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Allogeneic chimeric antigen receptor T cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CART have been created using gene editing techniques that induce DNA double-stranded breaks (DSBs), resulting in unintended on-target editing outcomes with potentially unforeseen consequences. Cytosine base editors (CBEs) install C•G to T•A point mutations in T cells with between 90-99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared to CRISPR-Cas9. Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using four simultaneous base edits. We show that CBE, unlike CRISPR-Cas9, does not impact T-cell proliferation, lead to aberrant DNA damage response pathway activation or result in karyotypic abnormalities following multiplexed editing. We demonstrate 7CAR8 to be highly efficacious against T-cell acute lymphoblastic leukemia (T-ALL) using multiple in vitro and in vivo models. Thus, CBE is a promising technology for applications requiring multiplexed gene editing and can be used to manufacture quadruple-edited 7CAR8 cells with high potential for clinical translation for relapsed and refractory T-ALL.

Chimeric antigen receptor (CAR) T-cell therapy can induce durable remissions of relapsed/refractory B-acute lymphoblastic leukemia (ALL). However, case reports suggested differential outcomes mediated by leukemia cytogenetics. We identified children and young adults with relapsed/refractory CD19+ ALL/lymphoblastic lymphoma treated on 5 CD19-directed CAR T-cell (CTL019 or humanized CART19) clinical trials or with commercial tisagenlecleucel from April 2012 to April 2019. Patients were hierarchically categorized according to leukemia cytogenetics: High-risk lesions were defined as KMT2A (MLL) rearrangements, Philadelphia-chromosome (Ph+), Ph-like, hypodiploidy, or TCF3/HLF; favorable as hyperdiploidy or ETV6/RUNX1; and intermediate as iAMP21, IKZF1 deletion, or TCF3/PBX1. Of 231 patients aged 1-29, 74 (32%) were categorized as high-risk, 28 (12%) as intermediate, 43 (19%) as favorable, and 86 (37%) as uninformative. Overall CR rate was 94%, with no difference between strata. There was no difference in relapse free survival (RFS, p=0.8112), with 2-year RFS for the high-risk group of 63% (95%CI 52-77). There was similarly no difference seen in OS (p=0.5488) with 2-year OS for the high-risk group of 70% (95%CI 60-82). For patients with KMT2A-rearranged infant ALL (n=13), 2-year RFS was 67% (95%CI 45-99), and OS was 62% (95%CI 40-95), with multivariable analysis demonstrating no increased risk of relapse (HR 0.70, 95%CI 0.21-2.90, p=0.7040), but a higher proportion of relapses associated with myeloid lineage switch, and a 3.6-fold increased risk of all-cause death (95%CI 1.04-12.75, p=0.0434). CTL019/huCART19/tisagenlecleucel are effective at achieving durable remissions across cytogenetic categories. Relapsed/refractory patients with high-risk cytogenetics, including KMT2A-rearranged infant ALL, demonstrated high RFS and OS probabilities at 2 years.