Vaccination - a Novel Strategy to Improve the Persistence of CD19CAR Transduced T-Cells in Relapsed Paediatric ALL: Preliminary Results from the CD19TPALL Study
Authors: Pule Martin , Ghorashian Sara , Clifton-Hadley Laura , Smith Paul , Saiagh Soraya , Veys Paul , Goulden Nicholas , Wright Gary , Biagi Ettore , Altvater Bianca , Dreno Brigitte , Champion Kim , Cummins Michelle , Hough Rachael , Sauer Martin G. , Rossig Claudia , Amrolia Persis 
Published: October 2019

Abstract
Introduction
Patients with acute lymphoblastic leukaemia (ALL) relapsing after allogeneic stem cell transplantation (SCT) have a dismal prognosis. Recent clinical trials with T cells engineered to express 2nd generation CD19 chimeric antigen receptors (CARs) incorporating co-stimulatory domains for improved persistence and expansion report unprecedented anti-leukemic responses. However, responses are associated with Cytokine Release Syndrome (CRS) due to supra-physiological activation of the redirected T-cells. As an alternative, we studied use of donor-derived Epstein Barr virus (EBV)-specific T cells (CTL) transduced with a 1st generation CD19CAR as effectors, relying on signalling through the endogenous T cell receptor (TCR) to drive more physiological proliferation and persistence. This has enabled us to investigate a novel strategy to facilitate the expansion/persistence of CD19CAR T cells by vaccination with irradiated donor-derived, EBV transformed lymphoblastoid cell lines (LCL). We are conducting a European multi-centre phase I/II study of this approach in patients with pediatric ALL relapsing after SCT and report our interim findings.
Methods
Donor-derived EBV-specific CTL were generated from 80mls donor blood by repetitive stimulation with LCL, followed by transduction with an SFG retroviral vector encoding a CD19CAR consisting of the FMC63 single chain Fv linked to a CD3ζ endodomain. Patients were eligible for CD19CAR CTL therapy either pre-emptively if they became MRD-positive (> 5 x 10-4 in BM) within the 1st year post-SCT or prophylactically at day 60-70 post-2nd SCT. All patients had early withdrawal of immunosuppression and received lymphodepletion with fludarabine 90 mg/m2. Patients with detectable residual disease also received cytoreduction with vincristine/dexamethasone prior to infusion of cryopreserved CD19CAR CTL. Persistence of CAR CTL was measured by quantitative PCR and flow cytometry of blood. Disease status was assessed by morphology and IgH MRD analysis on bone marrow samples. The study design incorporated an interim analysis, allowing for vaccination with irradiated LCL if CD19CAR CTL were not detectable in 50% of patients at 2 months post-infusion.
Results
So far, 20 patients have been recruited (14 pre-emptive, 6 prophylactic arm) and 7 patients treated (3 pre-emptive, 4 prophylactic). The infused cell dose was 2 x 108/m2 in 6 patients and 4 x 107/m2 in the other. CD19CAR expression varied from 12.1-58.9%.
No grade 3-5 toxicity was noted. In particular, no CRS, neurotoxicity or graft versus host disease (GVHD) attributable to CD19CAR CTL was seen. B-cell depletion was transient, lasting 1-2 months.
In terms of disease response, 2 patients treated prophylactically remain in MRD negative remission after 3 and 17 months’ follow-up. A further patient showed transient clearance of BM MRD following immunotherapy in association with EBV viremia. He subsequently relapsed but has stable disease after retreatment with CD19CAR CTL with LCL vaccination. The other 4 patients had disease progression between 2 weeks and 3 months post-CD19CAR CTL infusion. At a median follow-up of 8 months, 2 patients have died of relapse, 3 are alive with disease and 2 remain disease-free.
A planned interim analysis of the initial 6 patients treated with CD19CAR CTL alone showed poor expansion/persistence of CD19CAR CTL which were only detectable in the blood in 1 patient up to 28 days post-infusion. This may reflect that only 1 patient had EBV viremia at the time CD19CAR CTL were infused. In view of this, a second trial cohort received subcutaneous vaccination with irradiated, donor-derived LCL at 2 days before and at 1 and 2 months following CD19CAR CTL infusion to provide signalling through the endogenous EBV-specific TCR. So far, 2 patients have been vaccinated and a 3rd is planned shortly. Data on the effect of vaccination on CD19CAR CTL expansion/persistence will be presented.
Conclusions
This ongoing study shows safety of adoptive immunotherapy with donor EBV CTL transduced with a 1st generation CD19CAR in paediatric patients with ALL relapsing post allo-SCT. However, in the absence of a co-stimulatory domain in vivo expansion and persistence of transferred CTL is poor. We are investigating whether vaccination with irradiated, donor-derived EBV LCL improves persistence and efficacy of CAR transduced T cells and initial data on this approach will be presented

Disclosures
Pule: Cellectis: Martin Pule's laboratory receives funding for contract research from Cellectis Therapeutics Other.