injected with 100 g TRS-H. T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo. Subject terms:Immunization, Cellular immunity, Tumour immunology == Introduction == Clinical therapies that aim at antibody-mediated redirection of T-cells towards cancer cells are a successful therapeutic strategy for liquid cancers with a few registered products available for patients (e.g. blinatumomab, mosunetuzumab and Tebentafusp) [13] or are in process of approval or clinical evaluation [4]. All these make use of the principle to engage CD3+T-cells in Liraglutide the killing of cancer cells and bypass the need for tumor-specific T-cells, however have not been as potent for solid tumors as for hematological cancers [4,5]. The lower efficacy towards solid tumors can be attributed to several challenges, such as, the quality of the tumor-infiltrating T-cells (TILs) in the tumor microenvironment (TME) and on-target off-tumor toxicities caused by low expression levels of the tumor-associated antigens (TAAs) expressed on healthy tissues [4,68]. Moreover, targeting of all CD3 expressing T-cells may lead to an important side effect: the unwanted and excessive release of cytokines called the cytokine-release syndrome (CRS) [9]. Therefore, it may be beneficial to redirect a more limited group of CD8+T-cells that are known to be very potent instead of the entire CD3+T-cell population. Some viruses are widely prevalent in the human population such as the human herpesviruses cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DLL1 [10,11]. These Liraglutide viruses have coevolved with humans and can persist as a lifelong, (largely) asymptomatic, latent infection with occasional reactivations [12]. In EBV and CMV infections, T-cell immunity plays a pivotal role in the clearance of the virus and can lead to an unusual large number of potent CD8+T-cells [11,13]. Virus-specific T-cells are present in the TME but can only act as bystanders as there are no target antigens expressed by the tumor [14,15]. It was previously shown that intratumoral injection of virus-derived peptides can overcome the immunosuppressive TME and can trigger an effective antiviral T-cell response against the tumor [16]. Therefore, this group of T-cells might be attractive candidates to be redirected towards the cancer cells. To be able to redirect those virus-specific T-cells, immunogenic EBV or CMV T-cell major histocompatibility class I (MHC-I) epitopes were conjugated to tumor-targeting antibodies. With several antibody conjugation and delivery strategies it was proven that antibodies can efficiently deliver viral epitopes to cancer cells [1721] and we recently compared three approaches to generate these antibody-epitope conjugates (AECs) for cetuximab (CTX) and trastuzumab (TRS) [22]. It was demonstrated that AECs generated by means Liraglutide of a genetic fusion resulted not only in the most well-defined, but also the AECs with the highest stability. In this study, we explored the possibilities to increase the epitope-to-antibody ratio (EAR) and investigated the in vivo functionality of the genetically fused AECs in a xenograft mouse model. == Materials and methods == == Antibodies and tetramers == Cetuximab (CTX) and pembrolizumab were obtained from Merck (Germany, Darmstadt). Trastuzumab (TRS) and all genetically modified antibodies were produced at Genmab via transient expression in ExpiHEK293 FreeStyle cells as described before [23] and purified by Protein A affinity chromatography. If required, protein aggregates were removed via Size Exclusion Chromatography to yield protein product witha> 95% monomeric content as analyzed on HPLC-SEC. All antibodies were stored in phosphate-buffered saline (PBS). For the AECs used in vivo experiments mutations were introduced (P329G, L234A, and L235A) to disrupt possible interactions with Fc-receptors [24]. The following antibodies were used for flow cytometry: cetuximab, trastuzumab, goat anti-human IgG-A488 (Jackson immunoResearch, UK, Cambridgeshire, #109-546-098) or -PE (Jackson immunoResearch, #109-116-098), goat anti-mouse-FITC (Jackson immunoResearch, 115-096-062), mouse anti-HLA-A2 (produced in-house from clone BB7.2 [25]), mouse anti-human EGFR (Santa Cruz Biotechnology, #sc-120), and mouse anti-human Her2 (R&D, UK Liraglutide Abingdon, #MAB9896). Peptide MHC multimers complexes (tetramers) were generated in-house as described before [26] and labeled with PE-conjugated streptavidin. == Cell lines and cell culture == All adherent cell lines Liraglutide were cultured.
