Universal' Fratricide-Resistant CAR T Cells Against T Cell Leukemia Generated By Coupled & Uncoupled Deamination Mediated Base Editing

Chimeric antigen receptor (CAR) T cell approaches to target T cell malignancies have been hampered by the fundamental issue of 'T v T' fratricide when T lineage antigens such as CD3 or CD7 are targeted. Genome-editing can be employed to efficiently eliminate expression of shared target antigens, and multiplexed approaches can deliver simultaneous removal of additional molecules relevant to generating 'universal' T cells, such as the αβ T cell receptor, β₂m and CD52, the target antigen for Alemtuzumab. However, these strategies have generally relied on nuclease mediated double stranded DNA cleavage and repair by non homologous end joining (NHEJ), and this can trigger apoptosis and generate predictable and unpredictable chromosomal translocations. Base editing using CRISPR guided chemical deamination offers the possibility of highly precise, seamless, cytidine to uridine deamination (resulting in C→T or G→A substitutions) which can be directed to create stop codons or to disrupt splice donor/acceptor sites. This enables simultaneous genetic disruption of critical antigens or receptors required for the generation of 'off the shelf' cell banks that can be infused in combination without fratricidal effects.

A CRISPR-CAR coupled lentiviral configuration with a single guide RNA (sgRNA) specific for the constant region of TCR β chain (TRBC) was incorporated into the 3'ΔU3LTR of the vector under the control of a minimal U6 pol III polymerase promoter. The configuration supported high level base conversion and efficiently disrupted TCRαβ expression. When followed by stringent magnetic bead mediated depletion of residual TCRαβ T cells, highly homogenous CAR⁺TCR^- populations with <1% residual TCRαβ T cells were obtained, an important consideration in the application of mismatched allogeneic T cells. Combinational delivery during electroporation of additional, uncoupled sgRNAs against additional molecules relevant to overcoming HLA barriers supported up to 92% triple knockout of TCR/CD52/β₂M in CAR expressing cells.

CRISPR guide RNAs targeting CD7 and/or TRBC (for TCR/CD3 disruption) were delivered by electroporation to primary T cells in combination with codon-optimized (co) base editor 3 (BE3) or coCas9 mRNA, ahead of transduction with sin-lentiviral vectors expressing CARs against CD3 or CD7. Simultaneous disruption of TCR/CD3 and CD7 was confirmed by flow cytometry and verified by direct sequencing of the target loci. Efficient multiplexed editing enabled co-culture of T cell populations expressing anti-CD3 and anti-CD7 specific CARs, with fratricidal elimination of residual non-edited populations. Chromium release and flow based cytotoxic functional responses were similar between cells generated using coCas9 and coBE3. However, digital droplet PCR of coCas9 edited cells detected low frequency (1-3%) predicted translocation events, while these were barely detectable in base edited cells. In vivo anti-leukemic functionality of base edited CAR T cells was verified by serial imaging of NOD/SCID/γc mice engrafted with luciferase labelled Jurkat T cells modified to express CD3, or CD7, alone or in combination. Effector inoculation with T cells expressing anti-CD3 and anti-CD7 CARs inhibited leukemic expansion and luciferase signal.

Additional characterisation of T cells following multiple deamination mediated editing is underway to investigate wider RNA and DNA effects of chemical deamination. The strategies are readily scalable through the adaption of existing semi-automated manufacturing processes. A time limited therapeutic application of 'universal' anti-T CAR T cells is planned to deliver leukemic clearance and deep molecular remissions ahead of conditioning and programmed allogeneic stem cell transplantation for donor derived multilineage immune reconstitution.