Open access Protocol Tolerogenic dendritic cell-based treatment for multiple sclerosis (MS): a harmonised study protocol for two phase I clinical trials comparing intradermal and intranodal cell administration Barbara Willekens,‍ ‍ 1,2 Silvia Presas-Rodríguez,3,4 MJ Mansilla,5,6 Judith Derdelinckx,1,2 Wai-Ping Lee,7 Griet Nijs,7 Maxime De Laere,2 Inez Wens,2 Patrick Cras,1 Paul Parizel,8 Wim Van Hecke,9 Annemie Ribbens,9 Thibo Billiet,9 Geert Adams,10 Marie-Madeleine Couttenye,11 Juan Navarro-Barriuso,5,6 Aina Teniente-Serra,5,6 Bibiana Quirant-Sánchez,5,6 Ascensión Lopez-Diaz de Cerio,12,13 Susana Inogés,12,13 Felipe Prosper,12,14 Anke Kip,15 Herman Verheij,15 Catharina C Gross,16,17 Heinz Wiendl,16,17 To cite: Willekens B, Presas- Marieke (SM) Van Ham,18,19 Anja Ten Brinke,18,19 Ana Maria Barriocanal,20,21 Rodríguez S, Mansilla MJ, et al. Anna Massuet-Vilamajó,22 Niel Hens,23,24 Zwi Berneman,2,7 Eva Martínez-Cáceres,5 Tolerogenic dendritic cell- Nathalie Cools,2,7 Cristina Ramo-Tello,3 On behalf of the RESTORE consortium based treatment for multiple sclerosis (MS): a harmonised study protocol for two phase I clinical trials comparing intradermal and intranodal Abstract Strengths and limitations of this study cell administration. BMJ Open Introduction Based on the advances in the treatment 2019;9:e030309. doi:10.1136/ of multiple sclerosis (MS), currently available disease- bmjopen-2019-030309 ►► The concept of the clinical trials is built on recent modifying treatments (DMT) have positively influenced advances in the understanding of tolerance induc- ►► Prepublication history for the disease course of MS. However, the efficacy of DMT is tion via cell-based therapy, and the effort made to this paper is available online. highly variable and increasing treatment efficacy comes precisely silence myelin-antigen-specific, putatively To view these files, please visit with a more severe risk profile. Hence, the unmet need for deleterious immune responses in the disease. the journal online (http://​dx.​doi.​ safer and more selective treatments remains. Specifically org/​10.​1136/​bmjopen-​2019-​ ►► The use of cryopreserved tolerogenic dendritic cells restoring immune tolerance towards myelin antigens may 030309). (tolDC) allows production of batches of tolDC, stored provide an attractive alternative. In this respect, antigen- in ready-to-use aliquots for the required administra- specific tolerisation with autologous tolerogenic dendritic BW and SP-R contributed tions during the treatment period, thereby reducing equally. cells (tolDC) is a promising approach. variability and global production cost. NC and CR-T contributed Methods and analysis Here, we will evaluate the clinical ►► Harmonisation of clinical, MRI and immunological equally. use of tolDC in a well-defined population of MS patients in evaluations of the patients will enable us to compare two phase I clinical trials. In doing so, we aim to compare NC and CR-T are senior authors. results between two phase I clinical trials evaluating two ways of tolDC administration, namely intradermal BW and SP-R are first authors. the safety and feasibility of autologous tolDC admin- and intranodal. The cells will be injected at consecutive istration in patients with active MS. Received 08 March 2019 intervals in three cohorts receiving incremental doses ►► The patients are not randomised across the tri- Revised 09 July 2019 of tolDC, according to a best-of-five design. The primary als, limiting direct comparison of both routes of Accepted 23 July 2019 objective is to assess the safety and feasibility of tolDC administration. administration. For safety, the number of adverse events including MRI and clinical outcomes will be assessed. For feasibility, successful production of tolDC will be © Author(s) (or their determined. Secondary endpoints include clinical and MRI Introduction employer(s)) 2019. Re-use Multiple sclerosis (MS) is the leading cause of outcome measures. The patients’ immune profile will be permitted under CC BY. assessed to find presumptive evidence for a tolerogenic non-traumatic disability in young adults and Published by BMJ. effect in vivo. typically presents between the age of 20–40 For numbered affiliations see end of article. Ethics and dissemination Ethics approval was obtained years, in the prime of a patient’s personal and for the two phase I clinical trials. The results of the trials professional life. The heterogeneity of the Correspondence to will be disseminated in a peer-reviewed journal, at disease course in MS, that is, relapsing-remit- Nathalie Cools; scientific conferences and to patient associations. ting or progressive, remains a challenge for ​nathalie.​cools@​uza.​be Trial registration numbers NCT02618902 and patient management and design of clinical NCT02903537; EudraCT numbers: 2015-002975-16 and Cristina Ramo-Tello; trials. According to Lublin et al,1 MS is cate- 2015-003541-26. ​cramot@​gmail.​com gorised as relapsing or progressive. In both Willekens B, et al. BMJ Open 2019;9:e030309. doi:10.1136/bmjopen-2019-030309 1 Open access forms, disease activity (defined by clinical relapse and/or (CD80, CD83 and CD86) and anti-inflammatory cytokine MRI lesions) and disease progression are measured. To profile, even following rechallenge with an inflammatory date, more than 10 disease-modifying treatments (DMT) stimulus such as lipopolysaccharide. Furthermore, tolDC are approved for clinical use in patients with active relaps- from MS patients are capable of inducing stable and ing-remitting MS (RRMS). This has dramatically influ- antigen-specific T cell hyporesponsiveness. After in vitro enced the management of MS patients in daily practice stimulation with myelin-derived peptide-pulsed vitamin and has positively influenced prognosis of of a subset of D3-treated tolDC, T cells were unresponsive to the myelin MS patients. Indeed, whereas earlier epidemiological peptides used, while retaining their capacity to respond studies indicated that 50% of patients develop secondary to an unrelated antigen. Moreover, T cell hyporespon- progressive MS after 15 years when untreated,2 only 18% siveness was robust, as T cells were not reactivated after of patients treated with modern MS therapeutics were rechallenge with immunostimulatory DC.21–23 reported to evolve to a secondary progressive course after Also in vivo, the administration of bone marrow-de- a median duration of 16.8 years in a recent cohort.3 None- rived vitamin D3-treated tolDC pulsed with MOG40-55 has theless, DMT are not specific nor selective for MS, that been shown to induce antigen-specific T cell tolerance is, they work in an immunomodulatory or immunosup- in experimental autoimmune encephalomyelitis (EAE), pressive way by sequestering or depleting lymphocytes, the animal model of MS. Mice treated with MOG40-55- although that some therapies can induce immune recon- pulsed bone marrow-derived vitamin D3-treated tolDC stitution as well. Moreover, treatment-related side effects before disease induction showed reduced incidence of or risks can be severe,4 leaving a significant and unmet the disease. Furthermore, when the treatment was used need for safer and more disease-selective treatments. therapeutically in mice already showing clinical signs of The crux of MS is the patient’s own immune cells the disease, the severity of the disease was significantly attacking self-antigens in the central nervous system reduced.24 However, repetitive injections of tolDC were (CNS), caused by a loss of tolerance against myelin necessary for a prolonged clinical effect in EAE. In this antigens. This is manifested by inflammatory infiltrates, context, cryopreserved MOG40-55-pulsed tolDC demon- demyelination and axonal loss, resulting in the clinical strated similar clinical benefit compared with fresh tolDC, symptoms of the disease.5 6 Previously, it was shown that underlining the therapeutic potential and clinical appli- proteins expressed in the myelin sheath, protecting cability of cryopreserved tolDC.21 25 neuronal axons of the CNS, are an important target of the Recently, a number of phase I studies investigating the autoreactive T cell response.7–9 Also in our hands, ex vivo safety and feasibility of tolDC therapy for autoimmune T cell reactivity against a mix of seven immunodominant diseases such as rheumatoid arthritis, type I diabetes or myelin-derived peptides could be demonstrated in RRMS Crohn’s disease were completed (table 1).18 The first patients as compared with healthy controls and patients results are highly encouraging since none of the trials with other neurological disorders.10 Although the under- found safety concerns related to tolDC administration lying cause of the loss of tolerance towards myelin anti- in these patients. TolDC were well tolerated and autoim- gens has not been elucidated yet, one ultimate aim in the munity was not exacerbated in the patients treated.26–29 treatment of MS is to reestablish antigen-specific immune Nevertheless, numerous questions remain, and the effi- tolerance towards CNS structures.11–15 In this perspective, cacy of antigen-specific tolDC therapy may depend on it is postulated that tolerance-inducing antigen-specific many factors, of which the route of administration is therapy can be an innovative and promising strategy for among the most important. When considering the route the treatment of autoimmunity. This approach is based of delivery of DC, one needs to take into account that on the possibility that autoreactive B and T cells, driving different routes lead to different sites of accumulation myelin destruction and damage in the CNS, will be erad- of the vaccinated DC. In most clinical studies to date, ex icated by inducing tolerance to myelin-derived peptides, vivo generated DC were injected either intravenously, without interfering with protective immunity. Several intradermally or subcutaneously. In humans, migration approaches involving the induction of antigen-specific of DC towards the secondary lymph nodes is superior tolerance have reached the clinical development phase after intradermal injection compared with after subcuta- and demonstrated promising results in phase I/II clin- neous injection of DC,30–32 whereas migration of intrave- ical trials (extensively reviewed by Willekens and Cools).12 nously injected DC has not been monitored so far. Direct Given the fact that dendritic cells (DC) play a key role delivery of DC to lymph nodes via intranodal injection in controlling the immune response by steering the showed promising results in therapies using immunostim- outcome of antigen presentation to T cells, the use of ulatory DC in cancer,33 34 but has not been evaluated for tolerance-inducing or tolerogenic dendritic cells (tolDC) tolDC. It was demonstrated that brain-derived antigens may provide prospect for the treatment of MS.16–20 can be drained from the interstitial or cerebrospinal fluid Recently, we demonstrated the potential of 1α,25-dihy- via the lamina cribrosa and nasal mucosa to the cervical droxyvitamin D3 to generate tolDC from MS patients.21–23 lymph nodes, indicating that the cervical lymph nodes Indeed, vitamin D3-treated tolDC from MS patients display could be one of the first stations for the antigenic presen- a maturation-resistant phenotype, that is, they maintain tation at the peripheral level.35 36 Hence, we hypothesise their low expression levels of costimulatory molecules that intranodal injection of tolDC directly interferes with 2 Willekens B, et al. BMJ Open 2019;9:e030309. doi:10.1136/bmjopen-2019-030309 Table 1 Overview of clinical trials using tolDC as therapeutic intervention in autoimmune diseases Primary Number of Cell product and control Administration outcome Reference Indication Study design patients condition Dose mode measure Results Immunological effects 6 6 6 Zubizarreta MS and NMO open-label, 8 MS and 4 autologous tolDC loaded 50×10 , 100×10 , 150×10 , intravenous safety and well tolerated ↑ IL-10 production in peptide- et al44 dose-escalation, NMO with either myelin peptides and 300×106 tolDC in tolerability no serious stimulated PBMCs and ↑ in the 2019 phase Ib or AQP4 total, separated in three adverse events frequency of Tr1 independent doses administered every 2 weeks Bell Inflammatory unblinded, 9 autologous tolDC loaded 1×106, 3×106 or 10×106 tolDC intra-articular flare of disease no target knee no consistent et al28 arthritis dose-escalation, with autologous synovial arthroscopically vs saline only in the target knee flares within immunomodulatory effects in 2017 randomised, fluid as a source of within 5 days of 5 days of peripheral blood phase I autoantigens treatment treatment Willekens B, et al. BMJ Open 2019;9:e030309. doi:10.1136/bmjopen-2019-030309 Benham Rheumatoid open-label, 34 autologous DCs modified a low dose of 1×106 DCs and intradermal safety mild adverse ↑ in effector T cells and an ↑ et al27 arthritis controlled, with a nuclear factor a high dose of 5×106 events ratio of regulatory to effector T 2015 phase I kappaB (NF-kappaB) cells; ↓ in serum interleukin-15 inhibitor exposed to (IL-15), IL-29, CX3CL1, and four citrullinated peptide CXCL11; ↓ T cell IL-6 responses antigens, designated to vimentin 447-455-Cit450 relative “Rheumavax,” to controls Jauregui- Crohn's open-label, 9 autologous tolDC first three cohorts: a single intraperitoneal safety no adverse Amezaga disease dose-escalation, injection of 2×106, 5 x 106 effects 6 et al29 phase I or 10 x 10 tolDC; last three 2015 cohorts: 3 bi-weekly injections (same dose escalation schedule) Giannoukakis Diabetes type 1 randomised, 10 autologous unmanipulated 10×106 cells once every intradermal safety no adverse ↑ in the frequency of peripheral et al26 double-blind, dendritic cells or tolDC 2 weeks for a total of four effects B220+CD11c- B cells 2011 phase I administrations CXCL, Chemokine Ligand; DC, dendritic cells; Il, Interleukin; MS, multiple sclerosis; NMO, neuromyelitis optica; PBMC, Peripheral Blood Mononuclear Cell; tolDC, tolerogenic dendritic cells; Tr1, Regulatory T-cell type. Open access 3 Open access the antigen presentation and consequently, the stimula- both patients and neurologists.38 While patients were tion and proliferation of autoreactive T cells. Further- not involved in the design of the clinical trial, they are more, this route of administration omits the need for the involved in the conduct of the study. In fact, in order migration requirements of the tolDC, thereby potentially to spread awareness and information about the clinical enhancing the efficacy of the vaccine. Although intran- trials and its results beyond the research community odal injection is more complex, requiring ultrasound with the public as a whole, a stakeholder committee was guidance, this technique is part of the daily practice at installed consisting of experts from major interest groups the endocrinology or radiology department of most refer- including members of the Flemish, Belgian and Spanish ence hospitals. MS societies as well as Flemish and Spanish MS patients. Until now, there are no available data showing superi- This stakeholder committee will accompany the clinical ority of one route over others for the administration of trials from the start to the end. It has several aims: (i) to peptide-loaded tolDC. Here, we will compare intradermal keep the stakeholder informed, (ii) to ensure that their injection to intranodal injection. Only the direct compar- views are considered, (iii) to challenge the project by the ison of these different routes of administration in two identification of potential emerging needs and (iv) to dose-escalation studies will allow us to determine if both play an active part in the dissemination and the use of the routes are equally safe. project’s results. In conclusion, our objectives are to evaluate safety, clin- ical feasibility and the immunological consequences of Study population peptide-loaded tolDC administered intranodal or intra- Eligibility and enrolment dermal in MS patients in two clinical trials. Harmonisation Patients with active relapsing-remitting and active progres- of the procedures for clinical, MRI as well as immune-mon- sive MS, diagnosed according to most recent McDonald itoring will enable us to compare results between trials of criteria,39 40 and who are not eligible for or do not want to which the study protocols will be discussed in detail here. be treated with currently available DMT, will be recruited. Patients are included after written informed consent and Methods and analysis enrolled in the study when the inclusion and exclusion Study design criteria are met (table 2). Two open-label, dose-escalation phase I clinical trials, MS-tolDC and TOLERVIT-MS, are designed in a coor- Determination of sample size and dose-escalation procedure dinated and comprehensive manner and run simultane- The studies will be conducted according to a ‘best of five’ ously in Belgium (Antwerp University Hospital, Edegem) and Spain (Hospital Germans Trias i Pujol, Badalona), design,41 an alternative of the traditional 3+3 design in respectively. Patient recruitment started mid-2017 and is that one additional patient is added when one or even two anticipated to end in 2020. dose-limiting toxicities (DLT) are observed among the first three patients. Another patient is added when two Primary objectives DLT are observed among four treated patients. Dose esca- To evaluate the safety of administering tolDC, the occur- lation is allowed if DLT are observed among none of three, rence and severity of adverse events (AE) will be recorded. one of four or two of five patients, but the trial will termi- To assess feasibility, successful production of tolDC nate if three or more DLT are observed. A DLT is defined according to good manufacturing practices (GMP) starting as a serious adverse event (SAE) that is attributable to the from a leukapheresis procedure will be assessed. study cells administered, or of which the severity prevents further escalation. Dose escalation decisions will be made Secondary objectives after all subjects in the cohort have completed at least 3 Preliminary efficacy measures, including clinical months of follow-up and when the results of the safety outcomes and brain MRI, will be evaluated. In addi- and tolerability analyses of the preceding dose regimen tion, whole blood lymphocyte phenotyping and cytokine are satisfactory in the judgement of the investigators and profiling will be assessed before and after completion the independent Data Safety Monitoring Board (DSMB). of the vaccination cycle, as well as the ability of tolDC to An overview of the dose escalation is provided in table 3. suppress pathogenic T cell responses. For this, myelin-spe- Altogether, each phase I study is intended to accrue a cific T cell reactivity will be determined before and after total number of 9–15 evaluable patients. completion of the vaccination cycle. All secondary objec- tives contribute to determination of proof of principle. Study medication Patient reported outcome measurements Generation of tolDC will be carried out according to the Multiple Sclerosis Quality of Life-54 37 will be evaluated to principles and guidelines of GMP laid down in Directive detect changes in general and disease-specific quality of life. 2003/94/EC. TolDC production will be performed in the GMP facilities of the Center for Cell Therapy and Regen- Patient and public involvement erative Medicine of the Antwerp University Hospital This study was inspired by the unmet need of finding (Belgium) and of the Cell Therapy Area of the Clínica a cure for MS, which is a key priority recognised by Universidad de Navarra (Spain). 4 Willekens B, et al. BMJ Open 2019;9:e030309. doi:10.1136/bmjopen-2019-030309 Open access Table 2 Inclusion and exclusion criteria. All the inclusion criteria must be fulfilled. The presence of any of the exclusion criteria shall exclude the patient Inclusion criteria Exclusion criteria MS according to most recent McDonald criteria Previous use of immunosuppressive or cytostatic treatment, including mitoxantrone, cladribine, alemtuzumab or bone marrow transplantation or stem cell transplantation at any time prior to enrolment Age 18–60 years Treatment with fingolimod or natalizumab or dimethylfumarate in the past 12 weeks or teriflunomide within the past 15 weeks or ocrelizumab/rituximab within the past 6 months prior to the first administration EDSS of 0–6.5 inclusive Pregnancy or planning pregnancy in the next 12 months and breast feeding First signs or symptoms at least 3 months prior to enrolment in the Drug or alcohol abuse study Active MS (relapsing and/or progressive): one relapse in the past year Inability to undergo MRI assessments and/or at least one enhancing lesion on brain MRI in the past year; at least one new or enlarging T2 lesion in comparison with a reference scan from maximum 1 year before Normal peripheral B-cell count after treatment with ocrelizumab   No evidence of relapse for at least 30 days prior to start of screening History of or actual signs of immunodeficiency or and throughout during the screening phase malignancies Positive T cell reactivity response to a mix of seven myelin-derived Concurrent clinically relevant cardiac, immunological, peptides pulmonary, neurological, renal or other major disease Able to sign informed consent and comply with the protocol Active or chronic infection (hepatitis B or C, HIV, syphilis assessments or tuberculosis) No wish to be treated with currently available DMT Splenectomy Appropriate venous access and *adequate cervical lymph nodes on   ultrasound mapping Use of adequate contraceptive measures. Women of childbearing   potential can only be included in the study following use of adequate contraceptive measures. Accepted methods of contraception include use of hormonal contraceptives (oral, intravaginal, intrauterine or transdermal), intrauterine devices, sterilisation or postmenopausal status, use of condoms with spermicide. *Only in TOLERVIT-MS. DMT, disease-modifying treatments; EDSS, Expanded Disability Status Scale; MRI, Magnetic Resonance Imaging; MS, multiple sclerosis. Clinical-grade autologous tolDC will be prepared from human AB serum, granulocyte macrophage colony-stim- a leukapheresis. CD14+ monocytes will be cultured in ulating factor, interleukin (IL)−4 and 1α,25 dihydroxyvi- GMP-grade cell culture medium supplemented with 2% tamin D3. At day 4, tolDC will be stimulated using an inflammatory cytokine cocktail, consisting of tumour necrosis factor-α, prostaglandin E2 and IL-1β. At day 6, Table 3 Outline of the cell doses and patient numbers, tolDC will be harvested, loaded with seven myelin anti- per phase I clinical trial, in the dose escalation cohorts for gens (MBP13-32, MBP111-129, MBP154-170, PLP139-154, MOG1-20, intradermal and intranodal administration of tolDC MOG35-55 and MBP83-99), and cryopreserved at −196°C. Cohort Treatment regimen Patient numbers Separate aliquots of the cell product will be prepared 1 6 i.d./i.n. injections of 5×10 6 N=3 (+1+1) for quality control and quality assurance. This includes tolDC (i) sterility testing, (ii) cell count, (iii) viability, (iv) flow 2 6 i.d./i.n. injections of 10×106 N=3 (+1+1) cytometric phenotyping and (v) induction of T cell hypo- tolDC responsiveness in an allogeneic mixed leucocyte reaction. 3 6 i.d./i.n. injections of 15×106 N=3 (+1+1) tolDC Trial intervention: tolDC administration In this study, six vaccine doses will be administered to N, number; i.d., intradermal; i.n., intranodal; tolDC, tolerogenic each participant according to the following immunisa- dendritic cells. tion schedule: a total of six vaccine doses (V), with V1-4 Willekens B, et al. BMJ Open 2019;9:e030309. doi:10.1136/bmjopen-2019-030309 5 Open access Figure 1 Study design. Treatment will start at V1, approximately 4 weeks after the leukapheresis. Patients will receive additionalinjections on week +2 (V2), week +4 (V3), week +6 (V4), week +10 (V5) and week +14 (V6). Patients will have follow up visits one month, and 3, 6 and 12 months following the last study treatment. at biweekly (±3 days) intervals and V5-6 at monthly inter- A proportion of the patients may experience MS relapses vals (±3 days). A complete overview of the study design during the study. A relapse is defined as a new or worsening is depicted in figure 1. Detailed assessments per visit are neurological symptom that occurs in the absence of fever shown in table 4. or infection, occurs at least 30 days following the onset of a In Belgium (MS-tolDC), tolDC vaccination occurs previous relapse, persists for at least 24 hours and includes through intradermal injection at five alternating sites an increase in Expanded Disability Status Scale (EDSS) (100 µL/site) in the posterior neck region to ensure score42 from the previous assessment matching one of the lymphatic drainage to superficial and deep cervical lymph following: (i) an increase of ≥1 on the total scale score; nodes (5–10 cm from the cervical lymph nodes). In addi- or (ii) an increase of ≥2 points on one of the appropriate tion, in Spain (TOLERVIT-MS), an expert physician will Functional System Scores (FSS) or (iii) an increase of >1 inject tolDC directly in the cervical lymph nodes under point on two or more of the appropriate FSS. Patients will echographic guidance. If the patient’s anatomical condi- be instructed to notify their neurologist as soon as possible tions do not allow it (eg, small lymph nodes), the vaccine but within 72 hours the latest and will be examined within will be distributed in more nodes (max. 500 µL/node). 7 days of onset of the symptoms. In case of an MS relapse, a course of high-dose steroids can be administered at the Primary outcome measures discretion of the treating neurologist. Safety Study treatment must be discontinued for a given To evaluate the safety of administered tolDC, occurrence patient if the investigator determines that continuing of AE will be recorded using clinical outcome measures, would result in a significant risk for that patient. In case that is, physical (skin, pain and adenopathies) and neuro- of terminating the study treatment, patients will still be logical examination (relapses and worsening disability) monitored for safety issues for the duration of the study. and non-clinical outcome measures, that is, brain MRI, Feasibility biochemical and haematological safety. The frequent To evaluate the feasibility, successful production of tolDC MRI monitoring will allow us to monitor the safety of after leukapheresis will be evaluated. This includes the tolDC in MS patients objectively by measuring T1-en- production of sufficient numbers of tolDC and compli- hancing lesions and new and/or enlarging T2 lesions. ance of the final investigational product to the prespeci- In this way, unexpected disease activity can be detected fied release criteria following cryopreservation. timely. The severity of AE will be defined according to the WHO toxicity grading scale. The relationship of an AE Secondary outcome measures to the investigational product will be determined by the Clinical evaluation neurologists and the independent DSMB on the basis of The following clinical and laboratory assessments will their clinical judgement. be performed during a complete physical examination: 6 Willekens B, et al. BMJ Open 2019;9:e030309. doi:10.1136/bmjopen-2019-030309 Table 4 Study calendar. The different visits, examinations, tests and the tolDC administration are detailed on the tabular study schedule overview. Screening Treatment period Safety follow-up Baseline V1 Ts (T0) V1 +1 day V2 V3 V4 V5 V6 F1 F3 F6 F12 Informed consent • Inclusion and exclusion criteria • IFN-γ EliSPOT (T cell reactivity assay) • • •+1w •+1w • Leukapheresis • TolDC preparation • TolDC treatment • • • • • • Patient evaluation:  Vital signs (HR, BP, …) • • • • • • • • • • • • Willekens B, et al. BMJ Open 2019;9:e030309. doi:10.1136/bmjopen-2019-030309  ECG • • • • • • • • • • • •  Blood analysis for safety (1×5 mL EDTA tube and • • • • • • • • • • • • 2×10 mL serum tubes)  Urine pregnancy test • • • • • • • • • • • • Full neurological examination • • (EDSS, 9-HPT, T25FW, SDMT, MSQOL-54) Pain score after treatment injection • • • • • • MRI • • • • •+1w •+1w •+1w • • • Immunomonitoring • •+1w •+1w • (10×10 mL heparin tubes+1 serum tube) Biobanking for follow-up immunomonitoring (Serious) Adverse events and concomitant Continuous medication BP, Blood Pressure; ECG, Electrocardiogram; EDSS, Expanded Disability Status Scale; 9-HPT, 9 Hole Peg Test; MRI, Magnetic Resonance Imaging; MSQOL-54, Multiple Sclerosis Quality of Life-54; SDMT, Symbol Digit Modalities Test; T25FW, Timed 25 Foot Walk; tolDC, tolerogenic dendritic cells. Open access 7 Open access vital signs, ECG, routine blood and urine samples, urine lesion load and (iv) percent change in brain volume. In pregnancy test in female patients of reproductive poten- order to compare MRI outcome measures from different tial and serological screening tests, according to the study studies, a standardised acquisition is guaranteed by (i) a schedule (figure 1 and table 4). Concurrent drug use or uniform MRI protocol, (ii) the use of standard operating new AE will be reviewed and recorded. procedures for image acquisition and upload and (iii) EDSS is based on a standardised neurological exam- training of local MRI operators and study coordinators ination and measures impairments in eight functional for acquisition and upload. systems, including vision, brainstem, pyramidal, cere- bellar, sensory, bowel and bladder, mental (cerebral) and Immune-monitoring ambulation . It has been used for over 20 years as a clinical To evaluate therapy-related changes in the immune cell outcome measure of MS disease progression and consists profile, peripheral blood (10×10 mL heparin tubes) will of a 10-point scale of disease severity ranging from 0, that be sampled according to the time points depicted in is, no disability, to 10, that is, death from MS.42 All neurol- figure 1 and table 3, and analysed by multiparameter flow ogists involved in patient evaluation will be EDSS training cytometry prior to, during and after vaccination. In an certified. attempt to cover the main leucocyte subsets of peripheral EDSS will be supplemented by three well-known, blood, the following subsets and their activation status quantitative, continuous tests that evaluate ambulation will be enumerated in whole blood samples: CD4+ and (walking speed by the timed 25 foot walk test or T25FW, CD8+ T cell subpopulations, B cell subsets, natural killer arm dexterity and function by the 9 Hole Peg Test or (NK) cells, NKT cells and myeloid cells. Myelin-specific T 9-HPT, and cognition by the Symbol Digit Modalities Test cell reactivity will be determined before, during and after or SDMT). completion of administration of tolDC. For this, respon- The effect on disability progression will be character- siveness of T cells to myelin antigens will be investigated. ised by reporting the proportion of patients who are As a control, the responsiveness of T cells to unrelated free from disease progression. For this, disability will be antigens, for example, cytomegalovirus or tetanus toxoid, assessed based on a sustained clinically relevant change will be addressed. In doing so, we will be able to assess ex seen in any one of the disability assessments: EDSS, vivo the potential risk of inducing opportunistic infections T25FW, SDMT or the 9-HTP. Disease progression is by administering tolDC to MS patients. Finally, patient defined by: 3-month sustained increase from baseline in materials from the studies will be specifically biobanked. the EDSS score (1 point in patients with baseline EDSS We envisage to maximally performing batch measure- score 0 to 5.0; 0.5 point in patients with baseline EDSS ments and centralised immune monitoring analysis as score of 5.5–6.5) or 3-month sustained increase of at soon as a dose cohort has reached 3 months follow-up least 20% from baseline in the time taken to complete of all patients. For this, extensive immune cell profiling the T25FW or 3-month sustained increase of at least 20% as well as cytokine production of T cells will be analysed from baseline in the time taken to complete the 9-HPT. by multiparameter flow cytometry. Using lineage-specific Percent change from baseline after treatment in SDMT as well as activation markers, proportions and activation score will also be recorded. status of the different immune cell subsets will be deter- mined. Besides, antibody titers and memory B cell anal- MRI acquisition and evaluation ysis will be performed. MRI is the current gold standard for non-clinical moni- Patient reported outcome measurements toring of MS, and MRI-derived markers have been estab- MS QOL-5437 will be evaluated to detect changes in lished as standard outcome measures to monitor the general and disease-specific quality of life. treatment response in various MS clinical trials. MRI will be performed on the same 3T scanner throughout the Data management and monitoring study. The MRI protocol includes a 3PLANE scout, 3D Adequate and accurate patient records will be kept T1-weighted image pregadolinium and postgadolinium enabling the appropriate and required documentation (Dotarem, 20 mL) administration (voxel resolution of the study and subsequent verification of the collected 0.9×0.9×0.9, TR2300.0, TE2.29, TI 900.0) and a 3D FLAIR data. All data are completed in the electronic case report image (voxel resolution 0.4×0.4×0.9, TR5000.0, TE387.0, form for each patient enrolled in this study, including TI1800.0). The quantification of number of T2 lesions, patients who did not start with the investigational treat- T2 lesion load, number and volume of Gd-enhancing ment. A central data manager affiliated to a clinical lesions, based on 3D FLAIR images and 3D T1-weighted research organisation will perform source data verifica- images, is completely automatic avoiding inter-rater or tion. In doing so, study compliance will be monitored, intra-rater variability. The lesions are quantified within thereby assuring the protection of the rights, safety and different brain regions. Disease activity or progression on well-being of study subjects. MRI will be evaluated by pretreatment versus ongoing and post-treatment: (i) change in mean number of enhancing Analysis lesions; (ii) change in number of T1 Gd-enhancing and/ Given the design of the study (phase I) and its specific or new or enlarging T2 lesions; (iii) percent change in T2 primary end point (safety), no confirmatory statistical 8 Willekens B, et al. BMJ Open 2019;9:e030309. doi:10.1136/bmjopen-2019-030309 Open access testing will be performed. P-values will be calculated Dissemination but interpreted with caution. Study investigations will The results of the clinical trials will be published in a be exploratory and conclusions will be based on the peer-reviewed journal. In addition, the results will be complete set of patient evidence. presented at scientific conferences and to patient asso- Analyses of safety variables will be performed with the ciations. In particular for Spanish patient associations, safety population (all the patients treated with at least one dissemination of results will be handled by the Spanish dose of the cell product) by available data only analysis. Agency of Medicines and Medical Devices whose content For demographic and safety analysis, that is, the incidence is written in layman’s terms. On completion of the trial of AE, laboratory values and changes in vital signs, descrip- and after publication of the study results, data requests tive statistics will be calculated including frequencies for can be submitted to the researchers. categorical variables and mean and SD, or median and IQR for quantitative parameters. In brief, EDSS and other repeatedly measured variables will be analysed by means of Mixed Models for Repeated Measurements. In case Discussion model assumptions are violated (eg, non-Gaussian errors) Although the first phase I clinical trials have demonstrated or when dealing with ordinal efficacy variables, alterna- promising results with regard to safety of administering tive methods (including non-parametric methods and tolDC in other autoimmune diseases, numerous questions ordinal regression methods for repeated measurements) remain concerning which dose, treatment schedule or will be used. The percentage of patients with 1-point in route of administration is best with regard to safety, efficacy EDSS improvement will be estimated using a binomial and related costs of treatment with tolDC. In this collab- regression model including the treatment and the base- orative effort, each patient will receive six repetitive injec- line EDSS. The remainder of variables will be analysed tions of 5, 10 or 15×106 autologous myelin-derived peptide according to the appropriate statistical test: χ2 or Fisher’s mix-loaded tolDC, intradermal or intranodal, that is, four exact test to compare categorical variables, the dependent administrations once every 2 weeks and two administrations or independent t-test for continuous Gaussian-distributed once every 4 weeks. Previously, others evaluated the safety variables and the Wilcoxon or Mann-Whitney test for of intravenous administration of tolerogenic DC in MS and ordinal and non-Gaussian continuous data. The signifi- neuromyelitis optica patients (table 1).43 44 In the current cance level alpha will be set at 0.05 for two-tailed analysis. study, harmonisation of clinical, MRI and immunological Variables to assess the analysis of secondary and tertiary evaluations of the patients will enable us to compare results outcome measures will be performed with per protocol between two phase I clinical trials evaluating the safety and population. Continuous variables as a minimum will be feasibility of autologous tolDC administration in patients described by number of total and non-missing observa- with active MS. To our knowledge, this will be the first time tions (n) and the appropriate location-scale statistics that different routes of administration are set side by side including arithmetic mean, SD, minimum, median, Q1– for a cell therapy product. Q3 and maximum. Categorical variables will be presented In conclusion, our protocols envisage to restore toler- using the number of non-missing observations (n) or the ance to predefined myelin-peptide antigens using peptide- number of patients in the population (N) as applicable loaded tolDC. From the results of these two phase I clinical and percentages (%). Two-sided 95% (exact) CI will be studies, the optimal dose and administration route will be provided when relevant. selected for future phase II trials investigating the efficacy of this patient-tailored treatment in MS. Patient protection Author affiliations The study will be conducted in agreement with either 1 Department of Neurology, University Hospital Antwerp, Edegem, Belgium the Declaration of Helsinki or the laws and regulations of 2 Laboratory of Experimental Hematology, Vaccine & Infectious Disease Institute the country, whichever provides the greatest protection (VAXINFECTIO), University of Antwerp Faculty of Medicine and Health Sciences, of the patient. In the Belgian context, the Law of 7 May Wilrijk, Belgium 3 Multiple Sclerosis Unit, Department of Neurosciences, Hospital Universitari 2004 (‘Wet van 7 mei 2004 inzake experimenten op de Germans Trias i Pujol, Badalona, Spain menselijke persoon’) applies. The study will be conducted 4 Department of Medicine, Universitat Autònoma de Barcelona, Cerdanyola del in agreement with the ICH Harmonised Tripartite Guide- Vallés, Spain 5 line for Good Clinical Practice. Division of Immunology, LCMN, Hospital Universitario Germans Trias i Pujol and Research Institute, Badalona, Spain 6 Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma Informed consent de Barcelona, Bellaterra, Spain All patients will be informed of the aim of the study, the 7 Center for Cell Therapy and Regenerative Medicine, University Hospital Antwerp, possible AE, the procedures and possible hazards to which Edegem, Belgium 8 he/she will be exposed. They will be informed as to the Department of Radiology, University Hospital Antwerp, Edegem, Belgium 9 Icometrix NV, Leuven, Belgium strict confidentiality of their patient data, but that their 10 C-Clear Partners, Mortsel, Belgium medical records may be reviewed for trial purposes by 11 Department of Nephrology, University Hospital Antwerp, Edegem, Belgium authorised individuals other than their treating physician. 12 Haematology-Cell Therapy Area, clinica universidad de navarra, Pamplona, Spain Willekens B, et al. BMJ Open 2019;9:e030309. doi:10.1136/bmjopen-2019-030309 9 Open access 13 Immunology and Immunotherapy Department, Clinica Universidad de Navarra, Belgium. Judith Derdelinckx holds a PhD fellowship from the Research Foundation Pamplona, Spain Flanders (FWO). Dr Presas-Rodríguez is a neurologist who has received a grant of 14 Program of Haematology-Oncology, CIMA, Universidad de Navarra, Pamplona, Hospital Germans Trias i Pujol ('Germans Trias Talents 2016-2018') to work on this Spain project. Dr Willekens is a neurologist at the Antwerp University Hospital supported 15 Lygature, Utrecht, The Netherlands by a research fellowship (2016-2018) of the University of Antwerp to work on 16 Department of Neurology, University Hospital Munster, Munster, Germany this project and she currently holds a clinical PhD fellowship from the Research 17 Department of Neurology, University of Munster, Munster, Germany Foundation Flanders (FWO 1701919N). Spanish Patient association 'Treball de Vida' 18 Department of Immunopathology, Sanquin Research, Amsterdam, The Netherlands (Associació d'Afectats d'Esclerosi Múltiple del Barcelonès Nord i Maresme) and 19 Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, patient Ana Mª Calvo Marsal have donated funding to the Hospital Germans Trias i Amsterdam, The Netherlands Pujol MS Unit. 20 Clinical Research Polyvalent Unit, Clinial Trial Unit-Spanish Clinical Research Competing interests CCG received speaker honoraria and travel expenses for Network, Germans Trias i Pujol Health Sciences Research Institute, Badalona, Spain attending meeting from Genzyme, Novartis Pharma GmbH, and Bayer Health 21 Department of Pharmacology, Therapeutic and Toxicology, Universitat Autònoma Care. Her work is funded by the German Ministry for Education and Research de Barcelona, Cerdanyola del Vallès, Spain (BMBF; 01GI1603A) and the German Research Foundation (DFG; GR3946/3-1 22 Institut de Diagnòstic per la Imatge, Hospital Universitari Germans Trias i Pujol, and SFB128 A09). HW receives honoraria for acting as a member of Scientific Badalona, Spain Advisory Boards and as consultant for Biogen, Evgen, MedDay Pharmaceuticals, 23 Interuniversity Institute for Biostatistics and statistical Bioinformatics (I-BioStat), Merck Serono, Novartis, Roche Pharma AG, Sanofi-Genzyme, as well as speaker Universiteit Hasselt, Hasselt, Belgium honoraria and travel support from Alexion, Biogen, Cognomed, F. Hoffmann- 24 Centre for Health Economic Research and Modelling Infectious Diseases, Vaccine La Roche Ltd., Gemeinnützige Hertie-Stiftung, Merck Serono, Novartis, Roche & Infectious Disease Institute (VAXINFECTIO) & Center for Statistics, University of Pharma AG, Sanofi-Genzyme, TEVA, and WebMD Global. Prof. Wiendl is acting Antwerp Faculty of Medicine and Health Sciences, Wilrijk, Belgium as a paid consultant for Abbvie, Actelion, Biogen, IGES, Novartis, Roche, Sanofi- Genzyme, and the Swiss Multiple Sclerosis Society. His research is funded by the German Ministry for Education and Research (BMBF; 01FI1601E, 01GI1603A, and Acknowledgements We thank all the patients who contributed to the preclinical O1GI1603D), Deutsche Forschungsgesellschaft (DFG; SFB128 A09, A10, Z02, V and research by donating samples. Our gratitude goes to Marleen Breuls, MS nurse SFB1009 A03), Else Kröner Fresenius Foundation, Fresenius Foundation, Hertie and to the study nurses of the Neurology Department of the Antwerp University Foundation, NRW Ministry of Education and Research, Interdisciplinary Center Hospital, Linda Wagemaekers, Caroline Vinck and Maren Wyckmans as well as for Clinical Studies (IZKF) Muenster and RE Children’s Foundation, Biogen GmbH, Sandra Vidal, study nurse at Clinical Trial Unit in Research Foundation Germans GlaxoSmithKline GmbH, Roche Pharma AG, Sanofi-Genzyme. The institution of BW Trias i Pujol-IGTP. We thank the Spanish Clinical Research Network-SCReN for receives honoraria for acting as a member of Scientific Advisory Boards for Biogen, the support on Pharmacovigilance and Case Report Form implementation for the Merck Serono, Roche, Sanofi-Genzyme, Novartis and speaker honoraria and travel study. We thank also Jorge Luis Reverter, at Endocrinology Department at Hospital support from Biogen, Merck Serono, Roche, Sanofi-Genzyme, Novartis, TEVA. CRT Germans Trias i Pujol for the treatment administration. We thank the patients (MDB receives honoraria for acting as a member of Scientific Advisory Boards for Biogen, and PAM) and Belgian and Spanish MS patient organisations (Christiane Tihon, and Merck Serono, and speaker honoraria or travel support from Biogen, Merck Luc de Groote, Leyre Avellanal) as well as the experts (Ed Geissler, Antonio Ucelli, Serono, Roche, Sanofi-Genzyme and Novartis. SPR receives speaker honoraria or David Wraith, Catharien Hilkens, Tom Bosschaerts) for their participation in the travel support from Biogen, Merck Serono, Roche, Sanofi-Genzyme and Novartis. stakeholder committee. We thank the external scientific advisory board (Ed Geissler, PP is a medical advisory board member of Icometrix NV. The other authors report no Antonio Ucelli, David Wraith, Catharien Hilkens) and the data safety monitoring conflict of interest. board members (Christian Sindic, Ludo Vanopdenbosch, Joan Albert Arnaiz) for their contributions. Patient and public involvement statement See methods and analysis section Collaborators RESTORE Consortium collaborators: Naomi Ooms (Center for Cell Patient consent for publication Not required. Therapy and Regenerative Medicine, Antwerp, Belgium); Dirk Smeets (Icometrix, Ethics approval Ethics approval was obtained for both phase I clinical trials. Leuven, Belgium), Leone Bock, Denis Groot, Wim-Jan Koot, Janwillem Boiten, Jorg The study protocol was approved by the Federal Agency for Medicines and Health Janssen, Sjaak Peelen (Lygature, Utrecht, The Netherlands); A. Turksma, R. Vanlier, T. Products (FAMHP, Belgium), the Spanish Agency of Medicines and Medical Devices Rispens, D. DiBlasi, Iris Claessen (Sanquin, Amsterdam, The Netherlands); M. Puig- (Spain), the Ethics Committee of the Antwerp University Hospital (Belgium) and the Domingo, Laia Lagunas Vila, David Basanta Pons (Germans Trias y Pujol Research Ethics Committees of Hospital Germans Trias i Pujol and Clínica Universidad de Institute, Badalona, Spain); Itziar Astiasarán, Javier Mata Rodríguez (University of Navarra. Navarra, Spain); Antje Albring, Jana Arnholdt (University of Munster, Germany) Provenance and peer review Not commissioned; externally peer reviewed. Contributors BW, ZB, CRT, EMC, WVH and NC conceived and designed the study. WPL, GN, MDL, GA, AR, MC, IW and NC participated in logistical planning of the Open access This is an open access article distributed in accordance with the study. BW and SPR wrote the initial draft of the manuscript. BW, SPR, CRT, MJM, Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits IW, EMC and NC wrote and reviewed the manuscript. NH provided the statistical others to copy, redistribute, remix, transform and build upon this work for any support for the sample size estimates and the design of the statistical analysis. purpose, provided the original work is properly cited, a link to the licence is given, BW, SPR, MJM, JD, WPL, GN, MDL, IW, PC, PP, WVH, AR, TB, GA, MC, JNB, ATS, BQS, and indication of whether changes were made. See: https://​creativecommons.​org/​ ALDC, SI, FP, AK, HV, CCG, HW, SMVH, ATB, AMB, AMV, NH, ZB, EMC, NC and CRT licenses/​by/​4.​0/. made significant contributions to the development and conceptualisation of the protocol, reviewed the draft versions of this paper and have read and approved the final manuscript. Funding This work was supported by positive discussion through the A FACTT network (Cost Action BM1305: www.​afactt.​eu). COST is supported by the EU References 1. Lublin FD, Reingold SC, Cohen JA, et al. Defining the clinical course Framework Program Horizon 2020. 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