Biology subjects

Preece, R.

Publications and source records attributed to Preece, R..

5 recordsLinked to original sources

bioRxiv2026

Are entirely virus-free CAR T cells as good as lentiviral transduced universal cells?

Chimeric Antigen Receptor (CAR) T cells are now established as therapies for some haematological malignancies. While lentiviral or {gamma}-retroviral vectors are commonly used for CAR delivery due to their efficiency and stable integration, supply constraints have created bottlenecks to wider applications and access. Alternatively, genome editing tools such as CRISPR-Cas9 can insert CAR genes by homology-directed repair (HDR) into specific genomic loci. Universal donor CAR-T cells devoid of endogenous TCR{beta} after CRISPR-Cas9-mediated editing of the T cell receptor alpha (TRAC) locus are being investigated for more cost-effective, off-the-shelf therapies. Targeting insertion of CARs into the TRAC locus places transcription under the control of native regulatory machinery while simultaneously disrupting endogenous TCR{beta}, and this has been reported to reduce exhaustion and extend persistence in modelling studies using humanised mice. We compared anti-CD20 CAR-T cells, generated with CAR inserts at either TRAC or CD3{zeta} loci using entirely virus-free manufacture, and universal CAR20-T cells generated using existing lentiviral procedures and CRISPR/Cas9 knockout. While non-viral cell yields were lower than lentiviral products cytotoxic function in vitro was comparable between groups. Studies in humanised murine models of leukaemia inhibition found non-viral CAR20-T cells were generally less efficacious than LV-CAR20 and exhibited more exhausted phenotypes. Non-viral approaches offer the prospect of sophisticated editing and precise CAR insertion but careful preclinical evaluation and well-designed clinical trials benchmarked against lentiviral approaches are recommended.

molecular biology↗

bioRxiv2025

Allo-defensive, multiplex base-edited, anti-CD38 CAR T cells for "off-the-shelf" Immunotherapy

Chimeric antigen receptor (CAR) T cell therapies are being widely investigated in both autologous and allogeneic settings, with gene editing providing new strategies to address barriers to mismatched cell therapies. Currently universal donor derived T cell therapies require intensive lymphodepletion and are prone to host-mediated rejection. CD38, a transmembrane glycoprotein involved in cell activation and bioenergetics, is a promising immunotherapy target for haematological malignancies. Disruption of CD38 expression using base editing prevented fratricide between T cells expressing anti-CD38 CAR (CAR38). Additional base editing enabled generation of a universal donor CAR38-T cells, devoid of endogenous TCR{beta} and Human Leukocyte Antigen (HLA) molecules after disruption of T Cell Receptor Beta Constant (TRBC), Beta-2 microglobulin (B2M), and Regulatory Factor X5 (RFX5). Removal of cell surface HLA expression enabled evasion of anti-HLA antibodies in sera from sensitised donors and reduced allo-stimulation in mixed lymphocyte cultures (MLCs), while TCR{beta} disruption prevented allo-reactivity. In MLCs, CAR38 expression enabled potent allo-defense activity against CD38+ allo-reactive cells. Multiplex-base-edited CAR38-T cells exhibited antigen-specific anti-leukemic activity against human B, T, and myeloid malignancies and inhibited disease progression in humanised murine xenograft models. CAR38-T cells offer a potent off-the-shelf strategy against CD38+ haematological malignancies and plasma cells associated with autoimmunity.

immunology↗

bioRxiv2024

Base edited "universal" donor CAR T cell strategies for acute myeloid leukaemia

Acute myeloid leukaemia (AML) is often aggressive and life-threatening with limited curative options. Immunotherapies including chimeric antigen receptor (CAR) T cell approaches are under investigation, but high levels of disease heterogeneity remain a major hurdle to achieving durable responses. Targeting of multiple surface antigens may ensure complete immunological coverage of leukemic blast populations, but such antigens are often also present on healthy haematopoietic populations. To address likely aplasia, strategies can be designed to bridge CAR T cell therapies to allogeneic stem cell transplantation (allo-SCT), as demonstrated in recent anti-CD7 CAR T cell studies. Here we report that monotherapy using base edited (BE) universal donor CAR T cells against CD33, CLL-1, or CD7 delivered inhibition of AML in immunodeficient mice when antigen expression was homogenous, but combined use of BE-CAR33 and BE-CARCLL-1 T cells was required to address heterogenous CLL-1-/+CD33-/+ disease. We also demonstrate that removal of shared CD7 antigens enabled compatibility of BE-CAR33 with BE-CAR7 T cells, including in a patient-derived xenograft (PDX) model of AML. Therapeutic strategies envisage pick and mix applications of base edited universal CAR T cells in combinations determined by patient-specific antigen profiles. Such approaches also offer the possibility of deep, cell-based, de-bulking and preparative conditioning ahead of allo-SCT followed by donor-derived reconstitution.

immunology↗

bioRxiv2024

CD62L-selected umbilical cord blood universal CAR T cells

Umbilical cord blood (UCB) T cells exhibit distinct naive ontogenetic profiles and may be an attractive source of starting cells for the production of chimeric antigen receptor (CAR) T cells. Pre-selection of UCB-T cells on the basis of CD62L expression was investigated as part of a machine-based manufacturing process, incorporating lentiviral transduction, CRISPR-Cas9 editing, T-cell expansion and depletion of residual TCR{beta} T cells. This provided stringent mitigation against the risk of graft versus host disease (GVHD), and was combined with simultaneous knockout of CD52 to enable persistence of edited T cells in combination with preparative lymphodepletion using Alemtuzumab. Under compliant manufacturing conditions, two cell banks were generated with high levels of CAR19 expression and minimal carriage of TCR{beta} T cells. Sufficient cells were cryopreserved in dose-banded aliquots at the end of each campaign to treat dozens of potential recipients. Molecular characterisation captured vector integration sites and CRISPR editing signatures and functional studies, including in vivo potency studies in humanised mice, confirmed anti-leukaemic activity comparable to peripheral blood-derived universal CAR19 T cells. Machine manufactured UCB derived T cells banks offer an alternative to autologous cell therapies and could help widen access to CAR T cells.

molecular biology↗

bioRxiv2020

Base-edited CAR T Cells for combinational therapy against T cell malignancies

Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by T v T fratricide against shared antigens such as CD3 and CD7. Genome-editing can overcome such hurdles through targeted disruption of problematic shared antigens. Base editing offers the possibility of seamless disruption of gene expression through the creation of stop codons or elimination of splice donor or acceptor sites. We describe the generation of fratricide resistant, T cells by orderly removal of shared antigens such as TCR/CD3 and CD7 ahead of lentiviral mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base edited cells confirmed virtual elimination of chromosomal translocation events detected in conventional Cas9 treated cells. Interestingly, co-culture of 3CAR and 7CAR cells resulted in self-enrichment yielding populations that were 99.6% TCR-/CD3/-CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against their relevant target antigen in leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited the highest level of cytotoxicity against T-ALL targets expressing both target in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no evidence of promiscuous base conversion activity affecting CAR antigen specific binding regions which may otherwise redirect T cell specificity. Combinational infusion of fratricide resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allogeneic haematopoietic stem cell transplantation for T cell malignancies.

molecular biology↗