The scientific work conducted under the ERC POC project led to four series of results. Specifically, (i) it was observed that the HER2-ReHV named R-337 could confer some protection even upon systemic i.v.delivery even when administered to already established metastatic tumors. The developed model consisted of immunocompetent BALB/C mice made transgenic tolerant for the human HER2 (the murine strain was developed in the lab for this purpose). The mice bore lung tumors generated by i.v. injection of CT26-HER2 cells, or subcutaneous CT26-HER2 tumors. The anticancer efficacy of the HER2-ReHV was quantified as decrease in the number of lung nodules, or in the s.c tumor growth, and as immune T response to the tumor (Vannini et al., 2023a). (ii) studies on the blood clearance and biodistribution of systemically-administered HER2-ReHV in the above model were carried out in naïve mice and in mice preimmunized) to HSV (herein IMM), a situation that mimics the scenario in the HSV-seroprevalent human population. While HER2-ReHV only populated the tumor, and spare any other organ or tissue, In IMM mice the quantity of HER2-ReHV that populated the tumor and its antitumor efficacy were low. (iii) Our work ascertained that this was mainly due to Complement and to neutralizing antibodies (NAbs). The deleterious effect of complement was counteracted by means of cobra venom factor, especially in combination with Cyclophospamide. Efforts to remove NAbs from circulation were in part effective (Vannini et al., 2023a). (iv) We further explored to what extent the rapid clearance of the HER2-ReHV was the result of the unspecific absorption of the virus to cells expressing broadly distributed heparan sulphates and chondroitin sulphates (cumulatively GlycosAminoGlycans _GAGs) that act as a sink and drain off virus from circulation. To this end, we engineered a variant of R-337 named R-399 in which glycoprotein C (gC) was mutated so as to decrease its ability to interact with cell GAGs. Such modification slowed the blood clearance of R-399, improved the biodistribution and the systemic anticancer efficacy of R-399. This is the first evidence of the role of GAGs in biodistribution of HSV (Vannini et al., 2023c). Cumulatively, the points (i) to (iv) of the project provided instruments to improve the anticancer efficacy of systemically delivered HER2-ReHV. (v) A further key result was the design, analysis of cancer specificity and in vivo anticancer efficacy of a novel ReHV, named R-421, retargeted to nectin4, a novel member of Tumor Associated antigens, highly expressed in bladder and urethral cancer, in pancreas carcinoma. Our studies showed that R-421 is highly specific for the mentioned human cancers, does not exhibit off tumor and off-target activity, and exerts anticancer efficacy and anticancer vaccination (Vannini et al., 2023b). With respect to A#4, augment of the immunotheraputic response and in situ vaccination we observed that efficacy of HER2-ReHV is increased in combination with checkpoint inhibitors ICIs), irrespective of whether the ICIs are inserted in the viral genome, or are administered in combination regimens. We observed strong in situ vaccination to both the selected tumor antigen (e.g. HER2) and to agnostic tumor antigens.
The business part of the project was quite successful and indeed put the basis for the translation of ReHVs to clinical experimentation (Development) and ultimately for their future commercialization and for societal benefit. Ultimately, this should increase therapeutic options for specific metastatic diseases. In practice, we carefully detailed the plan of the activities required to bring ReHVs to the IND deposit for authorization of clinical experimentation; (ii) carried out a Market analysis finalized to the benchmarking and positioning of ReHVs in the competitive landscape. (iii) Designed a Business Plan and strategies to search for investors/partners and further funding. The overall result of the Business activities is that the University of Bologna, that is the owner of the IP, signed an Agreement with a international biotech company to bring ReHVs to IND deposit, and, subsequently to further development and deposit of protocols for clinical studies.
