Intrathecal administration of regulatory T cells for the treatment of multiple sclerosis

Abstract

The present invention relates to a pharmaceutical agent comprising CD3+CD4+CD25+CD127- regulatory T cells administered intrathecally in the treatment of multiple sclerosis.

Description

[Technical field]

[0001] Intrathecal administration of regulatory T cells for the treatment of multiple sclerosis

[0002] The present invention relates to a pharmaceutical product consisting of CD3+CD4+CD25+CD127- regulatory T cells for clinical use in therapy, the product being administered intrathecally in the treatment of multiple sclerosis. [Background technology]

[0003] Multiple sclerosis (MS) is an immune-mediated disease in which autoimmune T cells (Tconvs) sensitized to myelin sheaths disrupt the blood-brain barrier and destroy neurons in the central nervous system (CNS). + CD25 high CD127-FoxP3 + It is hypothesized that regulatory T cells (Tregs) may inhibit this destruction through their suppressive activity acting on Tconvs.

[0004] Treg lymphocytes, which account for approximately 1% of all peripheral blood lymphocytes, are important for maintaining the tolerance of autologous tissues. The lack of regulatory T cells leads to many autoimmune diseases and hypersensitivity disorders, as seen in patients with X-linked immunodeficiency syndrome, multiple endocrinopathy and enteropathy (IPEX), including multiple sclerosis.

[0005] Treg lymphocytes can be called "intelligent steroids" because, as steroids, they suppress inflammatory responses and act immunosuppressively; in contrast, the physiological suppressive effect of Treg cells is related only to pathological responses (e.g., directed against own tissues). Results of clinical trials, including our observations, have shown that treatment with Treg lymphocytes is safe and does not impair the immune response to foreign and dangerous antigens (viruses, bacteria, cancer cells). The prior art documents relevant to the invention of this application are as follows (including documents cited during the international phase after the international filing date and documents cited when the application entered the national phase in other countries). (Prior art document) (Patent Documents) (Patent Document 1) European Patent Application Publication No. 3131560 (Patent Document 2) International Publication No. 2017 / 105265 (Patent Document 3) International Publication No. 2018 / 024895 (Patent Document 4) U.S. Patent Application Publication No. 2014 / 170176 (Non-Patent Literature) (Non-patent document 1) N.MAREK-TRZONKOWSKA ET AL, "Administration of CD4+CD25highCD127- Regulatory T Cells Preserves ?-Cell Function in Type 1 Diabetes in Children", DIABETES CARE, Vol. 35, No. 9, 20 June 2012 (2012-06-20), page 1817-1820 Summary of the Invention

[0006] The present invention provides a method for administering a pharmaceutical product comprising CD3+CD4+CD25+CD127- regulatory T cells via intrathecal injection.

[0007] The subject of the present invention is a pharmaceutical product consisting of CD3+CD4+CD25+CD127- regulatory T cells.

[0008] The product is administered intrathecally for the treatment of patients diagnosed with multiple sclerosis.

[0009] The product is administered intrathecally.

[0010] This product is administered for multiple sclerosis. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 - Clinical outcomes in this study. Patients underwent protocol-planned neurological examinations throughout the study. Quality of life was assessed with the EQ.-5D questionnaire (EQ-5D), and physical / neurological status was monitored by the Timed 25-Foot Walk (FWT), components of the EDSS and MSFC scales, the 9-Hole Peg Test on the dominant (9-HPT P) and non-dominant (9-HPT L) arms, and the Paced Auditory Serial Addition Test (PASAT). Scores are presented as median (min-max) values ​​throughout follow-up, separating intravenous and intrathecal patients, and dots represent raw data. Figure 1 shows the S-CONSORT flowchart. [Diagram 2] Figure 2 - Demonstrating disease progression in the CNS using MRI Patients underwent protocol-planned MRI examinations throughout the study. The most significant changes are shown as indices of change in total plaque volume on FLAIR sequences, the volume of the five largest plaques on FLAIR sequences, and the number of plaques. Change indices on the "y" axis were calculated proportionally from day '0' (just before Treg administration) as 'I00' to the amount of change on subsequent examinations. Changes in values ​​of contrast-enhancing lesions and the number of microbleeds are shown as absolute numbers. Indices and absolute values ​​during the follow-up period are shown as median (minimum-maximum) separately for intravenous and intrathecal patients, and dots represent raw data. Differences between groups are connected with lines and marked with asterisks (*), and changes over time in a particular group are connected with lines and marked with hashes (#). Figure 2 - Demonstrating additional data on disease progression in the CNS using S-MRI Patients underwent planned MRI examinations throughout the study. The charts show the volumetric changes of specific structures of the CNS (gray matter, white matter, brain, brain cortex, brain parenchyma, cerebrospinal fluid) and T1 hypointensity of grey and white matter during the study period. Data are presented as indices of change on the 'y' axis, treating the individual values ​​of the variables from day '0' as 'I00' and calculating the proportional changes at the following tests. Indices are presented as median (min-max) during the follow-up period, separately for intravenous and intrathecal patients, and dots represent raw data. [Diagram 3]Figure 3 - Flow cytometry analysis showing Treg and Tconv levels throughout the study, representative flow histograms and gating strategy [A]. Analysis of the flow data started with the generation of forward vs. side scatter dot plots and a lymphatic gate (PI). This was used to create a CD3 vs. CD4 dot plot and a CD3+CD4+ T cell gate (P2). This gate was used to analyze Treg cells (dot plot in the left column) and CD4+Tconv cells (dot plot in the right column). A Treg gate (P3 left column) was established covering CD127-CD25high cells and FoxP3 expression in this gate was confirmed with a FoxP3 vs. Helios dot plot and used to create three gates: total Tregs FoxP3+ (P4 left column), thymic tTregs FoxP3+Helios+ (P5) and peripheral pTregs FoxP3+Helios- (P6). Levels of total Tregs (CD3+CD4+CD25highCD127 FoxP3+), thymic tTregs (CD3+CD4+CD25highCD127 FoxP3+Helios+), and peripheral pTregs (CD3+CD4+CD25highCD127-FoxP3+Helios) are shown in chart [B]. Additionally, dot plots of CD62L vs. CD45RA were generated to assess the proportion of naïve / memory cells by creating a TregFoxP3+ gate and a TconvFoxP3- gate. The levels of Tn (CD62L+CD45RA+-Q2), Tcm (CD62L+CD45RA-Q1), and Tern (CD62LCD45RA--Q3) in Tregs (CD3+CD4+CD25highCD127-FoxP3+) and Tconvs (CD3+CD4+CD25low / --CD127+FoxP3-) are shown in the chart [C]. The percentage of Treg FoxP3+ expressing the following markers: CCR10, CXCR4, CCR4, CD103, CCR8, CD18, CD39, CD73, CTLA-4, PD-1, 4-1BB, and OX40 was analyzed from all three Treg gates (P4 to P6-left column histograms in [A]). The example histogram shows the analysis of CD39 expression (Q2-1 quadrant of the left column histogram in [A]), but the same was performed for other markers.Using the files obtained for Treg analysis, we also assessed the expression of the same markers in Tconv cells. Tconv was detected by inverting the positions of the P3 and P4 gates originally used for Treg analysis. A Tconv gate covering CD127+CD25low / - cells was established (P3 right column histogram in [A]), and the lack of FoxP3 in this gate was confirmed in a FoxP3 vs. Helios dot plot and used to generate the Tconv FoxP3- gate (P4 right column histogram in [A]). The percentage of Tconv expressing the following markers: CCR10, CXCR4, CD103, CCR8, CD18, CD39, CD73, CTLA-4, PD-1, 4-1BB and 0X40 was analyzed from the P4 gate in the right column histogram. This example shows the analysis of CD39 expression (Q2-1 quadrant of the right column histogram in [A]). The levels of Tregs and Tconvs expressing markers (from P4 gate) are shown as heatmaps. The tree of clusters was aligned to find markers with contrasting expression between Tregs and Tconvs [Heatmap D, detailed levels are also shown in Fig. 3S]. A similar clustering analysis was performed comparing and contrasting thymic tTregs (CD3+CD4+CD25highCD127-FoxP3+Helios+ from P5 in the left column histogram of [A]) with peripheral pTregs (CD3+CD4+CD25highCD127-FoxP3+Helios- from P6 in the left column histogram of [A]) [Heatmap E]. The cutoff for positive signals in the flow analysis shown in [A] was established based on isotype controls and fluorescence minus one (FMO) gating. Arrows indicate the gating hierarchy. The number of dots was reduced to clearly show the populations, especially in the dot plots. The flow cytometer was operationally qualified (OQ) by an independent service operator and regular quality control was performed using CS&T beads (BD Bioscience, USA). Charts [B] and [C] show the percentage of cells at the time of Tregs treatment (day '0'), +3, +6, and +12 months after treatment, divided into / V. and tc. groups. Results are shown as median (min-max), and dots represent raw data.Asterisks [*] in the figures indicate significant differences. Figure 3S - Shows the levels of Treg and Tconv subsets throughout the study (percentage values ​​corresponding to the heatmaps in Figure 3). A. The percentage of Treg and Tconv subsets expressing chemokine receptors or integrins is shown separately for each study group at the time of Treg formulation administration (day '0'), +3, +6, and +12 months after administration. The percentages of CCR10+, CXCR4+, CCR4+, CD103+, CCR8+ and CD18+ cells within Tregs (CD3+CD4+CD25highCD127-FoxP3+) and Tconvs (CD3+CD4+CD25low / -CD127+FoxP3) are shown. Results are shown as median (min-max) and dots represent raw data. B. The percentages of Treg and Tconvs subsets expressing receptors and other molecules important for Treg function are shown for each study group at the time of Treg preparation administration (day '0') and at +3, +6 and +12 months after administration. The percentages of CD39+, CD73+, CTLA-4+, PD-1+, 4-lBB+ and OX40+ cells within Tregs (CD3+CD4+CD25low / -CD127+FoxP3) and Tconvs (CD3+CD4+CD25low / -CD127+FoxP3) are shown. Results are shown as median (min-max) and dots represent raw data. C. tTregs (CD3+CD4+CD25highCD127 FoxP3+Helios+) and pTregs (CD3+CD4+CD25highCD127 FoxP3+Helios) subsets are shown for each study group at day 0, +3, +6 and +12 months after Tregs treatment. Only significantly different subsets are shown: CCR10+, CD103+, CD39+, CD73+ and CTLA-4+. Results are shown as median (min-max) and dots represent raw data. [Figure 4]Figure 4 - Showing serum cytokine levels throughout the study Cytokine levels in the serum of patients who received Tregs intravenously or intrathecally are shown as heat maps. The cluster trees were arranged to find clusters of cytokines with contrasting levels between these two patient groups (detailed levels are also in Figure 4S). Cytokine levels are shown separately for each study group at the time of Tregs administration (day '0'), 14 days, +3 days, +6 days, +9 days and +12 months after administration. Asterisks [*] indicate cytokines whose levels were significantly different between these two patient groups. Figure 4S - Showing serum cytokine levels throughout the study (percentage values ​​corresponding to heat maps in Figure 4) Cytokine levels in patient serum are shown separately for each study group at the time of Tregs administration (day '0'), 14 days, 3 days, 6 days, 9 days and 12 months after administration. Cytokines with significantly different levels between groups are shown in bold. These include TGF-alpha, and MCP3, CXCL8 and IL1RA, which are associated with inflammation. Results are presented as median (min-max) and dots represent raw data. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention is illustrated by the following examples, but is not limited thereto.

[0013] Study Protocol The study was conducted in accordance with the principles of the Declaration of Helsinki. The protocol was registered in the EudraCT database under number 2014-004320-22 and was approved by the Institutional Review Board of the Medical University of Gdansk (nos. NKBBN / 414 / 2012 and NKBBN / 414-163 / 2017). Written informed consent was obtained from all participants at the time of recruitment and before starting any medical procedures.

[0014] Fourteen MS patients (aged 18–55 years) were divided into two groups, receiving Tregs intravenously (iv. n = 11) or intrathecally (tc. n = 3) (Table 1 and Figure IS). One patient from the iv. group dropped out of the study because she became pregnant during the follow-up period. Inclusion criteria were as follows: relapsing-remitting MS (diagnosis according to McDonalds criteria or revised McDonald criteria), with at least one relapse in the past year or at least two relapses in the past two years, Expanded Disability Status Scale (EDSS) score of up to 4, written informed consent available, and adequate venous access for blood sampling. The most important exclusion criterion was immunosuppressants, including interferon beta, administered up to 6 months prior to administration of Tregs preparations. The only exception was glucocorticoids, which could only be administered as a treatment for relapses. Other exclusion criteria were: other autoimmune diseases; diagnosed immunodeficiency; presence or history of active infections, including hepatitis B, hepatitis C, HIV, tuberculosis (TB), and systemic mycoses; history of malignancy; diagnosed cytopenias; elevated thrombotic activity or history of previous thrombosis; hospitalization due to cardiovascular events in the last 2 years prior to enrollment; elevated intracranial pressure defined as papilledema; retinopathy; arterial hypertension; presence or history of macroalbuminuria; undue patient anxiety related to the procedure; any medical condition that the investigator believes would prevent safe participation in the study; known active alcohol or drug abuse; positive pregnancy test (for female subjects); unwillingness to use effective contraception during the study and for 4 months after discontinuation, if appropriate; and intent to reproduce during the study or within 4 months after discontinuation, if appropriate (for male subjects).

[0015] Follow-up began at the time of Treg administration (day "0") and continued for 12 months, with visits at day +14, month +3, month +6, month +9, and month +12 after administration. End points measured included the amount and intensity of side effects of treatment, number of relapses per year, worsening of at least one point on the EDSS scale, change in the Multiple Sclerosis Functional Composite (MSFC) scale, changes in MRI according to the MAGNIMS 2015 consensus, quality of life questionnaire, peripheral blood lymphocyte immunophenotype, and changes in serum cytokine levels. [Table 1]

[0016] Production and administration of Tregs Tregs were prepared under GMP (good manufacturing practice) conditions, as in previous studies [10-13].

[0017] The venous peripheral blood (450 ml) of the patients was sorted using a HEPA-sealed FACS sorter (Influx, BD Bioscience, USA) with exchange sterile sample lines to identify CD3 + CD4 + CD25 high Cells were separated into CD127 lin doublets. The sorting itself was based on staining and gating of cells with GMP-grade monoclonal antibodies (fluorochrome / class / clone) from Miltenyi Biotec (Germany): anti-CD4 (Vio-blue, IgG1, M-T466), anti-CD25 (PE, IgG1, 3G10) and anti-CD127 (APC, IgG1, MB15-18C9). The mean Treg purity after sorting was “98%” (range 97–100%). Additionally, the following monoclonal antibodies (fluorochromes / classes / clones) from BDBiosciences (Poland): anti-CD3 (PacificBlue, IgG1, UCHT1), anti-CD4 (V-500, IgG1, RPA-T4), anti-CD8 (PerCP IgG1, SKI), anti-CD19 (PerCP, IgG1, 4G7), CD14 (PerCP, lgG2b, MfR9), anti-CD16 (PerCP-Cy5.5, IgG1, 3G8), anti-CD25 (PE, IgG1, M-A251), and anti-CD127 (APC, IgG1, hlL-7R-M21) were used to confirm the phenotype and impurities from the sorted Treg samples.

[0018] Intravenous administration involved expansion of Tregs for up to 14 days using clinical grade anti-CD3 / anti-CD28 beads (Miltenyi Biotec), interleukin 2 (aldesleukin, Novartis), and inactivated autologous serum [median (min-max) = 11 (10-14)]. Culture medium (X-Vivo20, Lonza) was supplemented with 10% serum and 1000Ul / ml IL2 throughout the entire period of expansion. Beads were added to cells at a 1:1 ratio early in the expansion, and then the 1:1 ratio was restored during passaging on days +7, +8, and +9. Cultures were washed from the beads and left in 10% serum and low levels of IL2 (100Ul / ml) for the final 24-48 hours of culture. Sentinel cultures with autologous CD4+ Tconvs were performed in 10% serum and low levels of IL2 (100Ul / ml) as a source of T responders for functional testing. The final product at the time of release had FoxP3 expression ≥90% [median (min-max) = 91% (90-97)]; CD62L expression ≥80% [median (min-max) = 87% (81-95)]; passed IFNy suppression assays and were negative for microbiology. Quality control of cultures was performed on day +7 and at the time of product release. IFNy suppression assays were performed as previously described

[14] . Briefly, samples of Tregs from expansion cultures (washed from beads and allowed to rest for at least 24 h) were co-cultured at a 1:1 ratio with autologous sentinel Tconv cells. Controls consisted of cultures of Tconvs or Tregs alone, stimulated or not stimulated to produce IFNy. Just before the assay, Tconvs were stained with the cell tracer CFSE (CFDA kit Thermo, USA) to distinguish Tconvs from Tregs and to show the ratio of IFNy-positive Tregs and Tconvs separately at the end of the assay. Stimulation and staining of cultures was performed using an intracellular staining kit (BDBiosciences, Poland) according to the manufacturer's instructions. Cultures were stimulated for 5 h with 50 ng / ml phorbol 12-myristate 13-acetate, 500 ng / ml ionomycin (Sigma, Poland) and 2 μl / ml of the cytokine leakage inhibitor GolgiPlug (BDBiosciences, Poland). Afterwards, cells were stained with anti-IFNy antibody.A positive readout of this assay was a suppression of IFNy production by Tconvs co-cultured with Tregs of at least 25% [median (min-max) = 69% (52-95)] when compared to IFNy production in cultures of Tconvs alone. IFNy production by Tregs never exceeded 2% of cells. Microbial safety was confirmed by negative microbiological culture results of the growth medium supernatant (BD Bactec system, BD Biosciences, Europe), negative endotoxin testing of the growth medium supernatant (Endosafe-PTS Endotoxin Cartridge / Cartridge reader, Charles River, USA), negative Gram staining of the growth medium supernatant (Gram Stain Kit, BD Biosciences, Europe) and the absence of genetic material of HBV, HCV, HIV-1 and HIV-2 in the product (Cobas MPX, Roche, Europe). Patients were followed up for any adverse symptoms related to possible product contamination until all microbial post-release results were confirmed to be negative. Ready-to-use Treg preparations had to be administered within 2 hours after release from the tissue establishment. Final dose was 40x10. 6 After release, the preparation was thoroughly washed, suspended in 250 ml of 0.9% NaCI for injection (Polfa, Warsaw) and administered slowly intravenously to the patient.

[0019] For intrathecal patients, 1 mln (1x10 6 Freshly isolated Tregs (without proliferation) were examined according to the release criteria above and then suspended in 10 ml of 0.9% NaCI. They were then slowly injected at L4 / L5 or L5 / S1 lumbar puncture with a puncture needle. There was a 6-hour bed regimen after the injection.

[0020] Clinical evaluation Apart from regular physical / neurological examinations at the visits, patients were monitored for disease progression according to the EDSS and MSFC scales

[15] by a board-certified neurologist and for quality of life according to the EQ-5D questionnaire

[16] .The following laboratory tests were performed (only significantly abnormal values ​​are shown): complete blood count, metabolic, renal and liver panel, C-reactive protein level, urinalysis.

[0021] MRI evaluation Brain MRI was performed according to the MAGNIMS 2015 standard protocol (3D 1D1-weighted, 3D T2-FLAIR, 3D T2-weighted, and gadolinium-enhanced 1D1-weighted images after single dose, all with non-gapped section thickness <3 mm, DWI sequences (<5 mm section thickness, 1,5 Tesla Magnetom Aera, Siemens, Germany)). MRI was performed at +3, +6, and +12 months post-dose. Assessment of lesions and their progression was performed using BrainMagix software (Brussels, Belgium) and a Philips Interspace Portal 10, and the total number of plaques and contrast-enhancing plaques were counted by two observers.

[0022] Immune response CD3 in peripheral blood + CD4 + CD25 high CD127-FoxP3 + Tregs and CD3 + CD4 + CD25 low / - CD127 + Immunophenotyping was performed using a 10-color panel to track FoxP3-Tconvs. In both populations, we tracked the expression of antigens important for the function of these subsets. In particular, we characterized the proportion of naive / memory subsets with the following phenotypes: naive / Tn (CD62L + CD45RA + ), Central Memory / Tcm(CD62L + CD45RA), and effector memory / Tem (CD62L The CD3 + CD4+ CD25 high CD127-FoxP3 + Tregs were further divided into peripheral [pTreg Helios(-)] and thymic [tTreg Helios(+)] subsets based on the expression of the transcription factor Helios

[17] (Figure 2S).

[0023] For this procedure, the following anti-human monoclonal antibodies (fluorochrome / class / clone) were purchased from BDBiosciences (Poland): anti-CD3 (PacificBlue / IgG1 / UCHTl or V500-C / IgG1 / clone SK7), anti-CD4 (PerCP or AlexaFluor700 / IgG1 / RPA-T4), anti-CD25 (PE or BV786 / IgG1 / M-A251), anti-CD127 (FITC or BUV737 / IgG1 / hlL-7R-M21), anti-CD45RA (PE-Cy7 / IgG1 / L48), Anti-CD73 (BUV737 / IgG1 / AD2), anti-CD279 (BV605 / IgG1 / EH12.1), anti-CD137 (BV650 / lgGl / 4B4-l), anti-CD134 (BV711 / IgG1 / ACT35), anti-CD152 (BV786 / IgG1 / BNI3), anti-CD18 (FITC / IgG1 / L130), anti-CD184 (PE-CF594 / IgG1 / 12G-5), anti-CD194 (BV605 / IgG1 / lGl), anti-CD39 (BV650 / IgG1 / TU66 or BUV737 / IgG1 / TU66), and anti-CD103 (BUV395 / IgG1 / Ber-ACT8) were used. Anti-CD62L (APC-Cy7 / IgG1 / 3B5) was from Invitrogen (USA); FoxP3 staining kit and anti-Helios (eFluor450 / IgG1 / 22F6) were from ebioscience / thermoFisher (USA); anti-CCR8 (PerCP / IgG1 / 91704) and anti-CCRIO (PE / IgG1 / 314305) were supplied by R&D / Biotechne (UK).

[0024] Serum levels of 38 cytokines: IFN alpha 2, IFN gamma, IL10, IL12p40, IL12p70, IL13, IL15, SCD40L, IL17, IL2, IL1RA, ILlα, ILlbeta, IL3, IL4, IL5, IL6, IL9, TNF alpha, TNF beta, EGF, FGF-2, TGF-alpha, G-CSF, GM-CSF, VEGF, FLT-3L, IL7, eotaxin, CX3CL-1, CXCL-1, MCP-3, CCL22, IL8, IP-10, MCP-1, MIP-1 alpha and MIP-1β were measured by Bead based Multiplex Assay on a luminex analyzer (Merck, USA). All assays were performed according to the manufacturer's instructions.

[0025] statistical analysis Data were calculated using the software Statistica 12.0 (StatSoft, Poland). Cluster analysis was performed with the ClustVis software (https: / / biit.cs.ut.ee / clustvis / #mathematics). Analyses were performed with non-parametric tests. P<0.05 was considered statistically significant.

[0026] result 1.1. Safety No serious adverse events were reported during the study. Patients who received Tregs intravenously (i.v.) experienced moderate adverse events. The most common side effects were recurrence and progression of lesions in the central nervous system. Interestingly, no side effects were observed in patients who received Tregs intrathecally (tc.) (Table 2). [Table 2]

[0027] Analysis of quality of life, as assessed by self-assessment using the EQ-5D form, showed no deterioration: results were similar in both groups throughout the follow-up period (all tests p>0.05, Figure 1).

[0028] 1.2. Efficacy – Clinical No differences were observed between the groups in clinical status as assessed by the EDSS scale throughout the study period [Kruskal-Wallis ANOVA: day 0: H = 0.18 p = 0.66; 6m: H = 0.36 p = 0.54; 12m: H = 0.029 p = 0.86] (Figure 1). However, the deterioration of the EDSS scale after 1 year ranged from 0 to 0.3 in the tc. group and from 0 to 1 in the iv. group. In the iv. group, 3 out of 10 patients showed a deterioration of more than 1 point on the EDSS. No such deterioration was observed in the intrathecal group. A total of 12 relapses were observed in the 5 patients who received intravenous treatment, with a frequency of 1 to 3 times per year. On the other hand, no relapses were observed in the tc. group.

[0029] Clinical status, assessed using the MSFC scale, did not change in any of the groups, and no differences were found between groups in any of the scale components over the study period (all tests p > 0.05, Figure 1 ).

[0030] 1.3.Efficacy – MRI MRI analysis revealed lower disease activity in the tc. group compared with the iv. group (Figure 2).

[0031] On FLAIR sequences, plaque volume in the CNS increased in the iv. group but remained unchanged in the tc. group [Friedman's ANOVA: iv.: x2 = 12.79 p = 0.005; tc.: x2 = 4.5 p = 0.21]. The difference between the two groups was significant at 6 and 12 months of follow-up [Kruskal-Wallis ANOVA: 3m: H = 1.65 p = 0.19; 6m: 1-1 = 6.14 p = 0.013; 12m: H = 5.33 p = 0.047]. Differences were also observed in the volume of the five largest plaques [Kruskal-Wallis ANOVA: 3m: H = 0.01 p = 0.91; 6m: H = 7.77 p = 0.005; 12m: H = 2.34 p = 0.067] and the number of new plaques [Kruskal-Wallis ANOVA: 3m: H = 3.76 p = 0.15; 6m: H = 5.10 p = 0.076; 12m: H = 4.61 p = 0.091] compared between groups. Interestingly, the increase in the total plaque volume during the follow-up period was not due to changes in the existing largest plaques [Friedman's ANOVA on the average volume from the five largest plaques: iv.: x2 = 3.66 p = 0.30; tc.: x2 = 3.90 p = 0.27], but rather to an increase in the number of plaques in the iv. group [Friedman's ANOVA on the number of plaques: iv. x2 = 20.77 p = 0.0001; tc. x2 = 5.5 p = 0.13]. Also, the iv. group had a significant decrease in contrast-enhancing T1 lesions at the end of the study. However, these lesions were not observed during the follow-up period in the tc. group [Friedman's ANOVA: iv.: x2 = 11.41 p = 0.009; tc.: total '0']. The volumes of the main structures of the CNS and the volumes of T1 hypointensities did not differ between both groups (Fig. 3S).

[0032] Immune response Treg subsets FoxP3 +The levels of Tregs and Tconvs did not change significantly during the follow-up period or between groups (p>0.05 for all tests, Fig. 3). However, in all patients, regardless of the route of Treg administration, Tregs differed from Tconvs in several measured subsets. Comparing Tregs and Tconvs across all patients, Tregs predominantly comprised the Tcm phenotype (more than 50%), whereas Tconvs predominantly comprised the Tn phenotype (more than 50%) (Fig. 3C and Table 1S). It was also revealed that Tregs expressed multiple receptors that were barely detected in Tconvs, including the chemokine receptors CCR10, CXCR4, and CCR4, the integrin CD103, the ectonucleotidase CD39, and two costimulatory molecules CTLA-4 and 4-1BB (Fig. 3S-A,B and Table 1S). The differences between Tregs and Tonvs in the expression of these receptors were confirmed by cluster analysis (Fig. 3D).

[0033] Furthermore, Tregs from approximately 20% of all patients did not express the transcription factor Helios, suggesting a peripheral origin of these cells (Fig. 3B). + Helios(+)tTregs and peripheral FoxP3 + We further analyzed the Helios(-)pTregs and Helios(-)pTregs. + cells, CD103 + cells, CD73 + cells, CD39 + A high proportion of pTreg cells were CTLA-4 + The percentage of tTreg and pTreg cells was higher (Fig. 3S-C and Table 1S). Cluster analysis confirmed that the differences between tTreg and pTreg were high expression of CCR10, CD103, and CD39, and low expression of CTLA-4 receptor (Fig. 3E). [Table 3] TIFF0007624683000004.tif235134

[0034] Cytokines The study also included an array of 38 cytokines measured in the serum of patients. Compared with i.v.-treated patients, i.t.-treated patients had higher levels of several factors related to inflammation, including MCP-3, IL1RA, and IL8. Interestingly, levels of the brain trophic factor TGFa were also higher in the tc.-treated group than in the iv.-treated group (Table 2S, Figure 4S). Levels of MCP-3 and IL1RA, located in the same cluster, were different between the tc.-treated and iv.-treated groups (Figure 4). Levels of the other measured cytokines did not differ between the study groups and within each group during the follow-up period. [Table 4]

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Claims

1. A pharmaceutical product comprising CD3+CD4+CD25+CD127- regulatory T cells for clinical use in multiple sclerosis, said pharmaceutical product being administered intrathecally.

2. Use of CD3+CD4+CD25+CD127- regulatory T cells for the preparation of a pharmaceutical product for intrathecal administration for the treatment of multiple sclerosis.

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