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 α 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 × 10 6 to 2.0 × 10 6 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.
Background 'Off-the-shelf' CAR T cell therapies are being investigated as alternatives to autologous CAR therapy, and can be generated using genome editing from allogeneic donors. Strategies to address HLA barriers include disruption of T cell receptor expression to prevent GVHD and removal of cell surface HLA expression to obviate host mediated rejection, as well as removal of CD52 to create a survival advantage in the presence of Alemtuzumab. In this study, lentiviral mediated CAR19 expression is linked to CRISPR editing through the incorporation of CRISPR guide RNA sequences within the vector 3'Long terminal repeat (LTR). The trial is in progress using pre-manufactured batches of TT52CAR19 T cells. Investigational medicinal product (IMP) Three allogeneic non-HLA matched donor derived CAR19 T cell banks were manufactured from steady state apheresis harvests from registry volunteer donors, using a semi-automated process under compliant conditions. Cells were activated with anti-CD3/CD28 (Transact) reagent and transduced with a lentiviral vector, TT52CAR19, and dual guide sgRNA cassettes targeting the T cell receptor alpha chain (TRAC) and CD52 loci. Next electroporation of Cas9 mRNA elicited transient genome editing, and automated magnetic bead depletion removed remaining TCRαβ+ T cells (mean 0.7%) and enriched CAR19+ T cells (mean 92.8%). Cells were cryopreserved in aliquots suitable for dose-banding and subjected to release testing, including flow cytometry, quantification of copy number (mean 3.83) and exclusion of replication competent lentivirus. Potency of each bank was confirmed in human:murine chimera experiments. Trial sponsorship & approvals The study is open at a single site and is sponsored by Great Ormond Street Hospital NHS trust and is supported by the Medical Research Council and National Institute for Health Research. Clinical trial authorisation was awarded by the MHRA after expert review, and ethical approval including gene therapy advisory committee (GTAC) review, and health research authority (HRA) approval. The study is open label, single arm and non-randomised. Study aims and Objectives The study aims to establish the safety and feasibility of TT52CAR19 for the induction of molecular remission in children with relapsed /refractory CD19-positive B-cell acute lymphoblastic leukaemia (B-ALL) within 28 days, ahead of planned allogeneic haematopoietic stem cell transplantation (allo-SCT). Assessments include time to remission, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of TT52CAR19 cells and tracking of immune recovery after allo-SCT is monitored. Recording of complications and toxicities, including possible genotoxic side effects from CRISPR/Cas9 modification provides safety profile information. Eligibility, infusion and outcomes The study plans to treat 10 children aged between 6 months and < 18 years with CD19+ B-ALL quantified at >10 -4 in marrow (by flow or PCR) who are ineligible for autologous therapy. To date 2/4 children screened were found eligible and proceeded to lymphodepletion comprising Fludarabine, Cyclophosphamide and Alemtuzumab followed by a single infusion of 0.8-2.0x10 6 CAR19 T cells and a maximum of 5x10 4/kg TCRαβ T cells. In both cases, there was no GVHD or CRS >grade 1 and recovery of neutrophil counts by d28. Molecular remission was achieved in one child, where TT52CAR19 cells persistence was tracked by chimerism and copy number until further conditioning and allo-SCT. This child remains in remission >6 months later. Conclusions Feasibility of pre-manufacturing off-the-shelf CRISPR/Cas9 edited CAR19 T cells is demonstrated and the trial has provided first in human safety data and preliminary indications of potent anti-leukaemic activity in one of two subjects dosed. Disclosures Qasim: Autolus: Current equity holder in publicly-traded company; Novartis: Honoraria; Servier: Research Funding; Tessa: Membership on an entity's Board of Directors or advisory committees.
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. Disclosures Qasim: UCLB: Other: revenue share eligibility; Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding.
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