T cells engineered to express engineered T cell receptor (eTCR) or chimeric antigen receptor (CAR) constructs have become a powerful tool in the field of immunotherapy. As of today, multiple T cell therapies are approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) showing impressive therapeutic effect and curative potential for patients with different types of blood cancers (https://www.cancer.gov/about-cancer/treatment/research/car-t-cells, 2022; Locke et al., 2017; Abramson et al., 2021; Anderson Jr et al., 2021; Berdeja et al., 2021; Moretti et al., 2022). While it's the midterm goal to make CAR T cell therapy more accessible by lowering manufacturing cost and to broaden its applicability to new tumor entities including solid tumors, the field is also refining techniques to engineer CAR T cells to produce safer and more potent cell products. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas may pave the way to this sophisticated and safer gene engineering and holds the promise to revolutionize future cell manufacturing.

More advanced engineered cell therapies employ either CARs with multiple targeting domains, additional genes to overcome the immunosuppressive tumor microenvironment in solid tumors, or genes designed to mitigate host rejection of allogeneic cell therapies (Moretti et al., 2022; Hawkins et al., 2021). These payloads are considerably larger than CARs employed in approved therapies and are typically delivered to targeted genomic loci using CRISPR/Cas-mediated editing via either adeno-associated virus (AAV) or dsDNA/ssDNA non-viral delivery templates. The packaging limit of AAV is ~5 kb and non-viral ssDNA and dsDNA templates introduce higher toxicity and lower efficiency as payload size increases. Although lentivirus (LV) has a relatively large payload capacity (up to 9 kb), it is not compatible with targeted delivery to a specific locus. Techniques that deliver similar performance with a smaller genomic payload could therefore improve both the potency and manufacturability of these more advanced engineered cell therapies. Careful selection of the targeted integration site may allow significantly reduction of overall construct size if it is placed under control of endogenous promoter and/or downstream signaling domains.

An intuitive target sequence for either eTCR or CAR constructs are TCR/CD3 complex-associated loci. The TCR/CD3 complex is one of the most intricate receptor structures (Call et al., 2002). Each receptor complex consists of six different subunits with TCRα and TCRβ chains being responsible for antigen recognition via peptide MHC (pMHC) binding (Germain, 2001; Garcia et al., 1996). The TCRαβ subunits are associated with CD3 hetero or homodimers formed by CD3εγ, CD3εδ, and CD3ζζ to initiate the downstream T cell signaling cascade (Edge, 2017). It has been shown that targeting a CAR or eTCR construct into the endogenous TCR locus optimizes their expression, safety, and function (Müller et al., 2021). Additionally, it was demonstrated that fusion molecules combining CARs scFv fragment and TCR/CD3 complex can replace full-length CARs (Baeuerle et al., 2019; Liu et al., 2021). Similarly, the eTCR or CAR sequence can be minimized to the antigen-recognition site only (either TRAV and TRBV for eTCR or VH and VL scFv sequence for CAR, respectively), if it is placed into TCR locus itself to take advantage of endogenous intracellular TCR signaling pathways (Mansilla-Soto et al., 2022; Eyquem et al., 2017).

Here, we demonstrate that the anti-CD19 scFv of a CAR construct can be integrated into the CD3ε locus generating a CAR-CD3ε fusion protein which preserves the native TCR structure. Moreover, by using the endogenous promoter and signaling cascade we reduce the genetic payload of the CAR to its minimum, the antigen-binding site. In this manuscript, we present the proof-of-concept data for this minimal CAR construct (miniCAR) and evaluate its benefits in both in vitro and in vivo studies.