INTRATHECAL ADMINISTRATION OF T REGULATORY CELLS IN THE TREATMENT OF MULTIPLE SCLEROSIS

The invention relates to the medicinal product consisting of CD3+CD4+CD25+CD127-T regulatory cells administered intrathecally in the treatment of multiple sclerosis.

The invention relates the medicinal product consisting of CD3+CD4+CD25+CD127− T regulatory cells for clinical use in the treatment. The product is administered intrathecally in the treatment of multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated disease in which autoimmune T conventional cells (Tconvs) sensitized against myelin sheath break blood-brain barrier and destroy neurons of the central nervous system (CNS). It is hypothesized that CD4+CD25highCD127−FoxP3+regulatory T cells (Tregs) may inhibit this destruction through suppressive activity exerted on Tconvs.

Tregs lymphocytes constitute for about 1% of all peripheral blood lymphocytes, but are important for maintaining the tolerance of their own tissues. Lack of regulatory T cells leads to a number of autoimmune diseases and hypersensitivity, as seen in the case of patients with X-linked immunodeficiency syndrome, polyendocrinopathy and enteropathy (IPEX). One of such autoimmune syndromes is also multiple sclerosis.

Treg lymphocytes can be called “intelligent steroids” because as steroids, they inhibit inflammatory reactions and act immunosuppressively, but in contrast the physiological suppressor effect of Treg cells concerns only pathological reactions (eg. directed against its own tissues). The results of clinical trials, including the inventors observations, indicate that therapy with Treg lymphocytes is safe and does not impair the immune response against foreign and dangerous antigens (viruses, bacteria, cancer cells).

The invention provides the way of administration of medicinal product consisting of CD3+CD4+CD25+CD127− T regulatory cells via intrathecal injection.

The subject of invention is the medicinal product consisting of CD3+CD4+CD25+CD127− T regulatory cells.

The product is administered intrathecally for the treatment of patients diagnosed with multiple sclerosis

The product is administered intrathecally.

The product is administered in multiple sclerosis.

The Present Invention are Illustrated by the Following Examples, Which are not its Limitation.

Protocol of the study

The study was conducted according to the Declaration of Helsinki principles. The protocol has been registered in the EudraCT database under the number 2014-004320-22 and received approval from the Institutional Review Board of the Medical University of Gdansk (no. NKBBN/414/2012 and NKBBN/414-163/2017). Written informed consent was received from all the participants at the recruitment, before any medical procedure was commenced.

Fourteen MS patients (18-55 yo) were recruited into the two groups treated with Tregs either intravenously (iv. n=11) or intrathecally (tc. n=3) (Table 1 andFIG.1S). One patient from iv. group dropped out of the trial due to pregnancy during the follow-up. The inclusion criteria were as follows: relapsing-remitting form of MS (diagnosed according to the McDonalds criteria or revised McDonald criteria) with at least 1 relapse during the last year or at least 2 relapses in the preceding 2 years, up to 4 points on the Expanded Disability Status Scale (EDSS), ability to provide the written informed consent, and an appropriate venous access for blood drawing. The most important exclusion criterion was any immunosuppression including interferon beta administered up to 6 months before the administration of the Tregs preparation. The only exception were glucocorticoids which could be administered as the treatment for relapses only. Other exclusion criteria included: other autoimmune diseases; diagnosed immunodeficiencies; presence or history of active infections, including hepatitis B, hepatitis C, HIV, tuberculosis (TB), systemic fungal infections; any history of malignancy; diagnosed cytopenias; elevated thrombotic activity or history of past thrombosis; hospitalization for cardiovascular events in the last 2 years before the inclusion; increased intracranial pressure defined as the papilledema; any retinopathy; arterial hypertension; presence or history of macroalbuminuria; excessive anxiety of the patient related to the procedures; any medical condition that, in the opinion of the investigator, may interfere with safe participation in the trial; known active alcohol or substance abuse; positive pregnancy test (for female subjects), unwillingness to use effective contraceptive measures during the study and for 4 months after discontinuation, when appropriate: intent to procreate during the study or within 4 months after discontinuation, when appropriate (for male subjects).

The follow-up started at administration of Tregs (day “0”) and lasted 12 months with the visits at: +14 days, +3 months, +6 months, +9 months, and +12 months post-administration. The endpoints measured included the amount and intensity of the therapy side effects, the number of annual relapses, worsening on the EDSS scale by at least 1 point, changes in the Multiple Sclerosis Functional Composite (MSFC) scale, changes in MRI according to the MAGNIMS 2015 consensus, changes in quality of Life Questionnaire (QOL), peripheral blood lymphocyte immunophenotype, and serum cytokines levels.

Manufacturing and administration of Tregs

The preparation of Tregs was manufactured under Good Manufacturing Practice (GMP) conditions similarly to our previous trials [10-13].

For intravenous administration, the expansion of Tregs was performed using clinical-grade anti-CD3/anti-CD28 beads (Miltenyi Biotec), interleukin 2 (aldesleukin, Novartis), and inactivated autologous serum for up to 14 days [median (min-max)=11(10-14)]. The medium (X-Vivo20, Lonza) was supplemented with 10% serum and 1000 UI/ml of IL2 throughout the entire expansion. The beads were added to the cells in the 1:1 ratio at the beginning of expansion and then during passages on days +7, +8 and +9 to restore 1:1 ratio. The culture was washed out from beads and left in 10% serum and low level of IL2 (100 UI/ml) for the last 24-48 h of the culture. The sentinel culture with autologous CD4+ Tconvs was performed in 10% serum and low level of IL2 (100 UI/ml) as a source of T responders for functional tests. The final product on release kept FoxP3 expression above 90% [median (min-max)=91%(90-97)]; CD62L expression above 80% [median (min-max)=87%(81-95)]; passed IFNγsuppression assay and microbiological tests were negative. The quality control of the cultures was performed on day +7 and on the release of the product. IFNγsuppression assay was performed as previously described [14]. Briefly, a sample of Tregs from the expansion cultures (washed out from the beads and left resting for at least 24 h) were cocultured with autologous sentinel Tconv cells in 1:1 ratio. The controls consisted of the cultures of Tconvs or Tregs only, either stimulated or not stimulated to produce IFNγ. Immediately prior to the assay, Tconvs were stained with cell tracer CFSE (CFDA kit Thermo, USA) in order to distinguish them from Tregs and therefore it was possible to give separately the proportions of IFNγ-positive Tregs and Tconvs at the end of the assay. The stimulation of the cultures and staining was performed with intracellular staining kit (BDBiosciences, Poland) according to the manufacturer description. The cultures were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate, 500 ng/ml of ionomycin (Sigma, Poland) and 2 μl/ml of cytokine leakage inhibitor GolgiPlug (BDBiosciences, Poland) for 5 h. Then, the cells were stained with anti-IFNγantibodies. The positive readout of the assay was the suppression of IFNγproduction by Tconvs cocultured with Tregs by at least 25% [median (min-max)=69% (52-95)], when compared to the production of IFNγin the cultures with Tconvs only. The production of IFNyby Tregs never exceeded 2% of the cells. The microbial safety was confirmed through negative results of microbiology cultures of supernatants from expansion media (BD Bactec system, BDBiosciences, Europe), negative endotoxin tests from supernatants of expansion media (Endosafe-PTS Endotoxin Cartridge/Cartridge reader, Charles River, USA), negative Gram staining of the supernatants from expansion media (Gram Stain Kit, BDBiosciences, Europe) and the absence of genetic material of HBV, HCV, HIV-1 and HIV-2 in the product (Cobas MPX, Roche, Europe). The patients were followed for any adverse symptoms related to the possible contamination of the product until all microbial post-release results were confirmed negative. The ready-to-use preparation of Tregs had to be administered within 2 hours of the release from the tissue establishment. The final dose was 40×106of Tregs/kg b.w. Upon release, the preparation was washed out completely, suspended in 250 ml of 0.9% NaCl for injection (Polfa, Warsaw), and then administered in slow intravenous infusion to the patient.

For patients treated intrathecally, 1 min (1×106) of freshly isolated Tregs (without expansion) was examined according to the release criteria described above and then suspended in 10 ml of 0.9% NaCl. Afterwards, it was administered in a slow injection during L4/L5 or L5/S1 lumbar puncture through a puncture needle. There was a 6-hour bed regimen post-injection.

Clinical Assessment

Apart from routine physical/neurological examinations at the site visits, patients were also assessed according to the EDSS and MSFC scales by certified neurologists [15] to monitor the disease progression and according to EQ-5D questionnaire to monitor the quality of life [16]. The following lab tests were performed (only significantly abnormal values are shown): complete blood count, metabolic, kidney and liver panels, C-reactive protein levels, urinalysis.

MRI Assessment

MRI of the brain was performed according to the MAGNIMS 2015 standard protocol (3D T1-weighted, 3D T2-FLAIR, 3D T2-weighted, and post-single-dose gadolinium-enhanced T1-weighted imaging, all with a nongapped section thickness of ≤3 mm, and a DWI sequence (≤5 -mm section thickness, 1,5 Tesla Magnetom Aera, Siemens, Germany). MRI was performed during visits at +3 months, +6 months, and +12 months post-administration. The assessment of lesions and their progression were made using BrainMagix software (Brussels, Belgium) and Philips Intellispace Portal 10, the total number of plaques and contrast-enhanced plaques were counted by two observers.

Immune responses

Immune phenotyping was performed using ten-color panels to follow CD3+CD4+CD25highCD127−FoxP3+Tregs and CD3+CD4+CD25low/−CD127+FoxP3−Tconvs in the peripheral blood. In both populations, the expression of antigens important for the functioning of these subsets was followed. We specifically determined the percentage of naïve/memory subsets based on the following phenotypes: naïve/Tn (CD62L+CD45RA+), central memory/Tcm (CD62L+CD45RA−), and effector memory/Tem (CD62L−CD45RA−). CD3+CD4+CD25highCD127−FoxP3+Tregs were further divided based on the expression of transcription factor Helios into the peripheral [pTreg Helios(−)] and thymic [tTreg Helios(+)] subsets [17] (FIG.2S).

Statistical analysis

Data were computed with the software Statistica 12.0 (Statsoft, Poland). Cluster analysis was performed with ClustVis software (https://biit.cs.ut.ee/clustvis/#mathematics). The analysis was carried out with nonparametric tests. P≤0.05 was considered statistically significant.

Results

No serious adverse events were reported throughout the trial. Moderate adverse effects were noted in patients treated with Tregs intravenously (iv.). The most common adverse effects were relapses and progression of lesions in the CNS. Interestingly, no adverse effects were noted in patients administered with Tregs intrathecally (tc.) (Table 2).

TABLE 2Adverse effects in the trialAdverse eventNumber of patients/eventsSeverityTregs administered intravenouslyRelapse of MS5/12 (3 patients experienced 3 relapses,Moderate1 patient experienced 2 relapses,and 1 patient experienced 1 relapse)Progression of changes5/5Moderatein the CNS on MRIProgression of visual impairment1/1ModerateLiver injury (increased ASPAT1/1Moderateand ALAT without clinicalsymptoms, unknown etiology)Tregs administered intrathecallyNo adverse events reported

The analysis of the quality of life revealed no deterioration in the self-assessment using EQ-5D form. The results were similar in both groups throughout the follow-up (all tests p>0.05,FIG.1).

The clinical status assessed using EDSS scale did not differ between the groups throughout the study [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] (FIG.1). However, one-year deterioration on the EDSS scale within the tc. group and within the iv. group was from 0 to 0.3 and from 0 to 1, respectively. In the iv. group, 3 out of 10 subjects revealed a deterioration higher than 1 point on the EDSS scale. No such a deterioration was seen in those treated intrathecally. Total of 12 relapses were noted in 5 patients treated intravenously with the frequency from 1 to 3 episodes per year during the follow-up. At the same time, no relapses were observed in the tc. group.

The clinical status assessed using the MSFC scale did not change in any group and did not differ between the groups in any of the scale components throughout the study (all tests p>0.05,FIG.1).

When compared to iv. group, the analysis of MRI scans revealed a lower activity of the disease in the tc. group (FIG.2).

The FLAIR sequence revealed that the total volume of plaques in the CNS throughout the follow-up increased in iv. group while it did not change in tc. group [Friedman's ANOVA: iv.: X2=12.79 p=0.005; tc.: x2=4.5 p=0.21]. The difference between the groups was significant at 6 and 12 month of the follow up [Kruskal-Wallis ANOVA: 3m: H=1.65 p=0.19; 6m: H=6.14 p=0.013; 12m: H=5.33 p=0.047]. The difference was also seen when the volume of the five biggest 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] were compared between the groups. Interestingly, it was the increasing number of the plaques in iv. group [Friedman's ANOVA for the number of the plaques: iv.: X2=20.77 p=0.0001; tc.: x2=5.5 p=0.13] rather than the changes of the existing the biggest plaques [Friedman's ANOVA for mean volume from 5 biggest plaques: iv.: X2=3.66 p=0.30; tc.: X2=3.90 p=0.27] were responsible for the increase in the total volume of the plaques during the follow-up. In addition, contrast-enhanced T1 lesions in iv. group decreased significantly at the end of the trial. This was not the case of tc. patients as these lesions were not seen in this group throughout the follow-up [Friedman's ANOVA: iv.: X2=11.41 p=0.009; tc.: all numbers ‘0’]. Neither the volume of the main CNS structures or the volume of T1 hypointensiveness differed between the groups (FIG.3S).

1.4. Immune response

There were no significant changes in the level of FoxP3+Tregs and Tconvs throughout the follow-up or between the groups (all tests p>0.05,FIG.3). However, Tregs differed from Tconvs in several measured subsets in all patients, regardless the route of administration of Tregs. When all patients were taken into account, when compared Tregs to Tconvs, Tregs contained mostly Tcm phenotype (50% or more), while Tconvs contained mostly Tn phenotype (50% or more) (FIG.3Cand Table 1S ). We have also found that Tregs expressed several receptors, such as chemokine receptors CCR10, CXCR4, CCR4, integrin CD103, ectonucleotidase CD39, and two costimulatory molecules— CTLA-4 and 4-1BB, which were almost undetectable on Tconvs (FIG.3S-A,B and Table 1S). The difference between Tregs and Tonvs in the expression of these receptors was confirmed with cluster analysis (FIG.3D).

In addition, around 20% of Tregs in all patients did not express the transcription factor Helios suggesting peripheral origin of these cells (FIG.3B). Having that in mind, we performed deeper analysis dividing Tregs into thymic FoxP3+Helios(+) tTregs and peripheral FoxP3+Helios(−) pTregs. When compared, tTregs contained higher percentage of CCR10+cells, CD103+cells, CD73+cells, and CD39+cells, while pTreg contained higher percentage of CTLA-4+cells (FIG.3S-Cand Table 1S). The cluster analysis confirmed that the higher expression of CCR10, CD103, CD39 and lower expression of CTLA-4 receptors differs tTregs from pTregs (FIG.3E).

The study included also the array of 38 different cytokines measured in the sera of patients. When compared to the intravenously treated patients, those treated intrathecally revealed higher levels of some factors associated with inflammation, such as MCP-3, IL1RA and IL8. Interestingly, also the level of brain trophic factor TGFα was higher in tc. group than in the iv. group (Table 2S,FIG.4S). The levels of MCP-3, IL1RA positioned in the same cluster differing tc. group from iv. group (FIG.4). The levels of other measured cytokines did not differ between the trial groups or within each group throughout the follow-up.

TABLE 2SThe significance of differences in the serum level of cytokinesbetween iv. patients and tc. patients. Only the statisticsof cytokines with significant differences is presented. (Kruskal-Wallis ANOVA, p ≤ 0.05 as significant in red)iv. patients vs. tc. patientscytokinetimeHpMCP-302.740.04814days0.480.483m3.800.036m0.020.889m5.830.0112m2.370.048IL8 (CXCL8)00.150.6914days0.220.633m0.200.656m2.890.0419m2.150.06912m2.920.046IL1RA00.970.3214days3.210.0373m2.140.086m0.070.779m4.110.02412m2.650.047TGF-α04.250.0314days0.940.543m2.210.086m3.500.039m3.050.0412m0.970.37

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