Prophylactic antigen-specific T-cells targeting seven viral and fungal pathogens after allogeneic haemopoietic stem cell transplant.

OBJECTIVES: Adoptive immunotherapy using donor-derived antigen-specific T-cells can prevent and treat infection after allogeneic haemopoietic stem cell transplant (HSCT). METHODS: We treated 11 patients with a prophylactic infusion of 2 × 107 cells per square metre donor-derived T-cells targeting seven infections (six viral and one fungal) following HSCT. Targeted pathogens were cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, varicella zoster virus, influenza, BK virus (BKV) and Aspergillus fumigatus. RESULTS: T-cell products were successfully generated in all patients with 10 products responsive to 6 or 7 infections. T-cell infusions were associated with increases in antigen-experienced activated CD8+ T-cells by day 30. CMV, EBV and BKV reactivation occurred in the majority of patients and was well controlled except where glucocorticoids were administered soon after T-cell infusion. Three patients in that circumstance developed CMV tissue infection. No patient required treatment for invasive fungal infection. The most common CMV and EBV TCR clonotypes in the infusion product became the most common clonotypes seen at day 30 post-T-cell infusion. Donors and their recipients were recruited to the study prior to transplant. Grade III/IV graft-versus-host disease developed in four patients. At a median follow-up of 390 days post-transplant, six patients had died, 5 of relapse, and 1 of multi-organ failure. Infection did not contribute to death in any patient. CONCLUSION: Rapid reconstitution of immunity to a broad range of viral and fungal infections can be achieved using a multi-pathogen-specific T-cell product. The development of GVHD after T-cell infusion suggests that infection-specific T-cell therapy after allogeneic stem cell transplant should be combined with other strategies to reduce graft-versus-host disease.

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.

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.

A phase III randomized trial of high-dose CEOP + filgrastim versus standard-dose CEOP in patients with non-Hodgkin lymphoma: 10-year follow-up data: Australasian Leukaemia and Lymphoma Group (ALLG) NHL07 trial.

Increasing dose intensity (DI) of chemotherapy for patients with aggressive non-Hodgkin lymphoma (NHL) may improve outcomes at the cost of increased toxicity. This issue was addressed in a randomized trial aiming to double the DI of myelosuppressive drugs. Between 1994 and 1999, 250 patients with previously untreated aggressive NHL were randomized to treatment with six cycles of 3-weekly standard (s) or intensive (i) chemotherapy: s-CEOP-cyclophosphamide 750, epirubicin 75, vincristine 1.4 mg/m(2) all on day 1, and prednisolone 100 mg days 1-5; i-CEOP-cyclophosphamide 1,500, epirubicin 150, vincristine 1.4 mg/m(2) all on day 1, and prednisolone 100 mg days 1-5. Primary endpoint was 5-year overall survival (OS). Relative to s-CEOP patients, i-CEOP patients achieved a 78% increase in the DI of cyclophosphamide and epirubicin. Despite this, there was no significant difference in any outcome: 5-year OS (56.7% i-CEOP; 55.1% s-CEOP; P = 0.80), 5-year progression free survival (PFS; 41% i-CEOP; 43% s-CEOP; P = 0.73), 5-year time to progression (TTP; 44% i-CEOP; 47% s-CEOP; P = 0.72), or complete remission (CR) + unconfirmed CR (CRu) rates (53% i-CEOP; 59% s-CEOP; P = 0.64). Long-term follow up at 10 years also showed no significant differences in OS, PFS, or TTP. The i-CEOP arm had higher rates of febrile neutropenia (70 vs. 26%), hospitalisations, blood product utilisation, haematological and gastrointestinal toxicities, and lower quality of life scores during treatment, although without significant differences 6-month later. In the treatment of aggressive NHL in the prerituximab era, increasing DI did not result in improved outcomes, while at the same time lead to increased toxicity.

Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation.

We investigated the use of adoptively transferred donor-derived cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) as immune reconstitution postallogeneic transplant in a phase 2 study. Fifty patients were infused with a single dose of 2 × 10(7)cells/m(2) after day 28 post-transplant. Twenty-six patients reactivated CMV posttransplant (only 5 post-CTL infusion) and 9 required therapy with ganciclovir or foscarnet (only 1 post-CTL infusion). There was 1 case of fatal CMV disease, attributable to high levels of antithymocyte globulin at the time of T cell infusion. We compared the patients in the phase 2 study with a group of contemporaneous controls also treated at the trial centers. There was no increase in acute or chronic graft-versus-host disease attributable to CTL infusion; overall and progression-free survival were similar in both groups. There was a reduction in the percentage of patients who required CMV directed antiviral therapy (17% vs 36%, P = .01) and in the total number of treatment days in the cohort receiving CTL (3.4 days vs 8.9 days, P = .03) without a reduction in CMV reactivation rates. We postulate that adoptively transferred cells are able to expand in response to viral antigen, limit viral replication, and prevent progression to tissue infection. This study was registered on the Australian Clinical Trial Registry as #ACTRN12605000213640 and #ACTRN12607000224426.