Mm01329177_m1 (Thermo Fisher Scientific). data set analyzed for the current study is available from the corresponding author on reasonable request. Abstract Background Oncolytic virus (OV)-based therapies have an emerging role in the treatment of solid tumors, involving both direct cell lysis and immunogenic cell death. Nonetheless, tumor-associated stroma limits the efficacy of oncolytic viruses by forming a barrier that blocks efficient viral penetration and spread. The stroma also plays a critical role in progression, immunosuppression and invasiveness of cancer. Fibroblast activation protein- (FAP) is highly overexpressed in cancer-associated fibroblasts (CAFs), the main cellular component of tumor stroma, and in this study we assessed whether arming oncolytic adenovirus (OAd) with a FAP-targeting Bispecific T-cell Engager (FBiTE) could retarget infiltrated lymphocytes towards CAFs, enhancing viral spread and T cell-mediated cytotoxicity against the tumor stroma to improve therapeutic activity. Methods The bispecific T-cell Engager against FAP was constructed using an anti-human CD3 single-chain variable fragment (scFv) linked to an anti-murine and human FAP scFv. This FBiTE was inserted in the oncolytic adenovirus ICOVIR15K under the control of the major late promoter, generating the ICO15K-FBiTE. ICO15K-FBiTE replication and potency were assessed in HT1080 and A549 tumor cell lines. The expression of the FBiTE and the activation and proliferation of T cells that induced along with the T cell-mediated cytotoxicity of CAFs were evaluated by flow cytometry (NSG) mice. Results FBiTE expression did not decrease the infectivity and replication potency of the armed virusFBiTE-mediated binding of CD3+ effector T cells Rabbit polyclonal to IGF1R and FAP+ target cells led to T-cell activation, proliferation, and cytotoxicity of FAP-positive cells FBiTE expression increased intratumoral accumulation of T cells and decreased the level of FAP, a marker of CAFs, in tumors. The antitumor activity of the FBiTE-armed adenovirus was superior to the parental virus. Conclusions Combination of viral oncolysis of cancer cells and FBiTE-mediated cytotoxicity of FAP-expressing CAFs might be an effective strategy to overcome a key limitation of oncolytic virotherapy, encouraging its further clinical development. Electronic supplementary material The online version of this article (10.1186/s40425-019-0505-4) contains Byakangelicin supplementary material, which is available to authorized users. and [14]and enhanced antitumor activity due to FAP depletion (NSG) mice (bred in house). Once tumors reached a median volume of 120?mm3, mice were randomized prior to treatment. To evaluate T-cell trafficking to the tumor, mice bearing A549 tumors were treated intratumorally with PBS, ICO15K, or ICO15K-FBiTE (1??109 vp/tumor). Four days later, 1??107 preactivated GFP- and CBG-luciferase-expressing T cells (LUC-T-cells) were intravenously injected to treated mice. Mice were given an intraperitoneal injection of 15?mg/mL D-luciferin potassium salt solution (Byosinth AG) and imaged daily for 7?days using IVIS Lumina XRMS Imaging System (PerkinElmer). For antitumor efficacy studies, mice were treated intratumorally with PBS or the indicated viruses (1??109 vp/tumor). Tumors were measured twice or thrice a week with a digital caliper and tumor volume was determined with the eq. V (mm3)?=?/6??W2??L, where W and L are the width and the length of the tumor, respectively. Immunohistochemistry To detect FAP and E1A-Adenovirus expression in tumors, immunohistochemistry (IHC) was performed using OCT-embedded sections (5?m thick) of freshly frozen tumor tissues. Sections were fixed with 2% of PFA at room temperature and endogenous peroxidases were blocked by incubation in 3% H2O2. Next, sections were blocked for 1?h with Byakangelicin 10% of normal goat serum diluted in 1% BSA, PBS-Tween. For FAP detection, primary antibody incubation was performed overnight at 4?C using a biotinylated polyclonal sheep anti-human/mouse FAP antibody (5?g/ml) or its isotype sheep IgG (R&D systems) in 5% of goat serum. For adenovirus detection, the primary antibody used was an anti-Ad2/5 E1A antibody (Santa Cruz Biotechnology) diluted 1/200 in PBS. The next Byakangelicin day, sections were incubated with ABC-HRP kit (Vectastain) for 30?min, followed by 5?min incubation with DAKO-DAB substrate (EnVision). Slides were dehydrated.