Genome-Edited T Cell Therapies

Abstract

Purpose of review: Alternative approaches to conventional drug-based cancer treatments have seen T cell therapies deployed more widely over the last decade. This is largely due to their ability to target and kill specific cell types based on receptor recognition. Introduction of recombinant T cell receptors (TCRs) using viral vectors and HLA-independent T cell therapies using chimeric antigen receptors (CARs) are discussed. This article reviews the tools used for genome editing, with particular emphasis on the applications of site-specific DNA nuclease mediated editing for T cell therapies.

Recent findings: Genetic engineering of T cells using TCRs and CARs with redirected antigen-targeting specificity has resulted in clinical success of several immunotherapies. In conjunction, the application of genome editing technologies has resulted in the generation of HLA-independent universal T cells for allogeneic transplantation, improved T cell sustainability through knockout of the checkpoint inhibitor, programmed cell death protein-1 (PD-1), and has shown efficacy as an antiviral therapy through direct targeting of viral genomic sequences and entry receptors.

Summary: The combined use of engineered antigen-targeting moieties and innovative genome editing technologies have recently shown success in a small number of clinical trials targeting HIV and hematological malignancies and are now being incorporated into existing strategies for other immunotherapies.

Keywords: CRISPR/Cas9; Chimeric antigen receptors; Clinical trials; Genome editing; Immunotherapy; T cell receptors; T cell therapies.

Fig. 1

Genome editing technologies. Introduction of double-stranded breaks enables the formation of Indels in the absence of a suitable repair template, leading to knockout of gene function. Several genome editing technologies are currently available, each with a specific mode of action. a Meganucleases are homing endonucleases that form dimers in order to cleave. The single nuclease domain is made up of the DNA recognition and cleavage domains. (from Bertoni C. Front. Physiol. 2014, 5:148) [10]. b ZFNs require dimerization of two Fok1 domains at targeted loci in order for scission to occur. Each zinc finger contacts three nucleotides of the target sequence. (from Didigu CA, Doms RW. Viruses 2012, 4(2), 309–324) [11]. c TALEN cleavage is also FokI mediated; however, each TALE contains 34 amino acid repeat sequences, with each RVD targeting a single base in the target sequence. (reprinted by permission from Macmillan Publishers Ltd.: Hyongbum K, Jin-Soo K. Nature Reviews Genetics 2014, 15, 321–334) [12]. d CRISPR/Cas9 technology is RNA-guided with Cas9 mediating double-stranded cleavage of the target site. The target site is flanked by a PAM sequence, with double-stranded cleavage occurring three bases upstream from this motif (from Agrotis A, Ketteler R. Front. Genet. 2015, 6:300) [13]

Fig. 2

Structure of TCRs and CARs. a The TCR is comprised of α and β chains that closely associate with the ε-δ-γ- and ζ-chains of the CD3 complex. Antigen-mediated activation of the α/β chains induces phosphorylation of the ITAMs by LCK. Subsequent activation of ZAP-70 and its downstream targets, LAT and SLP-76, induces an intracellular signaling cascade resulting in the upregulation of genes associated with T cell effector function. (reprinted from Lineberry N, Fathman GC: Immunity 2006, 24(5):501–503, with permission from Elsevier) [46]. b Design of the chimeric antigen receptor includes the single-chain variable fragment with antigen-binding affinity, fused to a spacer and transmembrane domain. Effector function is conferred via the TCR CD3ζ domain, while the addition of one (2nd generation) or two (3rd generation) costimulatory domains drives signal activation and amplification of various effector signaling cascades (with permission from Juno Therapeutics: Chimeric Antigen Receptor Technology (CARs) https://www.junotherapeutics.com/our-science/car-technology/) [47]