NG-641 is a modified variant of enadenotucirev (EnAd), an Ad11p/Ad3 chimeric group B adenovirus, which retains all the functional properties of enadenotucirev while also expressing transgenes designed to target breakdown of the stromal barrier and immune suppression within the tumor microenvironment. Enadenotucirev has potent and selective anti-tumor activity against a range of epithelial cancer cells, with a blood stability profile that enables systemic dosing; it has now been administered intravenously to over 100 cancer patients. As an approach to immunogene therapy targeting stromal rich tumors, we have created a transgene-modified variant of EnAd expressing a bi-specific T-cell activator molecule (FAP-TAc) recognizing fibroblast activating protein (FAP) on cancer associated fibroblasts (CAFs) and CD3 on T-cells. Production of FAP-TAc by virus infected tumor cells should lead to T-cell mediated killing of CAFs and thus modification of the tumor microenvironment to drive effective anti-tumor immunity. This local production approach also bypasses potential delivery and safety issues associated with systemic dosing of such molecules. To enhance the activity of the FAP-TAc molecule, particularly in tumors with poor immune cell infiltration (“excluded” or “immune desert” phenotypes), NG-641 also encodes the transgenes CXCL9, CXCL10 and IFNα to recruit T-cells and enhance the overall immune response and cancer cell killing.

Initial studies with different viruses expressing FAP-TAc alone showed that FAP-TAc activity generated by NG-641 infection was essentially the same as that with viruses bearing only the FAP-TAc transgene. Following infection of tumor cells with NG-641, the virus selectively secretes functional FAP-TAc molecules, as determined in cocultures of FAP+ fibroblasts and T cells. Production of CXCL9, CXCL10 and IFNα was confirmed by ELISA assays, with functionality evaluated by reporter cell, FACS and cell migration assays. T cell-activation mediated by FAP-TAc lead to cytokine secretion and cytotoxicity towards the fibroblasts, both of which were enhanced by expression of the IFNα transgene. Studies with primary human tumor samples (containing tumor cells, CAFs and infiltrated immune cells) also demonstrated potent activation of the endogenous T-cells, indicating that virus produced FAP-TAc is a potent T-cell activator despite suppressive influences of the tumor microenvironment.

In conclusion, we have shown that CAFs can be effectively targeted for T-cell mediated destruction by NG-641, a tumor stroma targeting transgene-bearing oncolytic virus. This is associated with strong activation of endogenous T-cells to kill CAFs even in the presence of an immunosuppressive microenvironment. Systemic dosing of such a virus to patients with stromal rich tumors may provide an effective approach for driving productive anti-tumor immunity.

Citation Format: Brian R. Champion, Matthieu Besneux, Marilena Patsalidou, Ana Silva, Manuela Zonca, Nalini Marino, Gianfranco di Genova, Sam Illingworth, Stefania Fedele, Lorna Slater, Fred Lilley, Darren Plumb, Katy West, Paul Cockle, Alice Brown. NG-641: An oncolytic T-SIGn virus targeting cancer-associated fibroblasts in the stromal microenvironment of human carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5013.