IMPROVED TREATMENT METHODS USING DMDS FOR THE TREATMENT OF AUTOIMMUNE DISEASES, AND BIOMARKER FOR PREDICTING AND/OR OPTIMISING SAID TREATMENT METHODS

Abstract

The invention pertains to improved treatment methods using DMDS, in particular cladribine, for the treatment of autoimmune diseases, in particular multiple sclerosis, and biomarkers, in particular immune cell subtypes, for predicting and/or optimising said treatment methods. The methods described rely on the use of immune cell subtypes, in particular T cells, B cells, NK cells subtypes and their ratios as present in blood samples from the patients for predicting and/or optimising the use of cladribine in the treatment of multiple sclerosis.

Claims

1.-7. (canceled)

8. A method to predict the response of a patient treated with cladribine for the treatment of an autoimmune disorder, a) wherein deviations of cell populations of: CD19.sup.+B cells of at least 70%, memory CD19.sup.+B cells of at least 70%, nave B cells of at least 70%, memory B cells of at least 70%, activated B cells, and/or activated CD69+ B cells of at least 45%, and/or combinations thereof; are indicative of early efficacy of said cladribine treatment; wherein: cell populations are determined in one or more body fluids samples of said patient at different stages of said cladribine treatment including determining said cell population in a body fluids sample from the patient collected before the start of treatment with cladribine and determining said cell population in a body fluids sample from the patient collected at at least a later point in time during or after treatment with cladribine, said deviations are given as decrease from the baseline levels; b) wherein deviations of cell populations of: CD19.sup.+B cells of at least 70%, CD38+ Plasma cells of at least 50%, Short lived plasma ells of at least 50%, Bregs of at least 70%, transitional B cells of at least 70%, nave T cells of at least 10%, T central memory cells of at least 40%, T central effector cells of at least 30%, TH1 cells of at least 35%, TH17 cells of at least 35%, and/or combinations thereof; are indicative of durable efficacy of said cladribine treatment at 1 year; wherein: cell populations are determined in one or more body fluids samples of said patient at different stages of said cladribine treatment including determining said cell population in a body fluids sample from the patient collected before the start of treatment with cladribine and determining said cell population in a body fluids sample from the patient collected at at least a later point in time during or after treatment with cladribine, said deviations are given as decrease from the baseline levels; c) wherein: a deviation of cell populations of memory B cells to a level above 40%, a deviation of cell populations of plasma cells to a level above 20%, a deviation of cell populations of Tnaive cells to a level above 40%, a deviation of cell populations of CD4+ Central memory T cells to a level above 20%, a deviation of cell populations of CD4+ Effector Memory T cells to a level above 10% a deviation of cell populations of the Treg/Teff cell ratio wherein the deviation is a decrease, a deviation of cell populations of the Breg/B memory cell ratio wherein the deviation is a decrease, a deviation of cell populations of the NKreg/NK eff cell ratio wherein the deviation is a decrease, and/or combinations thereof, are indicative of a risk of reactivation of multiple sclerosis; wherein: cell populations are determined in one or more body fluids samples of said patient at different stages of said cladribine treatment including determining said cell population in a body fluids sample from the patient collected before the start of treatment with cladribine and determining said cell population in a body fluids sample from the patient collected at at least a later point in time during or after treatment with cladribine, said deviations are from the baseline levels.

9. The method of claim 8, wherein the autoimmune disorder is multiple sclerosis.

10. A method for monitoring the need for adapting the treatment of an autoimmune disorder with a Disease Modifying Drug (DMD) of a patient in need thereof, wherein the patient has been treated with a DMD which is cladribine for the treatment of an autoimmune disorder, wherein deviations of cell populations as determined in one or more body fluids samples of said patient at different stages of said cladribine treatment including determining said cell population in a body fluids sample from the patient collected before the start of treatment with cladribine and determining said cell population in a body fluids sample from the patient collected at at least a later point in time during or after treatment with cladribine are selected from the list consisting in: a deviation of cell populations of memory B cells to a level above 40%, a deviation of cell populations of plasma cells to a level above 20%, a deviation of cell populations of Tnaive cells to a level above 40%, a deviation of cell populations of CD4+ Central memory T cells to a level above 20%, a deviation of cell populations of CD4+ Effector Memory T cells to a level above 10%, a deviation of cell populations of the Treg/Teff cell ratio wherein the deviation is a decrease, a deviation of cell populations of the Breg/B memory cell ratio wherein the deviation is a decrease, a deviation of cell populations of the NKreg/NK eff cell ratio wherein the deviation is a decrease, and/or combinations thereof; wherein said deviations are indicative of a need for a retreatment of said patient with cladribine.

11. The method of claim 10, wherein the autoimmune disorder is multiple sclerosis.

12. A method for the treatment of an autoimmune disorder with a Disease Modifying Drug (DMD) of a patient at risk of reactivation of multiple sclerosis, wherein the patient has been treated with a DMD which is cladribine for the treatment of an autoimmune disorder, wherein deviations of cell populations as determined in one or more body fluids samples of said patient at different stages of said cladribine treatment including determining said cell population in a body fluids sample from the patient collected before the start of treatment with cladribine and determining said cell population in a body fluids sample from the patient collected at at least a later point in time during or after treatment with cladribine are selected from the list consisting in: a deviation of cell populations of memory B cells to a level above 40%, a deviation of cell populations of plasma cells to a level above 20%, a deviation of cell populations of Tnaive cells to a level above 40%, a deviation of cell populations of CD4+ Central memory T cells to a level above 20%, a deviation of cell populations of CD4+ Effector Memory T cells to a level above 10%, a deviation of cell populations of the Treg/Teff cell ratio wherein the deviation is a decrease, a deviation of cell populations of the Breg/B memory cell ratio wherein the deviation is a decrease, a deviation of cell populations of the NKreg/INX eff cell ratio wherein the deviation is a decrease, and/or combinations thereof; are indicative that the patient is a patient at risk of reactivation of multiple sclerosis, wherein said treatment comprises administering to the patient a retreatment with cladribine, the administration of an additional and/or higher dose of cladribine or the administration of a different DMD.

13. The method of claim 12, wherein the autoimmune disorder is multiple sclerosis.

14. The method of claim 12, wherein the retreatment with cladribine comprises the oral administration of a formulation comprising cladribine, wherein the formulation is to be orally administered following the sequential steps below: (i) an induction period wherein said cladribine formulation is administered and wherein the total dose of cladribine reached at the end of the induction period is from about 1.7 mg/kg to about 3.5 mg/kg; (ii) a cladribine-free period of between about 8 and about 10 months wherein no cladribine formulation is administered; (iii) a maintenance period wherein said cladribine formulation is administered and wherein the total dose of cladribine reached at the end of the maintenance period is lower than the total dose of cladribine reached at the end of the induction period (i); and (iv) a cladribine-free period wherein no cladribine formulation is administered.

15. A method of monitoring the response of a patient having an autoimmune disease chosen among autoimmune neurological and neuroinflammatory diseases, to treatment with a disease-modifying drug (DMD) said method comprising: a) obtaining the absolute numbers of any of: total PBMCs; CD19.sup.+ B cells; CD19.sup.+ memory B cells; naive B cells; CD4.sup.+ Central memory T cells; CD4.sup.+ Effector memory T cells; Treg; Teff; Breg; memory B cells; CD16.sup.dim CD56.sup.bright NK cells; CD16.sup.bright CD56.sup.dim NK cells; transitional B cells; in a first and a second biological sample from the patient, wherein any of: an increase of the absolute number of CD19+ B cells or a decrease of 30% or less of the absolute number of CD19+ B cells, in the second biological sample compared to the first biological sample; an increase of the absolute number of CD19+ memory B cells or a decrease of 30% or less of the absolute number of CD19+ memory B cells, in the second biological sample compared to the first biological sample; and/or an increase of the absolute number of naive B cells or a decrease of 30% or less of the absolute number of naive B cells, in the second biological sample compared to the first biological sample; an increase of the absolute number of CD4+ Central memory T cells or a decrease of 20% or less of the absolute number of CD4+ Central memory T cells, in the second biological sample compared to the first biological sample; an increase of the absolute number of CD4.sup.+ Effector memory T cells or a decrease of 10% or less of the absolute number of CD4.sup.+ Effector memory T cells, in the second biological sample compared to the first biological sample; a decrease in the ratio of the absolute number of Treg over the absolute number of Teff in the second biological sample compared to said ratio in the first biological sample; a decrease in the ratio of the absolute number of Breg cells over the absolute number of memory B cells in the second biological sample compared to said ratio in the first biological sample; a decrease in the ratio of the absolute number of CD16.sup.dim CD56.sup.bright NK cells over the absolute number of CD16.sup.bright CD56.sup.dim NK cells in the second biological sample compared to said ratio in the first biological sample; a percentage of memory B cells over total PBMCs of between 0.05% and 1% in the second biological sample; a percentage of Breg cells over total PBMCs of between 3% and 14% in the second biological sample; a ratio of Transitional B cells:memory B cells of superior or equal to 1, in the second biological sample; is indicative of a sub-optimal response of the patient to the treatment.

16. The method of claim 15, wherein the autoimmune disease is multiple sclerosis.

17. A method of treating a patient having an autoimmune disease chosen among autoimmune neurological and neuroinflammatory diseases, to treatment with a disease-modifying drug (DMD) said method comprising: a) obtaining a first biological sample from the patient, b) administering the treatment to the patient; c) obtaining a second biological sample from the patient; d) determining in the first and the second biological sample the absolute numbers of any of: total PBMCs; CD19.sup.+ B cells; CD19.sup.+ memory B cells; naive B cells; CD4.sup.+ Central memory T cells; CD4.sup.+ Effector memory T cells; Treg; Teff; Breg; memory B cells; CD16.sup.dim CD56.sup.bright NK cells; CD16.sup.bright CD56.sup.dim NK cells; transitional B cells; wherein any of: an increase of the absolute number of CD19+ B cells or a decrease of 30% or less of the absolute number of CD19+ B cells, in the second biological sample compared to the first biological sample; an increase of the absolute number of CD19+ memory B cells or a decrease of 30% or less of the absolute number of CD19+ memory B cells, in the second biological sample compared to the first biological sample; and/or an increase of the absolute number of naive B cells or a decrease of 30% or less of the absolute number of naive B cells, in the second biological sample compared to the first biological sample; an increase of the absolute number of CD4+ Central memory T cells or a decrease of 20% or less of the absolute number of CD4+ Central memory T cells, in the second biological sample compared to the first biological sample; an increase of the absolute number of CD4.sup.+ Effector memory T cells or a decrease of 10% or less of the absolute number of CD4.sup.+ Effector memory T cells, in the second biological sample compared to the first biological sample; a decrease in the ratio of the absolute number of Treg over the absolute number of Teff in the second biological sample compared to said ratio in the first biological sample; a decrease in the ratio of the absolute number of Breg cells over the absolute number of memory B cells in the second biological sample compared to said ratio in the first biological sample; a decrease in the ratio of the absolute number of CD16.sup.dim CD56.sup.bright NK cells over the absolute number of CD16.sup.bright CD56.sup.dim NK cells in the second biological sample compared to said ratio in the first biological sample; a percentage of memory B cells over total PBMCs of between 0.05% and 1% in the second biological sample; a percentage of Breg cells over total PBMCs of between 3% and 14%; a ratio of Transitional B cells:memory B cells of superior or equal to 1, in the second biological sample; is indicative of a sub-optimal response of the patient to the treatment, administering to the patient with a sub-optimal response to the treatment a retreatment with cladribine, an additional and/or higher dose of cladribine or the administration of a different DMD.

18. The method of claim 17, wherein the retreatment with cladribine comprises the oral administration of a formulation comprising cladribine, wherein the formulation is to be orally administered following the sequential steps below: (i) an induction period wherein said cladribine formulation is administered and wherein the total dose of cladribine reached at the end of the induction period is from about 1.7 mg/kg to about 3.5 mg/kg; (ii) a cladribine-free period of between about 8 and about 10 months wherein no cladribine formulation is administered; (iii) a maintenance period wherein said cladribine formulation is administered and wherein the total dose of cladribine reached at the end of the maintenance period is lower than the total dose of cladribine reached at the end of the induction period (i); and (iv) a cladribine-free period wherein no cladribine formulation is administered.

19. The method of claim 17, wherein the autoimmune disease is multiple sclerosis.

20. A method of treating an autoimmune disorder in a patient in need thereof, said method comprising orally administering cladribine to said patient in an amount of 0.5 to 1.25 mg per kilogram of body weight of said patient in a time period between within 4 to 12 weeks, wherein, after said administration, said patient is having one or more deviations with regard to one or more of the cell populations, selected from the group consisting of: a) wherein the following deviations are biomarkers/correspond to early efficacy of said cladribine treatment, given as % decrease from the baseline levels: CD19.sup.+B cells (>70%+/10%), memory CD19.sup.+B cells (>70%+/10%), nave B cells (>70%+/10%), memory B cells (>70%+/10%), activated B cells, and/or activated CD69.sup.+ B cells (>45+/10%), and/or combinations thereof; b) Durable efficacy at 1 year (% decrease from baseline): memory CD19+, B cells (>70%+/10%), CD38+ Plasmacells (>50%+/10%), Short lived plasmacells (>50%+/10%), Bregs (>70%+/10%), transitional B cells (>70%+/10%), nave T cells (>10%+/5%), T central memory cells (>40%+/10%), T central effector cells (>30%+/10%), TH1 cells (>35+/10%), TH17 cells (>35+/10%), and/or combinations thereof; c) Durable efficacy: a NKreg/NKeff cell ratio (>1.5 fold), a Breg/Beff cell ratio (>/<fold), a Treg/Teff cell ratio (>/<fold), and/or d) Reactivation of disease decrease of Memory B cells from baseline (<30%+/10%), memory B cells (0.05 and 1% of PBMCs), a transitional B cell ratio in total PBMC, Bregs in whole blood or PBMCs from active or reactivating MS patients (3-14%), and/or e) Reactivation of Disease: memory B cells (repopulating to a level above 40% from baseline), plasma cells (repopulating to a level above 20% from baseline), Tnaive cells (repopulating to a level above 40% from baseline), CD4+ Central memory T cells (repopulating to a level above 20% from baseline), CD4+ Effector Memory T cells (repopulating to a level above 10% from baseline), a Treg/Teff cell ratio (decreased from baseline), and/or a Breg/B memory cell ratio (decreased compared to baseline), and/or a NKreg/NK eff cell ratio (decreased compared to baseline); and/or combinations thereof.

21. The method of claim 20, wherein the autoimmune disorder is multiple sclerosis.

Description

LEGEND OF THE FIGURES

[0535] FIG. 1: B cell differentiation pathways and expression of TrB-associated molecules.

[0536] FIG. 2: Main functions of human CD19+CD24hiCD38hi TrB cells discovered

[0537] FIG. 3. Study design of MAGNIFY-MS including the immune cell subtype analyses conducted as part of this sub-study. 9HPT, 9-hole peg test; BL, baseline; CUA, combined unique active; DMT, disease-modifying therapy; EDSS, Expanded Disability Status Scale; HIV, human immune deficiency virus; KFS, Kurtzke Functional System; MRI, magnetic resonance imaging; MS, multiple sclerosis; SDMT, symbol digit modalities test; T25FW, timed 25-foot walk; TB, tuberculosis.

[0538] FIG. 4. Median absolute values of B and T cell counts in patients treated with cladribine tablets. The first treatment course of cladribine tablets was administered at baseline and Month 1. BL, baseline; Q, quartile; TEMRA, terminally differentiated effector memory RA+; Th, T helper.

[0539] FIG. 5. Median absolute values of NK cell counts in patients treated with cladribine tablets. The first treatment course of cladribine tablets was administered at baseline and Month 1. BL, baseline; Q, quartile.

[0540] FIG. 6. Median serum concentration of immunoglobulins in patients treated with cladribine tablets. The first treatment course of cladribine tablets was administered at baseline and Month 1. BL, baseline; Ig, immunoglobulin; Q, quartile.

[0541] FIG. 7. Immune cells panels MAGNIFY-MS: Biomarkers for the identification of cell subsets

[0542] FIG. 8. Correlation matrix between numeric clinical attributes and B cell deconvolution scores. The areas of circles show the value of corresponding Pearson correlation coefficients. Positive correlations are displayed in blue and negative correlations in red. Color intensity (light to dark) and the size of the circle (small to big) are proportional to the correlation coefficients. Nonsignificant correlations (p-value<0.01) are marked with blank. Only EDDSS score at baseline is shown.

[0543] FIG. 9. Correlation matrix between numeric clinical attributes and CD8+ T cell deconvolution scores. The areas of circles show the value of corresponding Pearson correlation coefficients. Positive correlations are displayed in blue and negative correlations in red. Color intensity (light to dark) and the size of the circle (small to big) are proportional to the correlation coefficients. Nonsignificant correlations (pvalue<0.01) are marked with blank. Only EDDSS score at baseline is shown.

[0544] FIG. 10. Correlation matrix between numeric clinical attributes and CD4+ T cell deconvolution scores. The areas of circles show the value of corresponding Pearson correlation coefficients. Positive correlations are displayed in blue and negative correlations in red. Color intensity (light to dark) and the size of the circle (small to big) are proportional to the correlation coefficients. Nonsignificant correlations (p-value<0.01) are marked with blank. Only EDDSS score at baseline is shown.

[0545] FIG. 11. B cell subtypesAbsolute values displayed as cells/L

[0546] FIG. 12. ImmunoglobulinsAbsolute values displayed as g/L

[0547] FIG. 13: T cell subtypesAbsolute values displayed as cells/L

EXAMPLES

Example 1

[0548] Characterization of Peripheral Immune Cell Dynamics and Repopulation Patterns after Treatment with Cladribine Tablets in the MAGNIFY-MS Study

Introduction

[0549] Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), in which many immune cell subtypes play a role in pathogenesis with B and T lymphocytes being involved in demyelination and axonal damage (Hoglund and Maghazachi, 2014; Piancone et al., 2016). [0550] Cladribine tablets 10 mg (3.5 mg/kg cumulative dose over 2 years) is an immune reconstitution therapy (IRT) for MS that causes a selective and transient reduction in B and T cell counts, followed by a period of reconstitution (Giovannoni, 2017; Wiendl, 2017). [0551] Cladribine is a deoxyadenosine analog prodrug that is sequentially phosphorylated by deoxycytidine kinase (DCK) and deoxyguanosine kinase to its biologically active form, 2-chlorodeoxyadenosine triphosphate (Cd-ATP). The dephosphorylation and deactivation of Cd-ATP is catalyzed by 5-NT-ase. [0552] B and T lymphocytes possess a relatively high DCK/5-NT-ase ratio, making them particularly sensitive to cladribine (Giovannoni, 2017). [0553] Accumulation of cladribine within these immune cells leads to apoptosis through the inhibition of DNA polymerase. This effect on B and T cells interrupts the cascade of immune events that are central to the progression of MS (Deeks, 2018). [0554] The long-term immune cell dynamics following treatment with cladribine tablets has been evaluated for CLARITY, CLARITY Extension, PREMIERE and ORACLE-MS studies (Comi et al., 2019; Stuve et al., 2019). But it remains to be fully established as to the effect on which immune cell subtypes may be key for both onset and durability of the therapeutic effect of cladribine tablets in people with MS. [0555] It is against this background that the MAGNIFY-MS (NCT03364036) was designed and initiated in those with highly active relapsing MS. Here we report findings for a substudy of peripheral immune cell subset dynamics and immunoglobulin levels during the first 12 months after patients had initiated treatment.

Materials and Methods

Study Design and Participants

[0556] MAGNIFY-MS is an ongoing phase IV, open-label, single-arm study with a duration of 2 years (FIG. 3). [0557] Patients aged 18 years, with an Expanded Disability Status Scale (EDSS) score 5 and highly active relapsing MS were enrolled between May 28, 2018 and Apr. 23, 2019. [0558] In this study, highly active relapsing MS was defined as one relapse in the previous year and at least one T1 gadolinium enhancing (Gd+) lesion, or nine or more T2 lesions while on treatment with other disease-modifying therapies (DMTs), or two or more relapses in the previous year whether on DMT or not. [0559] Patients were excluded if they had previous exposure to DMT (fingolimod, natalizumab, alemtuzumab, mitoxantrone, or ocrelizumab), a lymphocyte count not within normal laboratory limits, presence of signs of progressive multifocal leukoencephalopathy, tested positive for human immunodeficiency virus, hepatitis B or C, or active/latent tuberculosis, had an active malignancy, or had an allergy or hypersensitivity to gadolinium and/or any other contraindication to perform magnetic resonance imaging (MRI). [0560] Enrolled patients were scheduled to receive cladribine tablets 3.5 mg/kg cumulative dose over 2 years, with 2 weeks of active treatment per course (Week 1 and Week 5 of each year).

Peripheral Blood Sampling

[0561] This exploratory analysis of MAGNIFY-MS involved longitudinal evaluation of peripheral blood immune cell subtypes in a subgroup of patients receiving cladribine tablets. [0562] Immunophenotyping was performed on blood samples collected at Baseline (pre-dose) and at the end of Months 3, 6, and 12. [0563] Further immunophenotyping was completed at Months 1 and 2 for TBNK and B cell panels. [0564] Immune cell subtypes were analysed by flow cytometry. [0565] Absolute cell counts and % change from baseline were assessed (Table 1). [0566] In parallel, serum levels of immunoglobulins (Ig) G and M were analyzed by nephelometric assay.

Statistical Analysis

[0567] The analysis of immune cell subtypes was an exploratory endpoint of the MAGNIFY-MS study, and were thus analysed descriptively. All patients that received at least one dose of cladribine tablets are included in the analysis.

Results

[0568] Between May 28, 2018 and Apr. 23, 2019, a total of 70 patients were enrolled to this MAGNIFY-MS sub-study, and 57 of these patients received treatment with cladribine tablets (13 patients withdrew from the sub study prior to starting treatment). [0569] The evaluable patient population was predominantly female (61.4%), aged 40 years (61.4%), and had a median EDSS score of 2.5 at baseline. [0570] Patient demographics and characteristics on entry to MAGNIFY-MS are shown in Table 2.

B Cell Subtypes

[0571] Changes in median CD19.sup.+, memory, and nave B cell counts were relatively rapid, as shown by percentage change from baseline to Month 1 of 77%, 74%, and 80%, respectively (FIG. 4, Table 3). [0572] Most B cell subtypes reached nadir by Month 2 after treatment, with CD38.sup.+ plasmablasts and short-lived plasma cells reaching nadir at Month 3. [0573] Thereafter, the reduction in memory B cells was sustained to Month 12. [0574] Regulatory B and transitional B cells recovered by Month 3, and increased over baseline levels. [0575] Nave B cells also recovered to near baseline levels at 12 months after starting treatment.

T Cell Subtypes

[0576] CD4.sup.+ and CD8.sup.+ T cells show a slow reduction in median cell counts, represented by a percentage change from baseline to Month 1 of 22% and 18%, respectively (FIG. 4, Table 3). [0577] Nadir was reached at Month 2, followed by a slight recovery toward baseline. [0578] Other T cell subtypes reached nadir between Months 3 and 6, after which they slowly began to recover toward baseline. [0579] Median Th17 and TEMRA T-cell counts show reductions at later time points, with Th17 reaching nadir at Month 6 (FIG. 4).

Natural Killer Cells

[0580] CD16.sup.+/CD56.sup.+ natural killer (NK) cell counts showed a decrease at Month 1 reaching nadir at Month 2 after treatment, represented by a change from baseline of 40% (Table 3, FIG. 5). [0581] Recovery of these subtypes towards baseline occurred from Months 3-12. [0582] CD16.sup.bright CD56.sup.dim NK cell counts decreased to lowest levels at Month 3 after treatment, while no effect on CD16.sup.low CD56.sup.bright NK cell counts was noted.

Immunoglobulins

[0583] Despite the sustained decrease of memory B cells and a decrease in CD38.sup.+ plasma cells in the first year of MAGNIFY-MS, IgG and IgM levels remained within the normal range (FIG. 6).

Intracellular Cytokines

Discussion

[0584] The characterization of immune cell dynamics in response to disease-modifying treatment (DMT) may provide a better understanding of the pathogenesis of MS as well as providing key information on the mechanism of action of such therapy. [0585] This sub-study of the MAGNIFY-MS population assessed immune cell dynamics over the first year following treatment with cladribine tablets, thus enabling an evaluation of the onset of lymphocyte reduction and repopulation dynamics with specific focus on CD19.sup.+ B cells, CD4.sup.+ and CD8.sup.+ T cells, and CD16.sup.+ NK cell subtypes. [0586] The dynamics of lymphocyte reduction indicate that B cells and NK cells reached nadir most rapidly, with almost all subtypes reaching nadir by Month 2. [0587] Most T cell subtypes reached nadir at Month 3, with CD8.sup.+ central memory, CD8.sup.+ effector memory, CD4.sup.+ and CD8.sup.+ nave, and CD4.sup.+ regulatory cells reaching nadir at Month 6. [0588] For NK cells, the CD16.sup.low CD56.sup.bright cell subtype reached nadir at Month 12. [0589] Regarding repopulation, most cell subtypes were recovering toward baseline by Month 12, with the exception of CD16.sup.low CD56.sup.bright NK cells. [0590] Perhaps surprising, given the reduction in memory B cells, is their sustained change from baseline at Month 12 in which there was an absolute percentage change of 6% from the nadir value of 93% at Month 2. [0591] However, whilst the median counts of memory B cells remain low, the numbers of nave B cells had recovered toward baseline. This is important as it is these nave B cells that are the main producers of interleukin-10, which may act to suppress undesired immune responses (Duddy et al., 2007). [0592] The CD56.sup.bright NK cell phenotype is regulatory (comprising 10% of the NK cell population) while CD56.sup.dim (comprising 90% of the NK cell population) is effector. A decrease in the number of effector cells, in parallel with an increase in regulatory cells, will drive towards an anti-inflammatory environment and may therefore contribute to the therapeutic efficacy of cladribine tablets. [0593] Our results indicate that whilst CD56.sup.bright cell counts were maintained there was a reduction in CD56.sup.dim with nadir reached at Month 3. At this 3-month time point it could be assumed that an anti-inflammatory environment is achieved, although further analyses would need to be conducted to confirm this. [0594] Our findings show that over the first year after treatment, the effects of cladribine tablets on B cells, T cells, and NK cells in patients with highly active relapsing MS are comparable to those observed in patients with clinically isolated syndrome and relapsing-remitting MS (Comi et al., 2019; Stuve et al., 2019). [0595] We observed no clinically relevant changes in IgG and IgM concentration over the 12 month study period. [0596] How do the results of this study compare to other DMTs? [0597] Alemtuzumab has been shown to decrease B cell, CD4.sup.+ and CD8.sup.+ T cell counts followed by a period of repopulation (Wiendl et al., 2020). [0598] The effects of alemtuzumab and cladribine was compared by Baker et al., and were found to be comparable with respect to B cell reduction (Baker et al., 2017). [0599] With fingolimod, total B cell counts were observed to decrease with treatment; an effect on nave B cell counts was less apparent (Claes et al., 2014). [0600] B cell reduction following treatment with ocrelizumab is rapid, with reductions in CD19.sup.+ cell counts occurring as early as 2 weeks after treatment (Montalban et al., 2016). [0601] Note that direct comparisons of peripheral immune cell subset dynamics and Ig levels across studies should be made with caution due to differing assessment schedules and baseline patient characteristics. [0602] Study strengths and limitations: [0603] Small sample size and a single-arm study, with no control group. [0604] Measurements for immune cell counts were made using only peripheral blood, yet cladribine is known to distribute into tissues such as those in the CNS shortly after administration (Hermann et al., 2019). This raises the possibility that this agent may act to reduce lymphocyte numbers within, and/or limit their recruitment into, the CNS as well as those circulating in the periphery, and further studies are warranted. [0605] Note how the immune cell subtype dynamics parallel the early onset of efficacy for MRI findings, reported separately.

Conclusions

[0606] The early onset and thereafter sustained therapeutic effect of cladribine tablets may be mediated through a specific pattern of decrease and reconstitution of B and T cell subtypes. [0607] The pronounced effect on B cells, especially memory B cells in the first 2 months after initiation of cladribine tablets treatment, suggests a contribution to early efficacy onset. [0608] Sustained depletion of memory B cells and the moderate decrease across T cell subtypes may contribute to the long-term therapeutic effect of cladribine tablets.

TABLE-US-00003 TABLE 1 Cell subsets and phenotypes Lymphocyte subset Phenotype CD4 nave T cells (CD3.sup.+, CD8.sup., CD4.sup.+, CD45RA.sup.+, CCR7.sup.+) CD4 central memory (CD3.sup.+, CD8.sup., CD4.sup.+, CD45RA.sup., CCR7.sup.+) T cells CD4 effector memory (CD3.sup.+, CD8.sup., CD4.sup.+, CD45RA.sup., CCR7.sup.) T cells CD8 nave T cells (CD3.sup.+, CD4.sup., CD8.sup.+, CD45RA.sup.+, CCR7.sup.+) CD8 central memory (CD3.sup.+, CD4.sup., CD8.sup.+, CD45RA.sup., CCR7.sup.+) T cells CD8 effector memory (CD3.sup.+, CD4.sup., CD8.sup.+, CD45RA.sup., CCR7.sup.) T cells CD8 terminally (CD3.sup.+, CD4.sup., CD8.sup.+, CD45RA.sup.+, CCR7.sup.) differentiated T cells Th1 cells (CD3.sup.+, CD8.sup., CD4.sup.+, CCR7.sup./.sup.+, CXCR3.sup.+) Treg cells (CD3.sup.+, CD8.sup., CD4.sup.+, CD25.sup.bright, CD127.sup.dim/) Th17 cells (CD3.sup.+, CD8.sup., CD4.sup.+, CD45RA.sup., CCR7.sup./.sup.dim, CCR6.sup.+, CD146.sup.+) Th2 cells (CD3.sup.+, CD8.sup., CD4.sup.+, CXCR3.sup., CCR6.sup.) CD19 B cells (CD45.sup.bright, SSC.sup.low, CD3.sup., CD14.sup., CD56.sup., CD19.sup.+) CD20 B cells (CD45.sup.bright, SSC.sup.low, CD3.sup., CD14.sup., CD56.sup., CD20.sup.+) Activated B cells (CD45.sup.bright, SSC.sup.low, CD3.sup., CD14.sup., CD56.sup., CD19.sup.+, CD20.sup.+, CD69.sup.+) Naive B cells (CD45.sup.bright, SSC.sup.low, CD3.sup., CD14.sup., CD56.sup., CD19.sup.+, CD20.sup.+, IgD.sup.+, CD27.sup.) Memory B cells (CD45.sup.bright, SSC.sup.low, CD3.sup., CD14.sup., CD56.sup., CD19.sup.+, CD20.sup.+, CD27.sup.+) Short-lived plasma (CD45.sup.bright, SSC.sup.low, CD3.sup., CD14.sup., cells CD56.sup., CD19.sup.dim, CD20.sup./.sup.dim, CD27.sup.bright) Breg cells (CD45.sup.bright, SSC.sup.low, CD3.sup., CD14.sup., CD56.sup., CD19.sup.+, CD24.sup.bright, CD38.sup.bright) CD38.sup.bright plasma cells (CD45.sup.bright, SSC.sup.low, CD3.sup., CD14.sup., CD56.sup., CD19.sup.dim, CD20.sup., CD38.sup.bright) Transitional B cells (CD45.sup.bright, SSC.sup.low, CD3.sup., CD14.sup., CD56.sup., CD19.sup.+, CD20.sup.+, IgD.sup.+, CD10.sup.+, CD27.sup.) NKp46 NK cells (SSC.sup.low, CD45.sup.+, CD19.sup., CD3.sup., CD16.sup.+CD56.sup.+, CD335.sup.+) CD16.sup.+ NK cells (SSC.sup.low, CD45.sup.+, CD19.sup., CD3.sup., CD16.sup.+CD56.sup.+) CD16.sup.bright CD56.sup.dim (SSC.sup.low, CD45.sup.+, CD19.sup., CD3.sup., NK cells CD16.sup.bright, CD56.sup.dim) CD16.sup.low CD56.sup.bright (SSC.sup.low, CD45.sup.+, CD19.sup., CD3.sup., CD16.sup./.sup.+, NK cells CD56.sup.bright) CD16.sup.CD56.sup.dim (SSC.sup.low, CD45.sup.+, CD19.sup., CD3.sup., CD16.sup. NK cells CD56.sup.dim) CD16.sup.+ CD56.sup. (SSC.sup.low, CD45.sup.+, CD19.sup., CD3.sup., NK cells CD56.sup., CD16.sup.+) Breg, B regulatory; NK natural killer; Treg, T regulatory.

TABLE-US-00004 TABLE 2 Patient demographics and characteristics at the time of entry to MAGNIFY-MS. Total Patients (N = 57) Female, n (%) 35 (61.4) Age in years, n (%) 40 35 (61.4) >40 22 (38.6) Time since onset of MS in months, mean SD 84.81 93.54 Time since diagnosis in months, mean SD 52.55 67.45 Time since first relapse in months, mean SD 52.62 80.75 Number of relapses within 12 months prior to baseline, n (%) 0 2 (3.5) 1 15 (26.3) 2 29 (50.9) >2 11 (19.3) EDSS score at baseline, n (%) 3 42 (73.7) >3 15 (26.3) Median (range) 2.5 (0.0-5.0) EDSS, Expanded Disability Status Scale; MS, multiple sclerosis; SD, standard deviation.

TABLE-US-00005 TABLE 3 Percentage change from baseline of B cell and T cell subtypes. Month Month Month Month Month Subtype 1 2 3 6 12 CD19.sup.+ B cells 77% 90% 80% 60% 35% n = 46 n = 44 n = 46 n = 35 n = 42 Memory B cells 74% 93% 93% 90% 87% n = 45 n = 44 n = 46 n = 34 n = 42 Activated B cells 64% 81% 73% 53% 45% (CD69.sup.+) n = 45 n = 44 n = 46 n = 34 n = 42 CD38.sup.+ plasma cells 11% 66% 71% 51% 51% n = 45 n = 44 n = 46 n = 34 n = 42 Short-lived plasma cells 28% 65% 78% 58% 51% n = 45 n = 44 n = 46 n = 34 n = 42 Nave B cells 80% 90% 75% 43% 5% n = 45 n = 44 n = 46 n = 34 n = 42 Transitional B cells 61% 63% +28% +34% +36% n = 45 n = 44 n=46 n=34 n=42 Regulatory B cells 45% 16% +176% +171% +50% n = 45 n = 44 n=46 n=34 n=42 CD4.sup.+ T cells 22% 51% 54% 51% 40% n = 46 n = 44 n = 46 n = 35 n = 42 CD8.sup.+ T cells 18% 39% 50% 43% 36% n = 46 n = 44 n = 46 n = 35 n = 42 CD4.sup.+ Th1 cells 51% 45% 35% n = 46 n = 34 n = 42 CD4.sup.+ Th17 cells 35% 34% 24% n = 45 n = 34 n = 41 CD4.sup.+ CM/EffM T cells 56%/35% 52%/35% 40%/15% n = 46 n = 34 n = 42 CD8.sup.+ CM/EffM T cells 39%/29% 46%/22% 29%/16% n = 46 n = 34 n = 42 CD4.sup.+ nave/CD8.sup.+ nave 63%/73% 64%/70% 53%/58% T cells n = 46/n = 46 n = 35/n = 34 n = 42/n = 42 CD4.sup.+ Treg cells 30% 38% 21% n = 46 n = 34 n = 42 CD8.sup.+ TEMRA 26% 15% 19% n = 46 n = 34 n = 42 CD16.sup.low CD56.sup.bright NK 7% 2% 9% cells n = 46 n = 34 n = 42 CD16.sup.+/CD56.sup.+ NK cells 34% 40% 34% 17% 14% n = 46 n = 44 n = 46 n = 35 n = 42 CD16.sup.bright CD56.sup.dim 40% 19% 12% NK cells n = 46 n = 34 n = 42 Key: Nadir value (in italics) Values above baseline level (underlined) CM, central memory; EffM, effector memory; NK, natural killer; TEMRA, terminally differentiated effector memory RA.sup.+; Th, T helper; Treg, T regulatory.

Magnify-MS 1 Year Blood Biomarker Substudy: Methods

[0609] MAGNIFY-MS is an ongoing Phase IV (NCT03364036, see FIG. 3), open-label, single-arm, multicenter, 2-year study. Patients with highly active relapsing MS received cladribine tablets, with 2 weeks active treatment per course (Week 1 and Week 5 of each year). Subjects meeting the eligibility criteria during a pre-baseline screening period of up to 3 months before baseline (Day 1 of treatment) will receive an initial treatment course in Year 1 and a retreatment course in Year 2. Subjects will attend visits for assessments at end of Months 1, 2, 3, 6, 12 following Baseline (Day 1 of treatment) and at end of Month 15, 18, 24 following second treatment course (Month 1 of Year 2), respectively. Subjects will attend visits for blood sample as per mandatory monitoring at pre-Baseline and at Months 2, 6, 12, 14, 18 and 24. The primary endpoint(s) will be assessed during the first 6 months, secondary and tertiary endpoints will be assessed up to Month 24. Subjects will be offered the chance to participate in optional sub-studies for exploratory analysis.

[0610] To assess the effect of Mavenclad on biomarkers in blood, an exploratory substudy has been planned to be completed in a subset patients. The goal of a blood biomarker substudy was to report on peripheral immune cell subset dynamics and immunoglobulin levels in the first 12 months of cladribine tablets therapy. The key objectives of performing exploratory research of markers of cladribine treatment response in MS are to define responders and non-responders to therapy and to better understand disease pathology. Validated biomarkers will help to define an optimal cladribine treatment response in MS and thus be of considerable value for taking treatment decisions and ensuring continued benefit from cladribine therapy. A reliable marker of cladribine treatment response in MS should correlate with cladribine bioactivity, be implicated in MS pathology and thus correlate with disease activity or severity. Given inter-subject variability and heterogeneity of MS pathology the combined analysis of several different markers will be most meaningful.

[0611] This blood biomarker sub-study of MAGNIFY-MS involved longitudinal evaluation of peripheral blood immune cells in patients receiving cladribine tablets. 57 patients were treated from the 70 patients enrolled. Whole blood and Peripheral mononuclear cells (PBMCs) were collected at months 1, 2, 3, 6, and 12 and Immunophenotyping was performed using the FACS (Fluorescence-activated cell sorting) technology. Absolute cell counts and % change from baseline were assessed for each immune cell subtype. Additional primary and secondary trial readouts are illustrated in FIG. 3. In addition to the described changes in biomarker over time the correlation of biomarkers with traditional MS related assessments (e.g. Relapse, Mill, EDSS) will be performed. The action of cladribine tablets on immune cells may be key for both onset and durability of its effect in people with MS.

Blood Analysis

[0612] Blood will be analysed for immune cell subtypes and intracellular cytokines using the FACS technology. Blood samples will be drawn specifically for the evaluation of potential immunological surrogate biomarker (see also FIG. 8). In order to distinguish different immune subtypes the following FACS Marker Panels were validated and applied by Covance.

T Cell Panel:

[0613] CD4 Nave, CD4 Central Memory, CD4 Effector Memory, CD8 Nave, CD8 Central Memory, CD8 Effector Memory, CD8 Terminally Differentiated, Th1, Treg, Th17, Th2

B Cell Panel

[0614] CD19 B cells, CD20 B cells, Activated B cells, Nave B cells, Memory B cells, Short-lived Plasma cells, Breg, CD38.sup.bright Plasma cells, Transitional B cells

NK Cells Panel

[0615] NKp46 NK cells, CD16 NK cells, CD16bright CD56dim NK cells, CD16low CD56bright NK cells, CD16-CD56dim NK cells, CD16+CD56 NK cells

TBNK Panel

[0616] T cells (CD45bright, SCClow, CD3+), CD4 T cells (CD45bright, SCClow, CD3+,CD4+), CD8 T cells (CD45bright, SCClow, CD3+,CD8+), B cells (CD45bright, SCClow, CD3,CD19+), NK cells (CD45bright, SCClow, CD3, CD16+/CD56+)
The following intracellular cytokines may be analysed/measured. [0617] T cells cytokines: IL-10, IL-17, GM-CSF, TNF-a, IFN-, IL-22, IL-4 [0618] B cells cytokines: IL-10, IL-6, GM-CSF

Example 2

Correlation of Plasma Cells and T Cells with MRI Efficacy at the Clarity 2-Year Timepoint (as Newly Determined from CLARITY 96 Weeks Study)

[0619] Plasmacell Correlation with MRI

[0620] Plasma cell signatures have weak positive, but significant, correlation with: MACT2 (Mean Active T2 Lesions), CACT2 (Cumulative Active T2 lesions), MRCULES (Mean CU Lesions), MNT1GEL (Mean New T1 Gd+ Lesions). The results are illustrated in FIG. 9.

CD8.sup.+ T Cell Correlation with MRI [0621] Naive CD8.sup.+T cells, CD8.sup.+ T-cell and CD8.sup.+ Tem signatures have positive correlation with:
CNT1GEL (Cumulative New T1 Gd+ Lesions), MNT1GEL (Mean New T1 Gd+ Lesions), CRCULES (Cumulative CU Lesions), MRCULES (Mean CU Lesions), CACT2 (Cumulative Active T2 Lesions), MACT2 (Mean Active T2 Lesions). The results are illustrated in FIG. 10.
Nave CD4.sup.+ and CD4.sup.+CM T Cell Signatures have Positive Correlation with MRI: [0622] most of the CD4+ cell related signature correlate well with MRI attributesespecially CD4.sup.+ naive T cells [0623] REL (number of relapses) is associated with naive CD4.sup.+ T-cell signature from CIBERSORT and CD4+ Tcm (from xCell). The results are illustrated in FIG. 11.

TABLE-US-00006 TABLE 1a Percentage change from baseline of B cell subtypes Month Month Month Month Month Subtype 1 2 3 6 12 CD19.sup.+ B cells 77% 90% 80% 60% 35% n = 46 n = 44 n = 46 n = 35 n = 42 Memory B cells 74% 93% 93% 90% 87% n = 45 n = 44 n = 46 n = 34 n = 42 CD38.sup.+ plasma cells 11% 66% 71% 51% 51% n = 45 n = 44 n = 46 n = 34 n = 42 Short-lived plasma 28% 65% 78% 58% 51% cells n = 45 n = 44 n = 46 n = 34 n = 42 Nave B cells 80% 91% 75% 43% 5% n = 45 n = 44 n = 46 n = 34 n = 42 Transitional B cells 61% 63% +28% +34% +36% n = 45 N = 44 n = 46 n = 34 n = 42 Regulatory B cells 45% 16% +176% +171% +50% n = 45 n = 44 n = 46 n = 34 n = 42 Key: NA, Not Available Nadir value in bold Values above baseline level

TABLE-US-00007 TABLE 1b Percentage change from baseline of T cell subtypes Month Month Month Month Month Subtype 1 2 3 6 12 CD4.sup.+ 22% 51% 54% 51% 50% n = 46 n = 44 n = 46 n = 35 n = 42 CD8.sup.+ 18% 39% 50% 43% 36% n = 46 n = 44 n = 46 n = 35 n = 42 CD4.sup.+ Th1 NA NA 51% 45% 35% n = 46 n = 34 n = 42 CD4.sup.+ Th17 NA NA 35% 34% 24% n = 45 n = 34 n = 41 CD4.sup.+ CM/EffM NA NA 56%/35% 53%/35% 40%/15% n = 46 n = 34 n = 42 CD8.sup.+ CM/EffM NA NA 39%/29% 46%/22% 29%/16% n = 46 n = 34 n = 42 CD4.sup.+ nave/CD8.sup.+ nave NA NA 63%/73% 69%/70% 53%/58% n = 46/n = 46 n = 35/n = 34 n = 42/n = 42 CD4.sup.+ Treg NA NA 30% 38% 21% n = 46 n = 34 n = 42 CD8.sup.+ TEMRA NA NA 26% 15% 19% n = 46 n = 34 n = 42

TABLE-US-00008 TABLE 1c Percentage change from baseline of NK cell subtypes Month Month Month Month Month Subtype 1 2 3 6 12 CD16.sup.low CD56.sup.bright NA NA 7% 2% 9% natural killer cells n = 46 n = 34 n = 42 CD16.sup.+/CD56.sup.+ 34% 40% 34% 17% 14% natural killer cells n = 46 n = 44 n = 46 n = 35 n = 42 CD16.sup.bright CD56.sup.dim NA NA 40% 19% 12% natural killer cells n = 46 n = 34 n = 42

Table Legend:

[0624] The percentage change from baseline was measured for B (1a), T (1b) and NK cell subtypes. [0625] There is early onset of action, with most B cell subtypes reaching nadir levels by Month 2. CD19+, Memory and nave B cells show an early onset at Month 1. Short lived plasma cells and C38.sup.+ plasma cells decrease at Month 2 and reach nadir at Month 3. In contrast transitional and regulary B cells are reduced up 61% and 45% at month 1 and then increase reaching levels higher than the basal values measured. [0626] T cell subtypes show a in general a similar profile in reduction with nadir values for CD4.sup.+ And CD8.sup.+ subtypes at Month 3 and 6 respectively. Interestingly regulatory CD4+ (Treg) and CD8+(TEMRA) shows a less prominent decrease of 38% and 26%. [0627] NK cell subtypes