Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients

Abstract

T-cell immunotherapy that takes advantage of Epstein-Barr virus (EBV)-stimulated immunity has the potential to fill an important niche in targeted therapy for EBV-related cancers. To address questions of long-term efficacy, safety, and practicality, we studied 114 patients who had received infusions of EBV-specific cytotoxic T lymphocytes (CTLs) at 3 different centers to prevent or treat EBV(+) lymphoproliferative disease (LPD) arising after hematopoietic stem cell transplantation. Toxicity was minimal, consisting mainly of localized swelling at sites of responsive disease. None of the 101 patients who received CTL prophylaxis developed EBV(+) LPD, whereas 11 of 13 patients treated with CTLs for biopsy-proven or probable LPD achieved sustained complete remissions. The gene-marking component of this study enabled us to demonstrate the persistence of functional CTLs for up to 9 years. A preliminary analysis indicated that a patient-specific CTL line can be manufactured, tested, and infused for $6095, a cost that compares favorably with other modalities used in the treatment of LPD. We conclude that the CTL lines described here provide safe and effective prophylaxis or treatment for lymphoproliferative disease in transplantation recipients, and the manufacturing methodology is robust and can be transferred readily from one institution to another without loss of reproducibility.

Trial registration: ClinicalTrials.gov NCT00058812.

Figure 1

Immunophenotypes of EBV-specific CTLs. (A) Phenotypic composition of CTL lines. Percentages of CD3⁺, CD4⁺, and CD8⁺ T cells, natural killer cells (CD3⁻CD56⁺), and residual CD19⁺ B cells are shown. Each symbol represents a cell line infused into a single subject. (B) Expression of naive, central memory, and effector memory surface markers on CD4⁺ and CD8⁺ populations of the 20 most recently generated EBV-CTL lines.

Figure 2

Incidence of LPD in patients receiving CD6⁺CD8⁺ T cell–depleted marrow with or without EBV CTL prophylaxis. Kaplan-Meier analysis was based on 90 patients who received CTLs prophylactically compared with 42 who were enrolled on the same transplantation protocol but who did not receive CTLs. These patients were treated on Institutional Review Board-approved transplantation protocols open to patients with hematologic malignancies at St Jude Children's Research Hospital (1993-2000) or Baylor College of Medicine (1998-2000) where they received marrow from a matched unrelated donor or mismatched family member that had been depleted of T cells with antibodies to CD6 and CD8 after conditioning with cyclophosphamide, cytarabine, total body irradiation, and antithymocyte globulin. All recipients received cyclosporine A at a dosage adjusted to attain plasma concentrations of 250 to 350 ng/mL. The control patients were enrolled on the same study but had either declined enrollment on the CTL prophylaxis study or were not eligible. The difference in incidence rates is highly significant.

Figure 3

Representative clinical response. An 18-month-old boy with XLP presented at day 87 after matched unrelated donor transplantation with pharyngeal swelling, an elevated lactate dehydrogenase level, and a rapidly increasing EBV-DNA load. (A) After receiving 2 × 10⁷ CTLs/m² on day 90, he showed rapid clinical improvement and a decrease in EBV DNA level to baseline. (B) Coincident with this response, there was an increase in EBV-specific activity, as measured with a γ-interferon EliSpot assay, whereas CMV activity remained undetectable.

Figure 4

Long-term detection of marked CTLs. Circles within bars represent detection of the retroviral integrant by real-time PCR analysis of either peripheral blood or a reactivated line; black X, assays where the marker gene was not detected. Each bar represents an individual patient. Patients were monitored every 1 to 2 weeks for 8 weeks and then 3 monthly for a year, and then annually.

Figure 5

Long-term functionality. Change in the frequency of gene-marked cells in 46 peripheral blood samples from 18 patients before and after ex vivo stimulation with EBV-LCLs. The median marking efficiency before prestimulation was 0.0008% (SEM = 0.0007%) compared with 1.49% (SEM = 0.40%) after stimulation (P < .001 by the Wilcoxon signed-rank test). In 20 samples that were initially negative for gene-marked cells, ex vivo stimulation with EBV-LCLs expanded the marked population to a level above the 1/10 000 threshold for quantitative RT-PCR detection.

Figure 6

Overall survival of the 114 patients receiving CTLs. The mean Kaplan-Meier estimate (with 95% confidence levels) at 3, 5, and 10 years was 70% (61%-78%), 69% (60%-77%), and 67% (57%-76%), respectively. Tic marks on the survival curves indicate patients still at risk; dashed lines outline the 95% confidence levels.