Genome-edited allogeneic donor "universal" chimeric antigen receptor T cells

Abstract

αβ T cell receptor (TCRαβ) T cells modified to express chimeric antigen receptors (CAR), are now available as authorized therapies for certain B-cell malignancies. However the process of autologous harvest and generation of patient-specific products is costly, with complex logistics and infrastructure requirements. Premanufactured banks of allogeneic donor-derived CAR T cells could help widen applicability if the challenges of HLA-mismatched T-cell therapy can be addressed. Genome editing is being applied to overcome allogeneic barriers, most notably, by disrupting TCRαβ to prevent graft-versus-host disease, and multiple competing editing technologies, including CRISPR/Cas9 and base editing, have reached clinical phase testing. Improvements in accuracy and efficiency have unlocked applications for a wider range of blood malignancies, with multiplexed editing incorporated to target HLA molecules, shared antigens and checkpoint pathways. Clinical trials will help establish safety profiles and determine the durability of responses as well as the role of consolidation with allogeneic transplantation.

Graphical abstract

Figure 1.

Genome-editing platforms for T-cell modification. (A-D) Editing tools for highly specific dsDNA cleavage comprise either protein-based DNA recognition molecules including (A) homing endonucleases (meganuclease), (B) zinc finger nucleases, (C) TALENs, (D) RNA-guided nucleases exemplified by CRISPR/Cas9. After DNA cleavage, repair by nonhomologous end joining offers the prospect of gene-knockout with indel signatures and alternative homologous repair based repair, which can be exploited for site-specific transgene insertion. The latter requires delivery of template DNA flanked by homology arms and allows placement of CAR genes under the transcriptional control of endogenous transcriptional machinery. Alternatively, (E) base editors use nickase-restricted Cas9 variants, fused to cytidine deaminase or adenosine deaminase for highly targeted C>T or A>G base conversion. Cytidine base editor (CBE) has been used for multiplexed gene knockout, avoiding translocations usually encountered following nuclease activity. (F) Emerging prime editing offers the prospect of gene editing through localized template repair by fusing reverse transcriptase to deactivated Cas9. dsDNA, double-stranded DNA.

Figure 2.

Concept of banked universal CAR T cells. (A) CAR T cells can be generated from healthy allogeneic donors for use in multiple recipients after genome editing to remove endogenous TCRαβ to prevent GVHD, disruption of HLA to reduce rejection or removal of CD52 to allow cells to persist in the presence of alemtuzumab, a serotherapy used as a part of augmented lymphodepletion. (B) Therapeutic effects sufficient to induce molecular remission are achievable within a period of 2 to 4 weeks, offering a bridge of consolidation with allogeneic SCT.

Figure 3.

Advanced engineering strategies. Combining CAR transfer with genome editing offers the prospect of enhanced CAR T cells. Viral vector CAR transduction has been combined with co-expressed guide RNAs and electroporation of mRNA coding for Cas9 or base editor (BE). Alternatively, Cas9/guide RNP complexes offer efficient editing, and a route to site-specific transgene insertion by homologous recombination if a suitable CAR template is provided. Other advances include the ability to remove shared antigens such as CD7 that would otherwise result in T-cell fratricide during manufacture, and simultaneous manipulation of checkpoint pathways to address exhaustion and promote activity.