**1. CARs: The Ultimate Tumour Killing Machines?**

An insufficiency of the breadth, or functionality, of the tumour reactive T cell repertoire can be overcome through the use of adoptive T cell therapy (ACT) in which T cells specific to the antigenic constituency of the tumour are generated ex vivo and re-introduced to the patient. These cells may be directly expanded from the tumour [1] or derived from peripheral blood in which novel specificity is conferred by expression of an ectopic T cell receptor (TCR) or a chimeric antigen receptor (CAR) [2]. This allows for T cells to be cultured to possess desirable phenotypes in vitro, and patients to be treated with preconditioning regimes to promote T cell engraftment and minimize suppression. T cells generated through these

**Citation:** Evgin, L.; Vile, R.G. Parking CAR T Cells in Tumours: Oncolytic Viruses as Valets or Vandals? *Cancers* **2021**, *13*, 1106. https://doi.org/10.3390/cancers 13051106

Academic Editors: Antonio Marchini, Carolina S. Ilkow and Alan Melcher

Received: 29 January 2021 Accepted: 3 March 2021 Published: 5 March 2021

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means have led to a large subset of complete responses in otherwise treatment refractory disease, with CAR T cells in particular experiencing unprecedented success against B lymphoid cancers [3].

While TCRs recognize intracellular antigens presented in the context of the major histocompatibility complex (MHC), the synthetic CAR confers specificity in an MHC unrestricted manner to cell surface, and now recently, soluble antigens [4]. The CAR design is modular with each domain contributing to the resulting functional outcome. In its most basic configuration, the CAR molecule is composed of an extracellular antigen binding domain (most commonly an scFv), an extracellular hinge region, a transmembrane domain, and an intracellular signaling domain (including the CD3ζ and costimulatory domains) [5].

The signaling region of the earliest iterations of CARs comprised only the CD3ζ endodomain and triggered effector function but had limited therapeutic potential [6,7]. A multi-step activation model is required to mount effective T cell responses, with signal one being derived from the TCR, signal two from co-stimulatory ligation, and signal three from cytokine exposure. Co-stimulatory domains have thus been included proximal to CD3ζ in second generation constructs to promote persistence and anti-tumour activity [8–10]. Third and fourth generation CARs include two or more co-stimulatory domains, or other transgenes including cytokines [11–14]. CD28 and CD137 (4-1BB) have been most rigorously explored preclinically and are included in the clinically approved constructs axicabtagene ciloleucel (Yescarta), and tisagenlecleucel (Kymriah), respectively [15–17]. Several other costimulatory domains have however been successfully evaluated including CD27, OX40, CD40L, and ICOS, and the incorporation of distinct costimulatory domains has been shown to have profound effects on phenotype, expansion kinetics, metabolism, and persistence [18–22]. Sophisticated synthetic biology circuits have been designed to incorporate additional regulatory tuning and recognition capacity [23]. The functional properties of each domain have been explored and are extensively reviewed elsewhere [24–27].

Retro- or lentiviral vectors are the primary means of introducing stable expression of the CAR into T cells, but random integration into the genome has the potential to lead to insertional mutagenesis and variegated expression of the CAR, thus prompting use of targeted means to introduce the CAR into genomic safe harbours such as the T-cell receptor α constant (TRAC) locus using CRISPR/Cas9 [28,29]. Although the CAR has primarily been introduced into autologous T cells to generate a bespoke patient product, disruption of the TRAC simultaneously enables the use of allogeneic T cells by preventing the development of graft vs. host disease (GVHD) [30].
