Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia

Ottaviano G, Georgiadis C, Gkazi SA, Syed F, Zhan H, Etuk A, Preece R, Chu J, Kubat A, Adams S, Veys P, Vora A, Rao K, Qasim W; TT52 CRISPR-CAR group.
Source: Transl Vis Sci Technol
Publication Date: (2022)
Issue: 26;14(668): eabq3010
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® LV-Unit

Capped, polyadenylated, and uridine-modified Cas9 mRNA was produced by TriLink. A CliniMacs Prodigy device with modified T cell transduction program was used (37), and cells were activated with TransAct anti-CD3/CD28 and cultured in TexMACS GMP medium supplemented with 3% human AB serum and human recombinant IL-2 (20 ng/ml). Electroporation used a customized Lonza 4D-Nucleofector® LV device. Depletion of residual TCRaß T cells used an antibiotin bead kit. 


Genome editing of allogeneic T cells can provide "off-the-shelf" alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor a chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator-like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3' long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 106 to 2.0 × 106 CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy.