Interferon‐γ capture T cell therapy for persistent Adenoviraemia following allogeneic haematopoietic stem cell transplantation (2013)

Adenovirus (ADV) is a particular problem after paediatric haematopoietic stem cell transplantation (SCT), and whilst antiviral drug therapy with Cidofovir can control viraemia, T cell reconstitution is required for viral clearance (Lion et al, 2003; Kampmann et al, 2005; Lindemans et al, 2010; Hiwarkar et al, 2012). Experience of adoptive transfer of ADV-specific T cells is limited and conventional approaches for generating donor-derived virus-specific T cells are time consuming and costly (Leen et al, 2009). A rapid approach whereby virus-specific T cells can be enriched within 24 h has been reported, and uses interferon-γ (IFN-γ) capture technology and magnetic bead-mediated selection (CliniMacs®, Miltenyi Biotec, Bergisch Gladbach, Germany) (Feuchtinger et al, 2006; Peggs et al, 2011). We previously demonstrated the feasibility of isolating ADV-T cells from healthy donors using γ-capture (Chatziandreou et al, 2007) and have now treated five children with intractable post-transplant ADV using T cells isolated either from their original stem cell donor or from third party haploidentical donors (Table 1). Four had received reduced intensity conditioning and had undergone in vivo T cell depletion with alemtuzumab as part of their conditioning regimen (Patients 1-4), and one had undergone ex-vivo T cell depletion, and CD34 stem cell enrichment as part of an unconditioned haploidentical transplant procedure (Patient 5). ADV reactivation in blood occurred within 28 d and all patients were treated with intravenous Cidofovir, and two cases (Patients 1 and 4) received additional therapy with Ribavirin. Peak viral loads in blood ranged from 5·6 × 104/ml to 22 × 106/ml before cell therapy. Three children received γ-captured T cells from their original stem cell donor (Patients 3-5) but in two children (Patients 1 and 2), assessment of donor cellular immunity failed to detect suitable IFN-γ secretion responses against hexon (the dominant ADV antigenic protein). In these subjects, parental donors did exhibit ADV-specific responses and donated third party-haploidentical T cells (Fig 1A). Flow cytometry of donations detected IFN–γ secretion responses of between 0·13–3·3%, which were enriched to between 19–64% after column selection. In all five cases, post-column T cell yields were sufficient to generate aliquots of 104 cells/kg [below threshold for graft-versus-host disease (GVHD)] and 105 cells/kg. PBSCT CD34 9·2 × 106/kg CD3 8·4 × 107/kg Ribavirin Cidofovir 9/10 MMUD TBI/CY/ Alemtuzamab PBSCT CD34 3·5 × 106/kg CD3 4·9 × 107/kg ADV resolved Bystander GVHD Vanishing bile ducts Died day 241 9/10 family donor Flu/Mel/ Alemtuzumab PBSCT CD34 14 × 106/kg CD3 1·0 × 108/kg Ciclosporin Prednisolone 4 mg/kg Viraemia persisted ADV pneumonitis Respiratory Failure Died day 56 MUD Flu/Mel/ Alemtuzumab PBSC CD34 7 × 106/kg CD3 1·6 × 109/kg Ciclosporin Prednisolone 1·0 mg/kg Ribavirin Cidofovir Haplo No conditioning PBSC CD34 6·6 × 106/kg CD3 1·7 × 104/kg Ciclosporin Prednisolone 0·5 mg/kg Adenovirus cell therapy was administered on average 80 d after the original stem cell graft (range 34–122), and in four children (Patients 1, 2, 4, 5) comprised a single dose of freshly selected cells at a dose of 104/kg. Three of these 4 children had previously developed skin GVHD (≤grade II) requiring ciclosporin and/or prednisolone prior to, and following, the administration of ADV-T cells. In three children, blood viraemia resolved and withdrawal of anti-viral drug therapy was possible within 4 weeks (Fig 1B). The fifth child (Patient 3) received a single dose of 105 cells/kg from the original mismatched (1A mismatch) family donor. Ciclosporin and 4 mg/kg prednisolone had been administered for ventilator-dependent pneumonitis, but despite the administration of ADV-T cells, the child died 56 d after SCT without evidence of antiviral reconstitution. It is possible that the underlying deficiency of Major Histocompatibility Complex class II expression and impaired antigen presentation in this patient was an important factor contributing to the poor outcome. There were no acute, infusion-related toxicities and no patients developed exacerbation of GVHD, other than in Patient 2 where we previously reported a phenomenon of bystander GVHD (Qasim et al, 2011). Patient 1 may have suffered exacerbation of bone marrow cytopenias following ADV-T cell therapy, but no causal link could be established in view of additional myelotoxic medications for concurrent cytomegalovirus viraemia. By six weeks, circulating ADV-specific T cells were detectable on the basis of IFN-γ secretion (Patients 1, 2, 4, 5) and based on differential human leucocyte antigen (HLA) 1/A2 staining following the infusion of haploidentical third party T cells it was possible to directly attribute ADV-specific responses to the infused populations (Patients 1, 2) (Qasim et al, 2011). Populations of IFN-γ secreting T cells were also detectable in Patients 3–5, where the original donor provided ADV-T cells, although in these cases it was not possible to unequivocally attribute recovery of immunity to captured ADV-specific cells. Of note, Patients 4 and 5 both cleared virus after the infusion of ADV-specific cells whilst continuing to receive ciclosporin and steroid treatment for GVHD. In this small study, we have demonstrated that a single infusion of 104 T cells/kg can be infused following IFN-γ based enrichment, and can mediate clearance of high level adenoviraemia, even in patients receiving on-going immunosuppression. Two limitations were notable, especially when dealing with unrelated donor transplants. Firstly, suitably potent IFN-γ responses against ADV were only detected in around half of healthy donors tested. We had reasoned that responses >0·05% above background would facilitate higher levels of purification and reduce the risk of GVHD through the inadvertent transfer of non-specific T cells, although this cut-off may be higher than required. A further limitation in the unrelated donor setting was the length of time taken to recall volunteer donors and obtain second donations. While it is technically possible to select and administer ADV-specific T cells within 24 h using γ-capture, it was an average of 80 d after SCT (range 34–122 d) before therapy was administered. Importantly, ADV viraemia had become established within 28 d of SCT in all five children, and persisted despite drug therapy. In the context of very high viral loads, it is likely that ADV has seeded in different organs and the risk of inflammatory complications after ADV-T cell administration is increased. Of note, both surviving patients had peak ADV levels <106 copies/ml, compared to log-fold higher levels in the non-survivors and we therefore advocate early intervention if at all possible. Since initiating this study, advances in the generation of virus-specific CTLs have included the availability of overlapping Hexon/Penton peptide pools capable of inducing accelerated ADV-T cell expansion in the great majority of healthy donors (Comoli et al, 2008). The experience presented here will help determine the optimal timing and dosing of virus-specific T cell therapy after SCT in forthcoming clinical trials of peptide-expanded ADV-specific T cells. ADV-specific T cells will be generated for all children identified as being at high risk of viral complications following SCT, and should be available within 28 d of transplantation, enabling early intervention soon after ADV reactivation is detected. WQ, PV, HBG designed the research, undertook the study and wrote the paper. KG, HZ, WI, SD, PH, AD undertook research. This work was supported by the Leukaemia and Lymphoma Research Fund, GOSHCC, Rosetree Trust and was undertaken at GOSH/UCL Institute of Child Health, which receives funding from the Department of Health's NIHR Biomedical Research Centre's funding scheme. The study received ethical approval from the local research and ethics committee and was performed with informed consent. We acknowledge the support of specialist nursing and laboratory staff, the kind assistance of donor registries/harvest centres and helpful advice from John Campbell at Miltenyi Biotech.