**8. Graffitiing Antigenic Specificity onto Tumours**

Oncolytic viruses exhibit a range of infectivity within tumours suggesting that highly susceptible cells would be killed, whilst cells refractory to oncolysis, but in which viral genes are expressed, could be targeted by CAR T. In solid tumours where no specific CAR targets have been identified, OVs could be used to deliver ectopic antigens to tumour cells [101,102]. This strategy could also be applied to re-target CAR T cells to antigen negative tumour cells, a common mechanism of treatment failure [103,104]. Proof of concept studies from Aalipour et al. and Park et al. used oncolytic vaccinia viruses to decorate cancer cells with CD19 and demonstrated targeting of CD19 CAR T to previously unrecognized tumour cells both in vitro and in vivo. Although CD19 delivery leads to unnecessary induction of B cell aplasia, the modular nature of the approach suggests that it could be extended to other antigens. One significant limitation would be heterogenous expression of the CAR target antigens and incomplete targeting by either modality. However, both OVs and CAR T have been demonstrated to elicit endogenous T and B cell responses to tumour associated antigens (TAA) through epitope spreading [80,105–107], and indeed Park et al. show that mice which were cured by combination therapy were partially protected against re-challenge with parental tumours which did not express the CAR antigen.

The immune adjuvanticity and transgene expression capabilities of OVs allow them to act as strong vaccines. TAAs can be expressed to a high degree in tumour cells, and upon cell lysis, taken up by dendritic cells or other phagocytic cells for presentation to T cells. Viruses may also initiate abortive non-lytic infections in non-transformed cells, such as APCs, which lead to the expression of viral genes and the subsequent priming of T cells. Thus, oncolytic vaccination against single TAAs, or even a library of antigens, provides stronger anti-tumour therapy than parental strains [108–110]. The size of the T cell pool is further magnified through the use of heterologous vectors for priming and boosting which encode the same antigen [111–113], or by adoptive transfer of transgenic antigen specific T cells [114–116]. Although CAR modified T cells acquire a novel specificity, they also retain the specificity conferred by their native TCR, and parallel work has sought to boost CAR T cells through the TCR. Transgenic T cells expressing both a CAR specific to HER2 and bearing a TCR specific for either the gp100 or OVA antigens are expanded by treatment with vaccinia expressing the cognate epitope, leading to accumulation of T cells in the tumour and eradication of large established tumours [117]. Clinical evaluation of a mixed infusion product containing Epstein-Barr Virus (EBV) TCR specific and open TCR repertoire GD2 CAR T demonstrated that virus specific CAR T circulate at a higher frequency than the counterpart control T cells [7]. Viral reactivation or vaccination further supports the expansion of virus specific CAR T cells [118,119].

Bispecific T-cell engager (BiTE) technology links an anti-CD3 scFv to a tumour antigen specific scFv, and thus bypasses both the TCR-MHC interaction and the CAR to engage effector function. In this way, OVs and CAR T cells engineered to express BiTEs can redirect endogenous T cells, or CAR T themselves, against a second tumour antigen specificity [120–123]. Putting these platforms together, Wing et al. demonstrated that oncolytic adenovirus expressing an EGFR-targeting BiTE improved the activation, proliferation, and cytokine production of CART cells targeting the folate receptor alpha (FR-α) and can help to overcome antigen heterogeneity [124]. Use of the virally expressed BiTE redirected both CAR T and CAR negative nonspecific T cells and provided superior anti-tumour

efficacy compared to each monotherapy. A multiplex strategy has also combined BiTE expression (specific to CD44v6) with cytokine (IL12) and checkpoint (anti-PDL1) delivery using the oncolytic and helper dependent adenovirus system in conjunction with HER2 CAR T to combat several mechanisms of tumour escape simultaneously [125].
