage of a clinically established antibody against human epidermal growth factor receptor 2 (HER2), a full-length trastuzumab-expressing oAd has been constructed [69]. Ad5/3-∆24-tras showed improved cytotoxicity in a panel of HER2 + cell lines and enhanced antitumor efficacy in a xenograft model of gastric cancer. Viral oncolysis by Ad5/3-∆24-tras activated CD11c + DCs in lymph nodes in a NK cell-dependent manner. The Fc-terminus of the antibody also labels target cells for recognition by innate immune cells, which may induce antibody-dependent cell-mediated cytotoxicity (ADCC). This approach therefore combines direct antitumor activity and the engagement of additional immune effector mechanisms.
