Therapies with genetically modified T cells that express chimeric antigen receptors (CARs) specific for CD19 or B cell maturation antigen (BCMA) are approved to treat certain B cell malignancies. However, translating these successes into treatments for patients with solid tumours presents various challenges, including the risk of clinically serious on-target, off-tumour toxicity (OTOT) owing to CAR T cellmediated cytotoxicity against non-malignant tissues expressing the target antigen. Indeed, severe OTOT has been observed in various CAR Review articlewe examine the implications of OTOT on the development of CAR T cell therapies targeting solid tumours, summarize OTOT evidence in preclinical and clinical studies, and discuss advances in CAR T cell engineering that might help to overcome OTOT in the clinic. Risks and mechanisms of OTOTOTOT stems from CAR T cell-mediated recognition and lysis of nonmalignant tissues expressing the target antigen, potentially causing severe adverse events 23,26 . Upon recognition of a target antigen, CAR T cell activation leads to the formation of an immune synapse between the CAR and the target cell 27 , triggering effector functions (Fig. 2). The release of perforin and granzymes 28 is assumed to be a principal mechanism of CAR T cell-mediated cytotoxicity. However, other mechanisms, such as upregulation of T cell-surface molecules to induce target apoptosis (such as FAS ligand) 29 or secretion of cytokines, including IFNγ and/or TNF, may also contribute to tissue destruction [29][30][31] (Fig. 2).To generate CAR T cells that are both safe and effective in patients with solid tumours, target antigen selection is crucial. Optimal antigen candidates, referred to as neoantigens, should be exclusively expressed on malignant cells and not on non-malignant cells. Such antigens could arise from tumour-specific non-synonymous mutations, insertions or deletions that alter the amino acid sequence of cell-surface proteins, aberrant expression of oncofetal antigens, or tumour-specific posttranslational modifications [32][33][34][35][36][37] . However, cell-surface neoantigens are rare, particularly in tumours with a low mutational burden 38 . EGFRvIII, found in 24-67% of glioblastomas, is one of the few identified examples 39,40 . Consequently, the majority of CAR T cell therapy targets for solid tumours are tumour-associated antigens (TAAs) that are also expressed on non-malignant tissues (Fig. 3).

Background Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. Results Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHC-negative CAR T cells resist allogeneic T cell targeting in vitro. Conclusions We outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics.

Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out (KO) or targeted transgene knock-in (KI). However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted KI and Cas9-derived base editing technology for DSB free KOs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two KOs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduced translocations to 1.5% of edited cells. Small insertions and deletion at the base editing target sites indicated guide RNA exchange between the editors. This was overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR KI and a Cas9-derived base editor enabled the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting T cell receptor- (TCR-) and MHC-negative CAR T cells resisted allogeneic T cell targeting in vitro. Thus, we demonstrate a solution for safer multiplex-edited cell products and a path towards off-the-shelf CAR therapeutics.