Study design and participant recruitment. This was an open-label phase I clinical trial. The objectives were (a) to determine if autologous LMP/EBNA1-specific T cells can be generated to clinical scale from the blood of patients with progressive MS and (b) to assess the safety and tolerability of adoptive transfer of LMP/EBNA1-specific T cells into patients with progressive MS. Participants were recruited through the MS Clinic at the Royal Brisbane and Women’s Hospital. Recruitment commenced on 27 November 2015.

The inclusion criteria were as follows: primary progressive or SPMS as defined by the revised McDonald Criteria (30); progressive neurological deterioration due to MS for at least 2 years; positive EBV serology; age 18 years or above; provision of informed consent; EDSS score of 5.0–8.0 (21); and life expectancy of at least 6 months. The exclusion criteria were as follows: serology or nucleic acid testing, indicating infection with human immunodeficiency virus, hepatitis B or C virus, syphilis, or human T cell lymphotropic virus; significant other disease; uncontrolled psychosis, uncontrolled depression, substance dependence, or any other psychiatric condition that might compromise the ability to participate in the trial; inability to provide informed consent; clinically significant abnormalities of full blood count, renal function, or hepatic function; any contraindication to MRI; prior cancers, except those diagnosed >5 years ago with no evidence of disease recurrence and with a clinical expectation of recurrence of <5%, or successfully treated nonmelanoma skin cancer, or carcinoma in situ of the cervix; immunomodulatory therapy (apart from short courses of corticosteroids) within the past year; and pregnancy, lactation, or unwillingness to use adequate contraception. Supportive and symptomatic care was continued during the trial.

Manufacturing and infusion of adoptive T cell therapy. Following the confirmation of eligibility, 400 ml blood was collected from each participant by venesection (venesection visit) to generate the investigational product (Figure 1). This product consisted of autologous LMP- and EBNA1-specific T cells in saline and was produced in the Q-Gen Cell Therapeutics facility at QIMR Berghofer Medical Research Institute, utilizing the recombinant adenoviral vector AdE1-LMPpoly. This vector contains multiple LMP1- and LMP2A-encoded CD8+ T cell epitopes fused to the EBNA1 gene with the glycine-alanine repeat deleted (17). To generate the T cell product, PBMCs were cocultured with autologous PBMCs (irradiated at 25 gray) infected with AdE1-LMPpoly (multiplicity of infection of 10:1) at a responder-to-stimulator ratio of 2:1, in RPMI 1640 containing 10% fetal bovine serum (growth medium; Thermo Fisher Scientific). On days 2, 5, 8, and 11, the cultures were supplemented with growth medium containing recombinant IL-2 (Proleukin, Novartis Vaccines and Diagnostics Inc.). On day 14, these T cell cultures were assessed for cell yield, viability, and T cell frequency. Specifications for release of the investigational product were (a) favorable sterility and adenovirus test results (analyzed at time of cryopreservation); (b) sufficient cells for 4 infusions; (c) cell viability ≥50%; and (d) proportion of CD3+ T cells ≥50%. Cryovials of the investigational product (1-ml aliquots) were stored in vapor-phase nitrogen.

Immediately prior to adoptive transfer, a single aliquot of cells was thawed rapidly using a 37°C water bath and then diluted in 19 ml 0.9% sodium chloride for intravenous infusion (Baxter Healthcare). The diluted cells were drawn into a 20-ml syringe and administered intravenously by a research nurse to the patient over 10–15 minutes within 4 hours of thawing. After the infusion of T cells was completed, a 20-ml saline flush was administered to minimize the number of cells remaining in the intravenous line. To reduce the risk of aggravating CNS inflammation, we chose an initial dose of 5 × 106 T cells, which was 25% of the median dose used to treat nasopharyngeal carcinoma (18), and escalated the dose gradually over the following 3 infusions to 1 × 107, 1.5 × 107, and 2 × 107 cells administered at 2-week intervals (weeks 1, 3, 5, and 7), as in the original patient we treated through the Special Access Scheme (20). The patients were admitted to the Royal Brisbane and Women’s Hospital and closely monitored for 24 hours after each infusion. We treated participants consecutively to allow time to detect treatment-related serious AEs. The stopping rule for the trial was as follows: If more than 1 grade IV or V AE or more than 1 EDSS deterioration ≥2.0 occurs in the first 4 patients that is directly related to the adoptive transfer of autologous T cells, the trial will be ceased. There was no interim analysis. We monitored the participants for 26 weeks after the first T cell infusion. Neither the patients nor the examining neurologists were aware of the level of EBV reactivity within the T cell product, thus minimizing the potential for bias. The follow-up of the last participant was completed on 17 January 2018.

Clinical assessments before and after T cell therapy. We assessed the patients at the venesection visit and at weeks 1, 3, 5, 7, 11, 15, 21, and 27. At the venesection visit we performed the following baseline assessments: clinical history and physical examination, including neurological examination and assessment of EDSS score; cognitive assessment; fatigue assessment; screening for depression; QOL assessment; blood testing; brain and spinal cord MRI acquired at 3 Tesla before and after the intravenous injection of gadolinium-containing contrast material (including 3-dimensional T1 and 3-dimensional T2 fluid-attenuated inversion recovery sequences for assessment of enhancing lesions and T2 lesions); and lumbar puncture for CSF analysis of intrathecal IgG production. Immediately prior to each adoptive transfer of T cells, O 2 saturation, heart rate, blood pressure, temperature, and respiratory rate were recorded. These observations were repeated every 5 minutes for the duration of the infusion, then hourly for 4 hours, and then every 4 hours until discharge, 24 hours after infusion.

Patients were asked about neurological symptoms in a face-to-face interview with a neurologist at all visits following enrollment. At these visits, the neurologist performed a clinical examination, including a detailed neurological examination, and assessed the EDSS score. To evaluate cognitive function, a clinical neuropsychologist used a comprehensive neuropsychological test battery to assess the patients at the venesection visit and at week 27. The main component of this test battery was the Minimal Assessment of Cognitive Function in MS, a reliable and validated approach for neuropsychological assessment of MS patients (25). The clinical neuropsychologist also administered the Computerized Test of Information Processing, which precisely measures reaction time. Because mood can affect cognition, the clinical neuropsychologist assessed depression with the Beck Depression Inventory – Fast Screen for Medical Patients questionnaire (26). In addition, we used the much briefer Montreal Cognitive Assessment (24) to measure cognitive function at the venesection visit and at weeks 1, 7, 15, and 27. To assess fatigue at the same time points, we utilized the Fatigue Severity Scale (22), a self-report scale commonly used to measure fatigue in MS, because it is relatively short, has good psychometric properties, and is sensitive to changes in fatigue. At all visits following enrollment we screened for depression by asking the patients the 2 questions used by Mohr et al. (27), namely, (a) “During the past 2 weeks, have you often been bothered by feeling down, depressed, or hopeless?” and (b) “During the past 2 weeks, have you often been bothered by little interest or pleasure in doing things?” We assessed QOL at the venesection visit and at weeks 1, 7, 15, and 27 with a single question from the MS QOL Instrument; this question asks “Overall, how would you rate your own quality of life?” on a scale from 0 (worst — as bad or worse than being dead) to 10 (best), as used by Cosio et al. (23). MRI of the brain and spinal cord before and after gadolinium and CSF analysis were repeated at the week 15 and week 27 follow-up visits.

The MRI scans were assessed for the number of gadolinium-enhancing lesions and for the development of new T2 lesions compared with baseline. Blood was collected for full blood count and serum biochemistry at all visits. This trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12615000422527).

Statistics. All statistical analyses were performed using GraphPad Prism version 7.04. Differences were considered significant at a level of P < 0.05. Changes in the Fatigue Severity Scale score after T cell therapy were statistically analyzed by the Wilcoxon matched-pairs signed-rank test to compare scores before and after T cell therapy (Figure 3A). To analyze changes in the scores for the individual components of the comprehensive neuropsychological test battery after T cell therapy, the scores for each component before and after T cell therapy were compared using either the paired 2-tailed t test or Wilcoxon matched-pairs signed-rank test, according to formal testing of data distribution using the D’Agostino and Pearson normality test. P values were then corrected for multiple comparisons using the Bonferroni correction. To display the changes in scores for all 15 components in the one graph (Figure 3C), the change in test score from week 1 to week 27 for each component was standardized by dividing the change in test score by the standard deviation of the week 1 group mean. This enabled the change to be expressed in standard deviations. To analyze the relationship between the clinical response to T cell therapy and the frequency of LMP/EBNA1-specific CD8+ T cells for each of the 4 cytokines (CD107a, IFN-γ, TNF-α, or IL-2) in the administered T cell product (Figure 5A) we used Fisher’s exact test. To assess the relationship between the clinical response to T cell therapy and the polyfunctionality of LMP/EBNA1-specific CD8+ T cells in the administered T cell product (Figure 5B) a multiple 2-tailed t test with the Holm-Sidak correction for multiple comparisons was used.

Study approval. The study was approved by the QIMR Berghofer Human Research Ethics Committee, the Royal Brisbane and Women’s Hospital Human Research Ethics Committee, and The University of Queensland Medical Research Ethics Committee. Participants provided written informed consent.