Oncolytic viruses (OVs) encoding a variety of transgenes have been evaluated as therapeutic tools to increase the efficacy of chimeric antigen receptor (CAR)–modified T cells in the solid tumor microenvironment (TME). Here, using systemically delivered OVs and CAR T cells in immunocompetent mouse models, we have defined a mechanism by which OVs can potentiate CAR T cell efficacy against solid tumor models of melanoma and glioma. We show that stimulation of the native T cell receptor (TCR) with viral or virally encoded epitopes gives rise to enhanced proliferation, CAR-directed antitumor function, and distinct memory phenotypes. In vivo expansion of dual-specific (DS) CAR T cells was leveraged by in vitro preloading with oncolytic vesicular stomatitis virus (VSV) or reovirus, allowing for a further in vivo expansion and reactivation of T cells by homologous boosting. This treatment led to prolonged survival of mice with subcutaneous melanoma and intracranial glioma tumors. Human CD19 CAR T cells could also be expanded in vitro with TCR reactivity against viral or virally encoded antigens and was associated with greater CAR-directed cytokine production. Our data highlight the utility of combining OV and CAR T cell therapy and show that stimulation of the native TCR can be exploited to enhance CAR T cell activity and efficacy in mice.

T cells modified to express intelligently designed chimeric antigen receptors (CARs) are exceptionally powerful therapeutic agents for relapsed and refractory blood cancers and have the potential to revolutionize therapy for many other diseases. To circumvent the complexity and cost associated with broad-scale implementation of ex vivo manufactured adoptive cell therapy products, alternative strategies to generate CAR T cells in vivo by direct infusion of nanoparticle-formulated nucleic acids or engineered viral vectors under development have received a great deal of attention in the past few years. Here, we outline the ex vivo manufacturing process as a motivating framework for direct in vivo strategies and discuss emerging data from preclinical models to highlight the potency of the in vivo approach, the applicability for new disease indications, and the remaining challenges associated with clinical readiness, including delivery specificity, long term efficacy, and safety.

Although patients benefit from immune checkpoint inhibition (ICI) therapy in a broad variety of tumors, resistance may arise from immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, OVs could potentially restore ICI responsiveness via recruitment, priming, and activation of anti-tumor T cells. Here we find that on the contrary, an oncolytic vesicular stomatitis virus, expressing interferon-ß (VSV-IFNß), antagonizes the effect of anti-PD-L1 therapy in a partially anti-PD-L1-responsive model of HCC. Cytometry by Time of Flight shows that VSV-IFNß expands dominant anti-viral effector CD8 T cells with concomitant relative disappearance of anti-tumor T cell populations, which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, combination OV and anti-PD-L1 therapeutic benefit could be restored. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, through encoding tumor antigens within the virus, oncolytic virotherapy can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

Oncolytic viruses (OVs) and adoptive T cell therapy (ACT) each possess direct tumour cytolytic capabilities, and their combination potentially seems like a match made in heaven to complement the strengths and weakness of each modality. While providing strong innate immune stimulation that can mobilize adaptive responses, the magnitude of anti-tumour T cell priming induced by OVs is often modest. Chimeric antigen receptor (CAR) modified T cells bypass conventional T cell education through introduction of a synthetic receptor; however, realization of their full therapeutic properties can be stunted by the heavily immune-suppressive nature of the tumour microenvironment (TME). Oncolytic viruses have thus been seen as a natural ally to overcome immunosuppressive mechanisms in the TME which limit CAR T cell infiltration and functionality. Engineering has further endowed viruses with the ability to express transgenes in situ to relieve T cell tumour-intrinsic resistance mechanisms and decorate the tumour with antigen to overcome antigen heterogeneity or loss. Despite this helpful remodeling of the tumour microenvironment, it has simultaneously become clear that not all virus induced effects are favourable for CAR T, begging the question whether viruses act as valets ushering CAR T into their active site, or vandals which cause chaos leading to both tumour and T cell death. Herein, we summarize recent studies combining these two therapeutic modalities and seek to place them within the broader context of viral T cell immunology which will help to overcome the current limitations of effective CAR T therapy to make the most of combinatorial strategies.

The chimeric antigen receptor (CAR) is a synthetic and modular molecule composed of both signaling and non-signaling domains that allows a T cell to recognize cell surface antigens and trigger cytolytic functionality. It is appreciated that the non-signaling structural components, including the linker, hinge, and transmembrane domains, can dramatically alter how the CAR molecule interacts with itself and other endogenous molecules in the immune synapse. Herein, we describe the current understanding of how the structural domains can alter CAR T cell therapeutic efficacy and highlight how knowledge of the target antigen characteristics can inform CAR design choices.

Although immune checkpoint inhibition (ICI) has produced profound survival benefits in a broad variety of tumors, a proportion of patients do not respond. Treatment failure is in part due to immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, we developed a vesicular stomatitis virus expressing interferon-ß (VSV-IFNß) as a viro-immunotherapy against HCC. Since HCC standard of care atezolizumab/bevacizumab incorporates ICI, we tested the hypothesis that pro-inflammatory VSV-IFNß would recruit, prime, and activate anti-tumor T cells, whose activity anti-PD-L1 ICI would potentiate. However, in a partially anti-PD-L1-responsive model of HCC, addition of VSV-IFNß abolished anti-PD-L1 therapy. Cytometry by Time of Flight showed that VSV-IFNß expanded dominant anti-viral effector CD8 T cells with concomitant, relative disappearance of anti-tumor T cell populations which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, the potent anti-viral response became amalgamated with an anti-tumor T cell response generating highly significant cures compared to anti-PD-L1 ICI alone. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, by chimerizing anti-viral and anti-tumor T cell responses through encoding tumor antigens within the virus, oncolytic virotherapy can be purposed for very effective immune driven tumor clearance and can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.