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Efficacy and safety of low-dose IL-2 (ld-IL-2) as a Treg enhancer for anti-neuroinflammatory therapy in newly diagnosed Amyotrophic Lateral Sclerosis (ALS) patients

Periodic Reporting for period 5 - MIROCALS (Efficacy and safety of low-dose IL-2 (ld-IL-2) as a Treg enhancer for anti-neuroinflammatory therapy in newly diagnosed Amyotrophic Lateral Sclerosis (ALS) patients)

Reporting period: 2021-03-01 to 2021-09-30

Background. Amyotrophic Lateral Sclerosis (ALS) is a neuro-muscular disorder affecting in the EU about 45,000 individuals, and causing 13,000 deaths /year. ALS is associated with inflammation making it a target for therapy. Regulatory T cells (Tregs) are key players in controlling inflammatory processes. In ALS, Treg numbers/function predicts rates of disease progression and survival. Low-dose interleukin-2 (ld IL-2) safely and specifically increases and activates Tregs, so ld IL-2 has the potential to significantly improve survival and deliver a therapeutic breakthrough in ALS.
Objectives. The MIROCALS project’s aims at developing a new therapy for ALS, and through a novel trial design, break the impasse in drug development in the field. The expected impact is to enhance quality of life and care for people with ALS, and provide a robust model for industry to encourage investment in ALS and other neurodegenerative diseases.
Method. MIROCALS is a double-blind, placebo-controlled, randomised clinical trial, comparing repeated 5 days cycles of Il2 to placebo in patients undertaking riluzole. Following a run-in period on riluzole, newly diagnosed patients were randomised to treatment groups and followed-up for 640 days. Five-days treatment cycles were repeated every 4 weeks for 18 months (19 cycles). Primary efficacy outcome was defined as survival, while secondary outcomes were rate of decline in function measured through functional scale (ALSFRS-R, monthly), and respiratory function test (Slow Vital Capacity, 6-monthly). Blood and CSF samples were collected at screening, randomisation, after the first cycle, at W17 (trough effect), and at W18 (max effect) and subsequently banked for further analysis (including core biomarker determination –pNFH, CCL2, cytometry).
Project status as per October 2022. 304 de novo ALS patients gave an informed consent to participate to the study, among which 220 patients were randomised to either IL-2 or placebo regimen and all followed-up for 640 days. By 22sd of July 2021, the last randomised patient completed his 18-month follow-up. Full database review was locked by July 2022. Statistical analyses are ongoing and full statistical report schedule for March 2023.
The first periods of the project, from September 2015 to June 2017, was dedicated to the clinical trial implementation, including regulatory and Ethics developments. Overall, 17 Clinical Centres were contracted, trained, and activated. All necessary tools for running the trial were developed, assembled, and distributed to all Clinical Centres. A Central laboratory (Biocytex, Marseille, France) was contracted to perform all core biomarker determinations in blood and CSF. SOPs for sampling and pre-analytical processing were drafted and centres trained to SOPs. For treatment preparation, a CMO was contracted (BathASU, Bath, UK), and SOPs for treatment logistics and handling drafted. In summary, all necessary tools to comply rigorously with regulatory constraints where set in place, and subsequently find appropriate by a contracted Independent Audit body (Qualilab, France).

From June 19th 2017 to October 29th 2019 (end of patients’ recruitment period), 17 centres included 304 patients of whom 220 were randomised (last randomised January 2020). All randomised patients were subsequently followed-up 3-monthly for 640 days until 30th October 2021 (cut-off date).
Over the trial period, at 2-weekly intervals, treatments packages were ordered by Centres, manufactured by CMO, transported to centres, and dispensed on a per-patient/per-cycle basis. By the end of the study, 3,678 individual treatment packages had been GMP manufactured, logistically managed and dispensed to patients.
Trial safety was monitored throughout the study by an Independent Data-Safety Monitoring board (DSMB). For the five DSMB Trial-safety assessments done, database management was performed, and data files ready for analysis were blindly prepared for the Independent DSMB statistician.
A biobank was constituted with (i) 30,800 plasma aliquots collected from 1,064 visits over 220 patients, (ii) 19,600 CSF aliquots from 613 visits over 220 patients, (iii) 8,200 PBMCs aliquots from 1031 visits over 220 patients.
For fresh-blood cytometry, 1161 blood samples were collected and analysed in real time. Data-bank on cytometry has been completed and database validation completed by July 2022. In addition, laboratory assays for deep immune & inflammatory phenotyping were also performed on biobank-samples. As of the October 2022, related tasks are completed (cytokine/chemokine database) or nearing completion (comprehensive cytometry).

For the analysis of Brain biomarker, we used antibodies (Abs) and immune complexes capture methods designed to test the humoral response to neurofilaments to two large ALS cohorts from the ALS biomarkers study, UK, and the Phenotype Genotype Biomarker study (CReATE), US. The results have shown that the Abs response to specific neurofilament isoforms can be used for clinical stratification of ALS. As of 30th September 2022 the data on the characterization of circulating protein aggregates in blood and their content in brain proteins including neurofilaments have been completed and statistical analysis ongoing. In addition free NFL determination was performed in blood and CSF (completed September 2022).

For genomics and transcriptomics analysis, preliminary activities were conducted to see what genes are expressed in the blood in response to treatment with riluzole and a low dose of interleukin-2 (ld-IL2) over a period of time, and to screen the patients for rare variants sequences in genes associated with ALS or T-lymphocyte regulation (the process the IL2 treatment is targeting). A total of 1,144 samples were processed for RNA extraction with quality controls showing a good RNA yield from all 17 centres. As of September the 30th 2022 the gene expression profiling over the entire cohort has been determined and statistical analysis is ongoing. In parallel, DNA extraction was performed for the 220 randomised patients and extracted DNA genotyping is now completed, and statistical analysis ongoing.

Last treatment for last patient in study was administered on June 28th 2021, and last status determined on the 30Th October 2021. Clinical Database cleaning was completed by July 13th 2022, allowing database lock and statistical analysis, which is ongoing as of October 2022.

In summary, MIROCALS project has been successful in setting-up and conducting to its planned end, a large randomised clinical trial recruiting 220 de novo ALS patients. All project defined parameters, clinical or laboratory, were successfully collected in a database, and as of the 30th September 2022, are undergoing statistical analysis. This unique resource should now help respond to all defined project objectives and beyond.
Living with ALS is a constant burden, due to muscle weakness and respiratory impairment. Would MIROCALS confirms the promises of ld IL2 therapeutic approach, finding a cure early in the course of the disease before severe disability occurs would dramatically improving quality of life for the patients and families. In addition, identification of biomarkers that could assess early responses to treatment and their validation would be a major achievement in ALS trial methodology, notably for investigation of novel therapies.