D. Bishop

Low-cost generation of Good Manufacturing Practice–grade CD19-specific chimeric antigen receptor–expressing T cells using piggyBac gene transfer and patient-derived materials

BACKGROUND AIMS Protocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification. METHODS We varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period. RESULTS Using optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19(+) cell lines and efficiently lysed both CD19(+) cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer. DISCUSSION We have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies.

Long-term control of recurrent or refractory viral infections after allogeneic HSCT with third-party virus-specific T cells

Donor-derived adoptive T-cell therapy is a safe and effective treatment of viral infection posttransplant, but it is limited by donor serostatus and availability and by its personalized nature. Off-the-shelf, third-party virus-specific T cells (VSTs) appear promising, but the long-term safety and durability of responses have yet to be established. We conducted a prospective study of 30 allogeneic hemopoietic stem cell transplant (HSCT) patients with persistent or recurrent cytomegalovirus (CMV) (n = 28), Epstein-Barr virus (n = 1), or adenovirus (n = 1) after standard therapy. Patients were treated with infusions of partially HLA-matched, third-party, ex vivo-expanded VSTs (total = 50 infusions) at a median of 75 days post-HSCT (range, 37 to 349 days). Safety, viral dynamics, and immune recovery were monitored for 12 months. Infusions were safe and well tolerated. Acute graft versus host disease occurred in 2 patients, despite a median HLA match between VSTs and the recipient of 2 of 6 antigens. At 12 months, the cumulative incidence of overall response was 93%. Virological control was durable in the majority of patients; the reintroduction of antiviral therapy after the final infusion occurred in 5 patients. CMV-specific T-cell immunity rose significantly and coincided with a rise in CD8+ terminal effector cells. PD-1 expression was elevated on CD8+ lymphocytes before the administration of third-party T cells and remained elevated at the time of viral control. Third-party VSTs show prolonged benefit, with virological control achieved in association with the recovery of CD8+ effector T cells possibly facilitated by VST infusion. This trial was registered at www.clinicaltrials.gov as #NCT02779439 and www.anzctr.org.au as #ACTRN12613000603718.

Haploidentical peripheral blood stem cell infusion in combination with chemotherapy for acute myeloid leukaemia in elderly patients

Elderly patients with acute myeloid leukaemia (AML) have a poor prognosis with standard chemotherapy. Two elderly AML patients treated with infusion of family‐derived partially human leukocyte antigen (HLA)‐matched peripheral blood stem cells following each cycle of chemotherapy entered morphological complete remission without graft versus host disease or major toxicity. Our results support this as a non‐toxic approach for inducing a graft versus leukaemia effect in patients not suitable for allogeneic transplantation. Additional resources required for donor assessment and harvest may be reduced by using banked partially HLA‐matched mononuclear cells from unrelated donors.

449. Multipathogen-Specific T Cells for Immune Reconstitution – A Decade of Manufacturing Development and Clinical Use

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

The LIM protein FHL3 binds basic Krüppel-like factor/Krüppel-like factor 3 and its co-repressor C-terminal binding protein 2

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.