449. Multipathogen-Specific T Cells for Immune Reconstitution – A Decade of Manufacturing Development and Clinical Use
Authors: Emily Blyth, Leighton Clancy, Renee Simms, Jane Burgess, Barbara Withers, Chun Kei Ma, David Bishop, Ming-Celine Dubosq, Kenneth Micklethwaite, David Gottlieb
Journal: Molecular Therapy (2015)

Since 2003 we have treated 101 patients with antigen specific T cells for treatment or prevention of opportunistic infections in the context of haemopoietic progenitor cell (HPC) and solid organ transplant. Over this time, we have modified laboratory procedures to progressively incorporate T cell therapy into standard transplant practice. We have increased the number of pathogen targets, utilised HPC products and streamlined culture techniques. We are now able to produce a multipathogen product within 3 weeks of HPC harvest in a manner consistent with good manufacturing practice and within the scope of systems established for HPC processing. Our approach could be used by most accredited transplant laboratories. We have developed robust, simple and inexpensive methods that use marrow or G-CSF mobilised HPC products as starting material for T cell expansion. This has major practical importance in minimising repeated donor screening, blood draws and apheresis. Our current procedure uses monocyte derived dendritic cells (mo-DC) isolated using a modified CD14 selection method that requires minimal quantities of CD14 selection reagent. Maturation of mo-DCs utilises GM-CSF, IL-4, PG-E2, IL-6 and IL-1β. Multiple antigens are presented to T cells by mo-DCs including peptide mixes for cytomegalovirus (CMV), Epstein Barr virus (EBV), BK virus and adenovirus, vaccines (Varicella Zoster and Influenza virus) and mycelial lysate (Aspergillus). Activated antigen specific T cells are expanded using IL-2. We use no T cell selection/depletion procedures, feeder layers, cytokine, tetramer capture or clinical grade sorting methodologies. The overall cost of manufacture of each product, including reagents, staffing and ongoing infrastructure costs is approximately AU$2400 per dose. The phenotypic composition of multipathogen products is mainly CD3 (mean 97%, range 90-97), with variable CD4:8 ratio, minimal contaminating B cells (mean 0.1%), monocytes (mean 0.3%) and NK cells (mean 3%). We have demonstrated antigen specificity using intracellular cytokine staining, MHC multimer analysis (CMV and EBV), interferon-γ Elispot and chromium (Cr51) release cytotoxicity assay. Products do not show alloreactivity by Cr51 release assay (Mean 0.8% specific lysis at E:T ratio of 20:1, range 0-5.3). Clinical results of the first 50 patients treated with CMV specific T cells have previously been reported and showed infusion to be safe and to reduce the need for CMV-directed antiviral therapy. Clinical outcomes of the remaining patients treated with multi-pathogen T cells specific for up to 8 pathogens will be reported when data is mature. Future areas of development will involve extension of the range of infectious pathogens targeted, addition of cells targeting malignant antigens and combination of these therapies with CD34+ stem cell selection. Developing progressively greater selectivity through graft engineering and immunotherapy will improve outcomes of transplantation in the future.