Abstract
Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with B cell malignancies. However, the complexity of production of patient bespoke T cell products is a major barrier to the broader application of this approach. We are investigating a novel strategy to enable "off-the-shelf"' therapy with mismatched donor CAR19 T cells. Transcription activator-like effector nucleases (TALEN)s can be used to overcome HLA barriers by eliminating the risk of graft-versus-host disease (GvHD) through disruption of T cell receptor expression, and by simultaneously targeting CD52, cells can be rendered insensitive to the lymphodepleting agent Alemtuzumab. Administration of Alemtuzumab can then be exploited to prevent host-mediated rejection of HLA mismatched CAR19 T cells. We manufactured a bank of such cells from volunteer donor T cells under GMP conditions on behalf of Cellectis S.A for final stage validation studies using a third generation self inactivating lentiviral vector encoding a 4g7 CAR19 (CD19 scFv- 41BB- CD3ζ) linked to RQR8, an abbreviated sort/suicide gene encoding both CD34 and CD20 epitopes. Cells were then electroporated with two pairs of TALEN mRNA for multiplex targeting of both the T cell receptor alpha constant chain locus, and the CD52 gene locus. Following ex-vivo expansion, cells still expressing TCR were depleted using CliniMacs alpha/beta TCR depletion, yielding a T cell product with <1% TCR expression, 85% of which expressed CAR19, and 64% becoming CD52 negative. This universal CAR19 (UCART19) cell bank has been characterized in detail, including sterility, molecular and cytometric analyses and human/murine functional studies ahead of submissions for regulatory approvals and Phase 1 testing in trials for relapsed B cell leukaemia. In the interim we received a request for therapy on a compassionate basis for an infant with refractory relapsed B-ALL, and with the agreement of Cellectis, we treated this first patient under UK special therapy regulations.
An 11 month girl with high risk CD19+infant ALL (t(11;19) rearrangement) relapsed in bone marrow 3 months after a myeloablative 8/10 mismatched unrelated donor transplant. Leukaemic blasts expressed CD19 but were CD52negative. Her disease progressed despite treatment with Blinatumomab (70% blasts in marrow) and we were unable to generate donor-derived CAR19 T cells on an existing study. Following institutional ethics review, detailed counseling, and parental consent, the patient received cytoreduction with Vincristine, Dexamethasone and Asparaginase followed by lymphodepleting conditioning with Fludarabine 90mg/m2, Cyclophosphamide 1.5g/m2 and Alemtuzumab 1mg/kg. Immediately prior to infusion of UCART19 cells, the bone marrow showed persisting disease (0.5% FISH positive). She received a single dose (4.5x106/kg) of UCART19 T cells without any significant toxicity. To date there has been no significant perturbation of cytokine levels in peripheral blood, and no indication of cytokine release syndrome. Although profoundly lymphopenic, UCART19 T cells were detectable by qPCR in the circulation by day 14 and at increased levels in both blood (VCN 0.35) and marrow (VCN 0.22) on day 28. The patient exhibited signs of count recovery and the bone marrow, while hypoplastic, was in cytogenetic and molecular remission. Chimerism was 90% donor, and a clearly demarcated population (7%) of third party cells indicated persistence of UCART19. A residual persistence of 3% recipient cells in the marrow suggests that leukemic clearance was not mediated by transplant mediated alloreactivity.
Within the short period of follow up available, our intervention comprising lymphodepletion and infusion of UCART19 T cells has induced molecular remission where all other treatments had failed. This first-in-man application of TALEN engineered cells provides early proof of concept evidence for a ready-made T cell strategy that will now be tested in early phase clinical trials.
Disclosures
Qasim: CATAPULT: Research Funding; CELLMEDICA: Research Funding; CALIMMUNE: Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding; CELLECTIS: Research Funding. Off Label Use: UCART19 T Cells are an unlicensed investigational medicinal product and in this case were used under MHRA special licence arrangements. Stafford:CELLECTIS: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Thrasher:CATAPULT: Patents & Royalties, Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding. Pule:AUTOLUS: Employment, Equity Ownership, Research Funding; CELLECTIS: Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties.
Localization of T cell receptor (TCR)- γδ+ T cells into human colorectal cancer: flow cytometric analysis of TCR-γδ expression in tumour-infiltrating lymphocytes