Manufacture of GMP-compliant functional adenovirus-specific T-cell therapy for treatment of post-transplant infectious complications.

BACKGROUND AIMS: In pediatric patients, adenovirus (ADV) reactivation after allogeneic hematopoietic stem cell transplantation (allo HSCT) is a major cause of morbidity and mortality. For patients who do not respond to antiviral drug therapy, a new treatment approach using ADV-specific T cells can present a promising alternative. Here we describe the clinical scale Good Manufacturing Practice (GMP)-compliant manufacture and characterization of 40 ADV-specific T-cell products, Cytovir ADV, which are currently being tested in a multi-center phase I/IIa clinical trial. This process requires minimal intervention, is high yield, and results in a pure T-cell product that is functional. METHODS: Mononuclear cells (2 × 10(7)) were cultured in a closed system in the presence of GMP-grade ADV peptide pool and cytokines for 10 days. On day 10, the T-cell product was harvested, washed in a closed system, counted and assessed for purity and potency. Additional characterization was carried out where cell numbers allowed. RESULTS: Thirty-eight of 40 products (95%) met all release criteria. Median purity of the cell product was 88.3% CD3+ cells with a median yield of 2.9 × 10(7) CD3+ cells. Potency analyses showed a median ADV-specific interferon (IFN)γ response of 5.9% of CD3+ and 2345 IFNγ spot-forming cells/million. CD4 and CD8 T cells were capable of proliferating in response to ADV (63.3 and 56.3%, respectively). These virus-specific T cells (VST) were heterogenous, containing both effector memory and central memory T cells. In an exemplar patient with ADV viremia treated in the open ASPIRE trial, ADV-specific T-cell response was detected by IFNγ enzyme-linked immunospot from 13 days post-infusion. ADV DNA levels declined following cellular therapy and were below level of detection from day 64 post-infusion onward. CONCLUSIONS: The clinical-scale GMP-compliant One Touch manufacturing system is feasible and yields functional ADV-specific T cells at clinically relevant doses.

Immunological characteristics and T-cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T-cell-depleted autologous stem cell transplantation.

Children with systemic Juvenile Idiopathic Arthritis (sJIA), the most severe subtype of JIA, are at risk from destructive polyarthritis and growth failure, and corticosteroids as part of conventional treatment can result in osteoporosis and growth delay. In children where there is failure or toxicity from drug therapies, disease has been successfully controlled by T-cell-depleted autologous stem cell transplantation (ASCT). At present, the immunological basis underlying remission after ASCT is unknown. Immune reconstitution of T cells, B cells, natural killer cells, natural killer T cells and monocytes, in parallel with T-cell receptor (TCR) diversity by analysis of the ß variable region (TCRVb) complementarity determining region-3 (CDR3) using spectratyping and sequencing, were studied in five children with sJIA before and after ASCT. At time of follow up (mean 11.5 years), four patients remain in complete remission, while one child relapsed within 1 month of transplant. The CD8(+) TCRVb repertoire was highly oligoclonal early in immune reconstitution and re-emergence of pre-transplant TCRVb CDR3 dominant peaks was observed after transplant in certain TCRVb families. Further, re-emergence of pre-ASCT clonal sequences in addition to new sequences was identified after transplant. These results suggest that a chimeric TCR repertoire, comprising T-cell clones developed before and after transplant, can be associated with clinical remission from severe arthritis.