Improved Therapies Using DMD for the Treatment of Autoimmune Diseases, and Biomarkers for Predicting and / or Optimizing Such Therapies

Abstract

The present invention relates to improved methods of treatment for autoimmune diseases, particularly multiple sclerosis, using DMD, in particular Cladribine, and biomarkers, in particular immune cell subtypes, for predicting and / or optimizing said methods of treatment. The described methods rely on the use of immune cell subtypes, in particular T-cell, B-cell, NK-cell subtypes, and their ratios present in a blood sample from a patient, to predict and / or optimize the use of Cladribine in the treatment of multiple sclerosis.

Description

[Technical field]

[0001] Introduction / Background of the Invention / Brief Summary of the Invention Multiple sclerosis (MS) is characterized by the presence of focal and diffuse inflammation and degenerative lesions in the CNS. Continuous modulation of the immune system, for example by DMDs (disease-modifying drugs), can potentially modify disease activity and also potentially affect the disease course, especially in the early phase. The therapeutic armamentarium has been developed substantially over the past 20 years and currently includes up to 16 different drugs, preferably disease-modifying drugs. In 2017, the European Medicines Agency (EMA) recommended the approval of cladribine, or more specifically oral cladribine or cladribine tablets, as an orally administered immunosuppressant for the treatment of highly active MS. The development of cladribine, preferably oral cladribine or cladribine tablets, began after the discovery that genetic deficiency of adenosine deaminase (ADA) - an enzyme involved in purine metabolism - causes lymphopenia in humans, specifically due to intracellular accumulation of toxic deoxyadenosine nucleotides in lymphocytes, and does not affect other tissues. Lymphocytes exhibit high levels of deoxycytidine kinase activity and low levels of nucleotidase activity, meaning that they rely on ADA activity to remove excessively triphosphorylated nucleotides. As a result, ADA-resistant purine nucleoside analogs, such as cladribine, can reproduce the lymphopenic effects of ADA deficiency. Upon entry into lymphocytes, ADA-resistant cladribine is triphosphorylated by deoxycytidine kinase to generate its lymphotoxic form, resulting in selective depletion of lymphocyte populations. [Background technology]

[0002] Some DMDs are administered as long-term treatment, where the drug is administered continuously to reach an optimized reduction in disease activity. Some other DMDs, such as cladribine, preferably oral cladribine or cladribine tablets, are administered in a short course of 2 weeks in the first and second years, or in an equivalent manner, preferably as described in the EU EMA SmPC and / or US / FDA USPI dosage and administration section for oral cladribine or cladribine tablets, allowing induction of immune reset (transient or permanent) in patients treated accordingly with cladribine, preferably oral cladribine or cladribine tablets, allowing immune reconstitution, potentially resulting in long-lasting drug-free remission. This important information was obtained from the CLARITY extension study, which investigated the sustained efficacy of cladribine, preferably oral cladribine or cladribine tablets, after the initial CLARITY trial. In this study, placebo-treated patients from the CLARITY trial received a cumulative dose of 3.5 mg / kg cladribine, preferably oral cladribine or cladribine tablets, in the extension phase. Patients who initially received a cumulative dose of 3.5 mg / kg or 5.25 mg / kg cladribine, preferably oral cladribine or cladribine tablets, were randomly reassigned in a 2:1 ratio to receive 3.5 mg / kg cladribine, preferably oral cladribine or cladribine tablets, or placebo, creating a total of five patient groups. Interestingly, relapse rates and disability were not significantly different between all groups, indicating that the efficacy of cladribine, preferably oral cladribine or cladribine tablets, is maintained over time, regardless of the order in which treatment, preferably cladribine, oral cladribine or cladribine tablets, is received. These results demonstrate that a short, "pulse-like" course of cladribine, preferably oral cladribine or cladribine tablets, delivered in two cycles produces long-lasting lymphopenia and / or response / efficacy, as well as maintaining potentially very durable drug-free remission.

[0003] It has been found that in the treatment of MS, and preferably also other inflammatory and / or demyelinating autoimmune diseases, cladribine, preferably oral cladribine or cladribine tablets, when used for short intermittent periods, can advantageously provide a highly effective immune reconstitution therapy of such disorders, thereby preferably inducing immune resetting and allowing the patient a treatment-free period, preferably an extended treatment-free period, potentially lasting for 2-5 years or 3-4 years and potentially beyond, counting from the last administration of cladribine, preferably oral cladribine or cladribine tablets.Long-term lymphocyte dynamics have been evaluated for the CLARITY, CLARITY extension, and PREMIERE trials, potentially indicating some kind of immune cell repopulation after treatment with cladribine tablets.

[0004] The design of the MAGNIFY-MS study aims to determine the onset of action of cladribine tablets (cumulative dose of 3.5 mg / kg over 2 years) in patients with highly active relapsing MS. Surprisingly, it has now been found that the action of cladribine, preferably oral cladribine or cladribine tablets, on immune cells may be important for both the onset and durability of its action in MS patients, and preferably also in patients with other inflammatory and / or demyelinating autoimmune diseases. Whether and how cladribine, preferably oral cladribine or cladribine tablets, rebalances, reshapes, and even permanently reprograms the immune system has not been understood or is incompletely understood so far. The inventors preferably address this lack of understanding in the present invention.

[0005] The use of disease-modifying therapies in autoimmune conditions is of great clinical interest; however, these therapies suffer from the inability to determine in a reasonable manner, for example using a test or preferably a blood test, whether the patient's immune system will respond appropriately to the treatment or not. In the present invention, the inventors preferably propose the use of various immunosubtype-based efficacy or retreatment biomarkers, preferably prognostic and / or predictive biomarkers, either alone or in combination. Preferably, these biomarkers are readily available in a body fluid sample of the individual and / or patient, preferably an individual or patient potentially to be treated or retreated with one or more DMDs, including but not limited to cladribine, preferably oral cladribine and / or cladribine tablets. Summary of the Invention

[0006] Summary of the invention / detailed description of the invention Meanwhile, global health organizations, while approving the use of cladribine tablets as outlined in the EU's EMA's SmPC and / or the US / FDA's USPI, follow their own dosing regimen, which is approximately 0.875 mg per kg of an individual patient's body weight administered within the first week of the first month that cladribine therapy is initiated (first treatment month), followed by an additional dose of approximately 0.875 mg per kg of an individual patient's body weight within the first week of the second month that cladribine therapy is initiated (second treatment month), followed by approximately 10 months of cladribine therapy. and then within the first week of the month following about 10 months during which no Cladribine tablets or other DMDs are administered to the patient (this period is preferably referred to as the first treatment year), during which no Cladribine tablets or other DMDs are administered to the patient during which Cladribine tablet treatment has been initiated (e.g., the 13th treatment month or the 1st treatment month of the 2nd treatment year), a further dose of about 0.875 mg per kg of the patient's body weight is administered, and then finally within the first week of the month following the 13th treatment month / the 1st treatment month of the 2nd treatment year. This unique treatment regimen has proven highly effective in treating MS, preferably relapsing-remitting MS (RRMS), i.e., a dramatic reduction in both relapse rate and / or MRI activity in the CNS of treated patients. Furthermore, it has been shown that the high efficacy of this treatment regimen / dosage of Cladribine tablets outlined above is maintained within 10 months after the last treatment of the second treatment year, and is sustained for one year (i.e., 3 years from the start of Cladribine tablet treatment) and the following year (i.e., 4 (far) years from the start of Cladribine tablet treatment) and longer after the second treatment year. Thus, patients receiving Cladribine tablet treatment in the first year (first treatment year) and now in the second year (second treatment year) have such reduced MS disease activity that they do not require retreatment with Cladribine tablets and are not required to re-treat with any other DMD for 3 to 6 years (and potentially longer) after the start of their initial Cladribine tablet treatment.

[0007] However, even if both the treatment regimen / dosage and / or the high efficacy associated therewith are comparable, if not better, than those associated with other DMDs, a detailed analysis of efficacy in the treated patient population indicates that there may be patients who benefit more or less from treatment with Cladribine tablets as described above, in terms of onset of action or duration of both early and long-term efficacy. On the other hand, this implies that there may be patients who could potentially benefit from intensified treatment with Cladribine tablets, such as early or relatively early retreatment with Cladribine tablets (earlier than according to the treatment regimen / dosage described above), or early switch to a different DMD, such as retreatment with Cladribine tablets within the first and / or second treatment years of Cladribine tablets treatment as described above, or a switch to a different one. On the other hand, this further implies that there may be patients who are potentially susceptible to MS disease relapse towards the end of the second treatment year with respect to cladribine tablet treatment, or for example during the next 1-6 years following the second treatment year with respect to cladribine tablet treatment, who would also benefit strongly from a relatively early retreatment with cladribine tablets or a relatively early switch to a different DMD. Similarly, the physician and / or patient may consider a relatively early retreatment with cladribine tablets or a relatively early switch to another DMD in order to prevent the risk of potential disease relapse, and thus potential harm, for patients who are retreated or early retreated with each DMD administered to said patients who do not actually require such retreatment. For example, patient data shows that there are patients who, once treated with cladribine tablets for 1 or 2 years according to the previously provided dosage / dosage regimen, do not appear to need cladribine tablets and / or other DMD treatments in the future, at least in the foreseeable future.

[0008] It would therefore be highly advantageous if there were a way to predict which patients would respond to treatment with cladribine tablets, both in terms of early to intermediate efficacy, and overall efficacy, including long-term efficacy, particularly at the end of the second treatment year, as well as for the year following the end of the second treatment year.

[0009] This high medical need is preferably well addressed by the present invention. According to the present invention, specific deviations of one or more cell populations, preferably one or more lymphocyte cell populations, preferably cell populations as described in more detail herein, are determined in one or more body fluids of an affected human or patient at different stages of treatment, preferably including determining said cell populations at least once before starting treatment with a disease modifying drug (DMD) and at least once at a later time point during or after treatment with said DMD, wherein the difference or deviation of said cell populations determined at different times is an indication of the efficacy of the treatment, preferably including early onset of efficacy, mid-term efficacy, long-term efficacy and / or sustained efficacy. However, said difference or deviation of said cell populations determined at different times is also preferably an indication of the ineffectiveness of the treatment, preferably including lack of (early) onset of efficacy, loss of (early) efficacy, lack of mid-term efficacy, loss of mid-term efficacy, lack of long-term efficacy and / or loss of long-term efficacy.

[0010] Thus, the method preferably comprises determining the cell population before initiation of treatment with the DMD, and at least one further time point, e.g. selected from treatment with the DMD, after treatment with the DMD, before re-treatment with the DMD, re-treatment with the DMD, and after re-treatment. Typically, (re)determining the cell population at at least one further time point, preferably at an early therapeutic stage of the treatment of the autoimmune disease with a disease modifying drug (DMD), preferably provides prediction or evidence of early efficacy and / or early onset of efficacy of the treatment of the autoimmune disease with the DMD, or early efficacy and / or lack of early onset of efficacy under these conditions. Typically, the method of the invention provides a method for predicting a patient's response, preferably an early response, to said treatment with DMD, when said one or more cell populations are determined before the start of said treatment with said DMD (i.e. at baseline or baseline level), and again during and after said treatment with said DMD, preferably within 1-12 months from the start of said treatment with said DMD, more preferably within 1-9 months from the start of said treatment with said DMD, and even more preferably within 1-6 months from the start of said treatment with said DMD, for example within 1 month, 3 months, or 6 months when said one or more cell populations are (again) determined. As outlined in more detail below, deviations between said one or more cell populations at different time points, or between their determinations at different time points, are indicative of efficacy or lack of efficacy, preferably early efficacy or lack of early efficacy. In the case of an indication of efficacy, the physician will understand that there is no need to re-treat said patient with the same DMD, administer additional and / or higher doses of the same DMD, or switch to a different DMD. However, if there are indications of lack of efficacy, the physician will realize (early) that an additional and / or higher dose of the same DMD, a switch to a different DMD, and / or the addition of a further DMD would be beneficial for the patient.

[0011] Likewise, the method of the invention preferably provides a method for predicting a patient's response, preferably a long-term response, to said treatment with DMD, if said cell population or populations are determined before the start of treatment with said DMD (i.e. at baseline or at baseline levels) and again during and after said treatment with said DMD, preferably after said treatment with said DMD, preferably within 12-60 months or 14-16 months after the start of the first treatment with said DMD, or preferably within 1-48 months from the last treatment with said DMD or more specifically from the date of the last administration of said DMD to said patient. Further determination of said cell population or populations may be repeated at regular intervals, for example at intervals of 6-24 months, more preferably 9-18 months, for example approximately once a year. Depending on said determination (again) of said cell population or populations as outlined above and / or below, or depending on the medium-term and / or long-term efficacy of said treatment as outlined in more detail below, deviations between said cell population or populations, or between their determinations, are indicative of efficacy or lack of efficacy, preferably early efficacy or lack of early efficacy. In the case of indications of efficacy, preferably mid-term and / or long-term efficacy, the physician will understand that there is no need to re-treat the patient with the same DMD, to administer an additional and / or higher dose of the same DMD, or to switch to a different DMD, but in the case of indications of lack of efficacy, the physician will understand the (early) information that an additional and / or higher dose of the same DMD, a switch to a different DMD, and / or the addition of a further DMD, and especially a re-treatment with the same DMD, would be beneficial for the patient.For example, in the treatment of MS with DMD cladribine or cladribine tablets according to the health authority approved treatment scheme (EMA's SmPC in force from 2017 to present (2021); FDA's USPI in force from 2019 to present (2021)) of two short treatment periods of about one month each from the start of treatment with cladribine (first treatment year), and two further short treatment periods of about one month each (second treatment year) starting approximately one year after the start of treatment with cladribine or cladribine tablets, most patients do not require retreatment with cladribine tablets before the end of the fourth year, which begins four or five years after the start of treatment with cladribine tablets, and sometimes even later. However, some patients benefit from relatively early retreatment with cladribine or cladribine tablets. The method of the present invention will therefore provide the physician with early information that retreatment would be beneficial at a time when disease activity or relapse is still undetectable. This may be achieved by carrying out the methods of the invention at one or more time points, such as within the first 12 months of initiating DMD treatment, or within 12-60 months, 24-60 months, and particularly 36-60 months of initiating DMD treatment.

[0012] Accordingly, preferred aspects of the present invention include, but are not limited to, the following: A. Biomarkers (immune signatures) for disease management of said autoimmune diseases, preferably autoimmune neurological and neuroinflammatory diseases, which preferably comprise monitoring the early response (preferably 1 to 3 months) and long term response (preferably 6 to 12 to 24 months and beyond, such as 3 to 15 years, preferably up to 3, 4, 5, 6, 7, 8, 9 or 10 years) of said autoimmune diseases, even more preferably in the context of Cladribine treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] As further defined herein, the immune signature of the present invention is defined by the presence and / or relative amounts of specific subpopulations of immune cells in a patient, more particularly as identified and / or quantified from a patient's biological sample.

[0014] By "biological sample" this refers herein to a sample obtained from a human subject. The biological sample may include tissue and / or biological fluid. As a non-limiting example, the biological sample may be selected from tissues, organs, cells, or any isolated fraction of a human subject, such as blood, plasma, lymph, saliva, urine, feces, tears, sweat, semen, or cerebrospinal fluid, synovial fluid, pleura, peritoneum, or pericardium, and any fraction or extract thereof. Preferably, the biological sample is a biological fluid that contains immune cells, preferably circulating immune cells. More preferably, the biological sample is blood, plasma, or serum. The biological sample may be pre-processed to preserve the integrity of the cells of interest and / or make them more accessible for further analysis. The biological sample may be subjected to, for example, centrifugation, purification, or other processing steps to facilitate access to or increase the concentration of the cells of interest. The blood sample may be pre-treated to enrich or isolate peripheral blood mononuclear cells (PBMCs), for example through the use of a density gradient centrifugation process using conventional techniques, or by leukoreduction.

[0015] Methods for identifying and enumerating immune cells or populations of immune cells are known to those skilled in the art as phenotyping or immunophenotyping, and are typically based on flux cytometry, also referred to herein as FACs or FACs analysis. Immunophenotyping is the analysis of a heterogeneous population of cells with the aim of identifying the presence, absolute number and / or ratio of various populations of interest. Fluorophore-conjugated antibodies are used to identify, characterize and quantify individual subpopulations of cells within a single heterogeneous cell population by detecting specific antigens, also referred to herein as biomarkers, expressed by these cells (usually functional membrane proteins involved in cellular communication, adhesion or metabolism). Briefly, a sample expected to contain immune cells, such as a biological sample, is contacted with a fluorophore-conjugated antibody specific for a biomarker expected to be present on the cell surface or secreted by the cells. The sample is then analyzed using an optical, laser-based device that analyzes the physical and fluorescent properties of the cells in suspension in real time as the cells flow through the device.Such methods are routine in the art and have been described by Pitoiset et al. (Deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses. Cytometry A. 2018 Aug;93(8):793-802. doi: 10.1002 / cyto.a.23570. Epub 2018 Aug 31. PMID: 3016889), Streitz et al. (Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study. Transplant Res. 2013 Oct 25;2(1):17. doi: 10.1186 / 2047-1440-2-17. PMID: 24160259; PMCID: PMC3827923.) or Hasan et al. (Milieu Interieur Consortium. Semi-automated and standardized cytometric analysis. Various fluorophores are used and described in detail in various publications to which the skilled artisan can refer, such as the article published by the same author, J. Immunol. 2015 Apr;157(2):261-76. doi: 10.1016 / j.clim.2014.12.008. Epub 2015 Jan 6. PMID: 25572534.

[0016] In the context of the present invention, the term "peripheral blood mononuclear cells", also "PBMSc" or "total PBMCs", is interpreted as commonly understood in the art and refers to any and all peripheral blood cells with a round nucleus, i.e. lymphocytes (T cells, B cells, NK cells) and monocytes. Methods for isolating PBMCs are well known in the art and these cells can be obtained from whole blood by dilution with PBS, gently layering on an equal volume of Ficoll in a Falcon tube and centrifuging at 400-500 g without brake for 30-40 minutes. Four layers are formed, each containing a different cell type - the top layer contains plasma, which can be removed by pipetting. The viability, concentration ratio and / or absolute number of isolated PBMCs can be measured by manual counting with trypan blue (TB) using a hemocytometer or by using an automated cell counter.

[0017] In the context of the present invention, the term "B cells" is interpreted as commonly understood in the art and refers to cells of the immune system generated in the bone marrow and involved in humoral responses. As is known in the art, B cells can be identified by the presence of specific phenotypic biomarkers secreted by these cells. In the context of the present invention, the term "B cells" refers to cells that are phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - Preferably, the term refers to B cells present in the circulating blood of a patient, also referred to as circulating B cells.

[0018] In the context of the present invention, the term "CD19 + "B cells" refers to B cells as defined herein, i.e., cells with the phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD19 +Preferably, the term refers to cells that have circulating CD19 + CD19, also known as B cells, present in the patient's circulating blood + Refers to B cells.

[0019] In the context of the present invention, the term "CD20 + "B cells" refers to B cells as defined herein, i.e., cells with the phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD20 + Preferably, the term refers to cells that have circulating CD20 + CD20, also known as B cells, present in the patient's circulating blood + Refers to B cells.

[0020] In the context of the present invention, the term "activated B cells" refers to B cells as defined herein, i.e., cells of the phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD20 + , CD19 + , CD69 + Preferably, the term refers to activated B cells present in the circulating blood of a patient, also referred to as circulating activated B cells.

[0021] In the context of the present invention, the term "naive B cells" refers to B cells as defined herein, i.e., B cells with the phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD20 + , CD19 + , IgD + , CD27 -Preferably, the term refers to naive B cells present in the circulating blood of a patient, also referred to as circulating naive B cells.

[0022] In the context of the present invention, the term "memory B cells" refers to B cells as defined herein, i.e., B cells with the phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD20 + , CD19 + , CD27 + Preferably, the term refers to memory B cells present in the circulating blood of a patient, also referred to as circulating memory B cells.

[0023] In the context of the present invention, the term "CD19 + "Memory B cells" are B cells as defined herein, i.e., B cells with the phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD19 + Preferably, the term refers to cells that have circulating CD19 + CD19 cells, also known as memory B cells, present in the patient's circulating blood + Refers to memory B cells.

[0024] In the context of the present invention, the term "short-lived plasma cells" refers to B cells as defined herein, i.e., cells with the phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD19 dim , CD20- / dim , CD27 brightPreferably, the term refers to short-lived plasma cells present in the circulating blood of a patient, also referred to as circulating short-lived plasma cells.

[0025] In the context of the present invention, the term "Breg" or "Breg cells" refers to B cells as defined herein, i.e., cells with the phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD19 + , CD24 bright , CD38 bright Preferably, the term refers to Breg cells present in the circulating blood of a patient, also referred to as circulating Breg cells.

[0026] In the context of the present invention, the term "CD38 bright "Plasma cells" are B cells as defined herein, i.e., cells with the phenotype CD45, as defined by conventional FACS analysis. bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD19 dim , CD20 - , CD38 bright Preferably, the term refers to cells that have circulating CD38 bright CD38 present in the patient's circulating blood, also known as plasma cells bright Refers to plasma cells.

[0027] In the context of the present invention, the term "transitional B cells" refers to B cells as defined herein, i.e., B cells with the phenotype CD45 bright , S.S.C. low , CD3 - , CD14 - , CD56 - and further having the phenotype CD19 + , CD20 + , IgD+ , CD10 + , CD27 - The term refers to cells having the following structure: circulating human transitional B cells. Methods for identifying circulating human transitional B cells are well known in the art and have been described in detail, among others, by Sims et al. (Identification and characterization of circulating human transitional B cells. Blood. 2005;105(11):4390-4398. doi:10.1182 / blood-2004-11-4284). Preferably, the term refers to transitional B cells present in the circulating blood of a patient, also called circulating transitional B cells.

[0028] In the context of the present invention, the term "CD4 naive" or "CD4 naive T cells" refers to T cells with the phenotype CD3 + , CD8 - , CD4 + , CD45RA + , CCR7 + Preferably, the term refers to CD4 naive T cells present in the circulating blood of a patient, also referred to as circulating CD4 naive T cells.

[0029] In the context of the present invention, the term "CD4 central memory" or "CD4 central memory T cells" refers to T cells with the phenotype CD3 + , CD8 - , CD4 + , CD45RA - , CCR7 + Preferably, the term refers to CD4 central memory T cells present in the circulating blood of a patient, also referred to as circulating CD4 central memory T cells.

[0030] In the context of the present invention, the term "CD4 effector memory" or "CD4 effector memory T cells" refers to T cells that are phenotypically CD3 + , CD8 - , CD4 + , CD45RA- , CCR7 - Preferably, the term refers to CD4 effector memory T cells present in the circulating blood of a patient, also referred to as circulating CD4 effector memory T cells.

[0031] In the context of the present invention, the term "CD8 naive" or "CD8 naive T cells" refers to T cells with the phenotype CD3 + , CD4 - , CD8 + , CD45RA + , CCR7 + Preferably, the term refers to naive CD8 T cells present in the circulating blood of a patient, also referred to as naive circulating CD8 T cells.

[0032] In the context of the present invention, the term "CD8 central memory" or "CD8 central memory T cells" refers to T cells with the phenotype CD3 + , CD4 - , CD8 + , CD45RA - , CCR7 + Preferably, the term refers to CD8 central memory T cells present in the circulating blood of a patient, also referred to as circulating CD8 central memory T cells.

[0033] In the context of the present invention, the term "CD8 effector memory" or "CD8 effector memory T cells" refers to T cells that are phenotypically CD3 + , CD4 - , CD8 + , CD45RA - , CCR7 - Preferably, the term refers to CD8 effector memory T cells present in the circulating blood of a patient, also referred to as circulating CD8 effector memory T cells.

[0034] In the context of the present invention, the term "CD8 TEMRA" or "CD8 TEMRA T cells" refers to T cells of the phenotype CD3 + , CD4 - , CD8 + , CD45RA + , CCR7 - Preferably, the term refers to CD8 TEMRA T cells present in the circulating blood of a patient, also referred to as circulating CD8 TEMRA T cells.

[0035] In the context of the present invention, the term "Th1" or "Th1 cells" refers to a subset of Th1 cells with the phenotype CD3 + , CD8 - , CD4 + , CCR7 - / + , CXCR3 + Preferably, the term refers to Th1 cells present in the circulating blood of a patient, also referred to as circulating Th1 cells.

[0036] In the context of the present invention, the term "Treg" or "Treg cells" refers to Tregs having the phenotype CD3 + , CD8 - , CD4 + , CD25 bright , CD127 dim / - Preferably, the term refers to Treg cells present in the circulating blood of a patient, also referred to as circulating Treg cells.

[0037] In the context of the present invention, the term "Th17" or "Th17 cells" refers to Th17 cells with the phenotype CD3 + , CD8 - , CD4 + , CD45RA - , CCR7 - / dim , CCR6 + , CD146 + Preferably, the term refers to Th17 cells present in the circulating blood of a patient, also referred to as circulating Th17 cells.

[0038] In the context of the present invention, the term "Th2" or "Th2 cells" refers to any Th2 cell with the phenotype CD3 + , CD8 - , CD4 + , CXCR3 - , CCR6 - Preferably, the term refers to Th2 cells present in the circulating blood of a patient, also referred to as circulating Th2 cells.

[0039] In the context of the present invention, the term "NKp46 NK cells" refers to cells with the phenotype SSC, as defined by conventional FACS analysis. low , CD45 + , CD19 - , CD3 - , CD16 + CD56 + , CD335 + Preferably, the term refers to NKp46 NK cells present in the circulating blood of a patient, also referred to as circulating NKp46 NK cells.

[0040] In the context of the present invention, the term "CD16 NK cells" refers to cells with the phenotype SSC, as defined by conventional FACS analysis. low , CD45 + , CD19-, CD3 - , CD16 + CD56 + Preferably, the term refers to CD16 NK cells present in the circulating blood of a patient, also referred to as circulating CD16 NK cells.

[0041] In the context of the present invention, the term "CD16 bright CD56 dim NK cells are phenotypically SSCs, as defined by conventional FACS analysis. low , CD45 + , CD19 - , CD3 - , CD56 dim , CD16 bright Preferably, the term refers to lymphocytes that have circulating CD16bright CD56 dim CD16, also known as NK cells, present in the circulating blood of patients bright CD56 dim Refers to NK cells.

[0042] In the context of the present invention, the term "CD16 dim CD56 bright NK cells are phenotypically SSCs, as defined by conventional FACS analysis. low , CD45 + , CD19 - , CD3 - , CD56 bright , CD16 - / + Preferably, the term refers to lymphocytes that have circulating CD16 dim CD56 bright CD16, also known as NK cells, present in the circulating blood of patients dim CD56 bright Refers to NK cells.

[0043] In the context of the present invention, the term "CD16 - CD56 dim NK cells are phenotypically SSCs, as defined by conventional FACS analysis. low , CD45 + , CD19 - , CD3 - , CD56 dim , CD16 - Preferably, the term refers to lymphocytes that have circulating CD16 - CD56 dim CD16, also known as NK cells, present in the circulating blood of patients - CD56 dim Refers to NK cells.

[0044] In the context of the present invention, the term "CD16 + CD56 - NK cells are phenotypically SSCs, as defined by conventional FACS analysis. low , CD45 + , CD19 - , CD3 - , CD56 - , CD16+- Preferably, the term refers to lymphocytes that have circulating CD16 + CD56 - CD16, also known as NK cells, present in the circulating blood of patients + CD56 - Refers to NK cells.

[0045] In the context of the present invention, the term "CD16 + CD56 + NK cells are phenotypically SSCs, as defined by conventional FACS analysis. low , CD45 + , CD19 - , CD3 - , CD56 + , CD16 +- Preferably, the term refers to lymphocytes that have circulating CD16 + CD56 + CD16, also known as NK cells, present in the circulating blood of patients + CD56 + Refers to NK cells.

[0046] B. Biomarkers (immune signatures) to aid in the decision to re-treat with cladribine or switch to DMD Current therapies for autoimmune diseases, preferably autoimmune neurological diseases and neuroinflammatory diseases, suffer from the inability to determine whether a patient is suitable for a particular treatment or treatment schedule, including the decision of when to treat and with which therapeutic agent or drug.Therefore, there is a high medical need to determine, preferably to predetermine or predict whether a patient's immune system will respond appropriately (or not) to a particular therapeutic agent, drug, treatment and / or treatment schedule.In this context, there is also a high need for biomarkers that allow prediction of disease activation or disease relapse, allowing clinicians to make appropriate decisions about whether to treat / re-treat or not, and if so, when to treat, when to re-treat and / or which therapeutic agent or drug to use.The present invention preferably addresses one or more of these medical needs.

[0047] Preferably, the compositions and / or methods are provided for individuals suffering from an autoimmune disease, preferably an autoimmune nervous system disease, preferably including one or more diseases selected from autoimmune demyelinating diseases, neuroinflammatory diseases, autoimmune muscle disorders (RMS and PMS, myasthenia gravis (MG), neuromyelitis optica (NMO), Marie-Tooth disease (CMT), adrenoleukodystrophy (ALD), acute disseminated encephalitis (ADEM), giant cell arteritis, multifocal motor neuropathy, polymyositis, diabetic neuropathy).

[0048] More preferably, compositions and / or methods are provided for individuals suffering from an autoimmune disease, preferably an autoimmune nervous system disease, a neuroinflammatory disease, preferably comprising an autoimmune demyelinating disease, an autoimmune nervous system disease and / or an autoimmune demyelinating disease, preferably comprising one or more diseases selected from multiple sclerosis (MS) and / or multiple sclerosis spectrum disorder (MSSD), neuromyelitis optica and / or neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis (MG), Marie-Tooth disease (CMT), adrenoleukodystrophy (ALD), acute disseminated encephalitis (ADEM), giant cell arteritis, multifocal motor neuropathy, polymyositis, diabetic neuropathy.

[0049] Compositions and / or methods are provided for individuals affected with MS (or other demyelinating diseases and AI neurological indications), divided into groups that provide the following individual information and / or prognosis: - responsive to treatment, preferably to treatment with one or more DMDs (disease-modifying drugs), and in particular to treatment with cladribine; lack of response to (future) treatment, preferably to (future) treatment with one or more DMDs (disease-modifying drugs), and in particular to (future) treatment with cladribine; Loss of response to (previous) treatment, preferably to (previous) treatment with one or more DMDs (disease-modifying drugs) and in particular to (previous) treatment with cladribine: and / or premature or early loss of response to (previous) treatment, preferably premature or early loss of response to (previous) treatment with one or more DMDs (disease-modifying drugs), and in particular premature or early loss of response to (previous) treatment with cladribine; Loss of response and disease relapse after long-term remission 3 and 4 years after cladribine treatment.

[0050] Particularly preferably, the effective immunomodulatory therapeutic effect of an individual therapeutic agent or medicinal product, preferably an effective immunomodulatory therapeutic effect of an individual DMD, and in particular an effective immunomodulatory therapeutic effect of Cladribine, in an individual individual is shown to be mediated through specific patterns of sustained depletion and / or reconstitution of specific B cell, specific T cell and / or NK cell subtypes in the RMS population.

[0051] These specific B cell, T cell and / or NK cell subtypes can be divided into either pathological subtypes with pro-inflammatory and disease induction and exacerbation potential, and regulatory subtypes with anti-inflammatory phenotypes that play a role in immune regulation and tolerance induction in MS and other autoimmune diseases, especially autoimmune inflammatory diseases. Immune subtypes can be identified by methods known and understood in the art, preferably by specific cell surface markers known and understood in the art, for example, by fluorescence-activated cell sorting (FACS) using specific CD markers, mass cytometry (CyTOF, time-of-flight cytometry), a variant of flow cytometry in which antibodies are labeled with heavy metal ion tags rather than fluorescent dyes, immune deconvolution methods using bioinformatics algorithms, or other methods known and understood by those skilled in the art. The patient's whole blood or PBMCs (peripheral blood mononuclear cells) isolated from the blood are used to identify immune cell subsets.

[0052] The increase or decrease in immune subsets can be at absolute levels, as a percentage of total peripheral blood mononuclear cells, B cells or T cells, e.g., CD19 + Or CD20 + B cells, CD4+ Or CD8 + In addition, the increase may be measured against healthy controls, against the same patient before treatment (baseline value), against a predetermined standard.

[0053] Surprisingly, in the context of the present invention, it has been found that an "immune signature" comprising a single immune cell type or a combination of multiple immune subtypes preferably serves as a biomarker for: Response to (future and / or past) treatment, preferably the treatments described herein; (Future) lack of response to treatment, preferably to the treatments described herein; and / or - (early or premature, late) loss of efficacy, response and / or responsiveness to (past and / or future) treatment, preferably a treatment as described herein.

[0054] For example, the response or lack of efficacy can preferably be monitored by measuring increased or decreased numbers of specific immune subtypes, respectively, or by an altered ratio of pathological versus regulatory B, T or NK subtypes, or by increased or decreased depletion, respectively, of said specific immune subtypes, or by an altered ratio of pathological versus regulatory B, T or NK subtypes relative to a predetermined standard.

[0055] According to the present invention, preferably the treatment of said autoimmune disease with DMD, preferably said treatment of MS with DMD, and in particular the treatment of MS with Cladribine, in an individual comprises: i) induces an early and sustained decline in memory B cells in the individual, preferably with a steepest drop or large decline in the first and / or second month from initiation of the individual's DMD treatment, which decline or increased levels are sustained for several months, preferably from month 1 through month 12 and 24 from initiation of the individual's DMD treatment; ii) induce a sustained increase in regulatory B cells in the individual, preferably a sustained increase in regulatory B cells above the baseline level before initiation of the treatment, wherein the sustained increase is preferably from 2 to 14 months, and particularly from 3 to 12 months, counting from the initiation of the individual's DMD treatment; and / or iii) induce a sustained increase in transitional B cells in the individual, preferably above the baseline level before the start of the treatment, where the sustained increase is preferably from 2 to 14 months, and in particular from 3 to 12 months, counting from the start of the individual DMD treatment. This characteristic pattern / immune signature preferably correlates with or is indicative of the development of a durable efficacy response to the DMD treatment, which efficacy response is preferably incorporated 2 or 3 months after the second monthly treatment course of cladribine, and preferably remains for at least 12 months. Thus, this characteristic pattern / immune signature persists for up to 12 months and is indicative or predictive of the durable effect of the DMD treatment in the individual. This characteristic pattern / immune signature is preferably shown to persist or be indicative of prolonged (6 to 12 months) DMD efficacy, as measured by MRI.

[0056] According to the present invention, preferably the treatment of said autoimmune disease with DMD, preferably the treatment of MS with DMD and in particular the treatment of MS with Cladribine in an individual, results in the suppression of plasmablasts / plasma cells (short-lived plasmablasts and CD38 cells involved in AB production and the formation of compartmentalized inflammation) from months 2 to 12. + This characteristic pattern is therefore a predictor of long-term response.

[0057] According to the present invention, preferably treatment of said autoimmune disease with DMD, preferably treatment of MS with DMD and in particular treatment of MS with Cladribine in an individual, is shown to induce a reduced ratio of plasma cell signatures determined by immune deconvolution methods at 96 weeks after initial cladribine treatment versus untreated controls in the CLARITY Phase 3 study, which correlates with MRI activity parameters that are predictive of long-term response at 96 weeks after cladribine.

[0058] According to the present invention, preferably the treatment of said autoimmune disease with DMD, preferably the treatment of MS with DMD and in particular the treatment of MS with Cladribine in an individual, preferably throughout a period of 3 to 12 months, counted from the start of the respective DMD treatment, is associated with an increased production of anti-inflammatory regulatory NK cells (CD16 low CD56 bright ), but not significantly affect the numbers of proinflammatory NK effector cells (CD16 bright CD56 dim ), which is due to the increased NKCD56 bright / NKCD56 dim The results show that this leads to a NK cell to NK cell ratio of 0.8 to 1.4 (0.8 vs. 1.4). An increase in this ratio of approximately 1.5-2.5-fold is indicative of a response to treatment.

[0059] According to the present invention, preferably, the treatment of said autoimmune disease with DMD, preferably said treatment of MS with DMD and in particular the treatment of MS with Cladribine in an individual, is achieved by the treatment of CD4 + and CD8 + It reduced nadir values ​​of naive, central and effector memory T cells at months 3 and 6, and limited re-expansion at month 12 in the MAGNIFY trial and week 96 in the Mavenclad CLARITY trial. As a novel biomarker to monitor durable responses beyond 10 months after the second year of cladribine treatment, naive T cells showed significant positive correlation with MRI parameters using immune deconvolution signatures to identify T cell subtypes at week 96 in the CLARITY trial.

[0060] According to the present invention, it is shown that the treatment of said autoimmune disease with DMD, preferably the treatment of MS with DMD and in particular the treatment of MS with Cladribine in an individual induces a sustained reduction of pathological memory B cells and plasmablasts / cells (>-70% and >-50% from baseline, respectively) from months 1-12 and months 3-12, respectively. Repopulation of these cells resulting in an increase in the number of memory B cells and plasma cells to <-40% and -20%, respectively, would illustrate the loss of efficacy serving as a retreatment marker. Thus, the present invention also relates to a method for monitoring the response of a patient with an autoimmune disease, preferably selected from autoimmune neurological and neuroinflammatory diseases, to treatment with a disease modifying drug (DMD), preferably Cladribine, comprising: a. in a first and a second biological sample from a patient: - total PBMCs; - CD19 + B cells; - CD19 + memory B cells; - Naïve B cells; - CD4 + central memory T cells; - CD4 + effector memory T cells; - Tregs; - Teff; - Breg; - Memory B cells; - CD16 dim CD56 bright NK cells; - CD16 bright CD56 dim NK cells; - Transitional B cells: or, - CD19 in the second biological sample compared to the first biological sample. + Increase in absolute B cell numbers or CD19 + A decrease of 30% or less in absolute B cell numbers; - CD19 in the second biological sample compared to the first biological sample. + Increase in absolute number of memory B cells or CD19 + A decrease of 30% or less in the absolute number of memory B cells; and / or - an increase in the absolute number of naive B cells or a decrease of no more than 30% in the absolute number of naive B cells in the second biological sample compared to the first biological sample; - CD4 in the second biological sample compared to the first biological sample. + Increase in the absolute number of central memory T cells, or CD4 + A decrease of 20% or less in the absolute number of central memory T cells; - CD4 in the second biological sample compared to the first biological sample. + Increase in absolute number of effector memory T cells or CD4 + a 10% or less decrease in the absolute number of effector memory T cells; - a decrease in the ratio of absolute Treg number to absolute Teff number in the second biological sample compared to that ratio in the first biological sample; - a decrease in the ratio of the absolute number of Breg cells to the absolute number of memory B cells in the second biological sample compared to that ratio in the first biological sample; - a ratio of CD16 in the second biological sample compared to that in the first biological sample; bright CD56 dim CD16 on absolute NK cell counts dim CD56 bright A decrease in the ratio of absolute numbers of NK cells; - the percentage of memory B cells relative to total PBMCs in the second biological sample between 0.05% and 1%; - a percentage of Breg cells relative to total PBMCs in the second biological sample of between 3% and 14%, preferably between 1% and 10%; - a ratio of transitional B cells:memory B cells greater than or equal to 1, preferably consisting of 1 to 3, in the second biological sample: Either is an indication of a suboptimal response of the patient to treatment.

[0061] Preferably, this first biological sample is a sample collected prior to a first administration or administration period for a particular DMD.

[0062] Preferably, the second biological sample is collected after the start of the first dose of the treatment and (i) during the course of the treatment, or (ii) after the last dose of the treatment. More preferably, the second biological sample is collected at least one month, preferably at least two months, from the first dose or the start of the treatment period for the particular DMD.

[0063] In one embodiment, the second biological sample is collected within 1-12 months of the first administration or start of the administration period for the individual DMD. In another embodiment, the second biological sample is collected within 13-24 months of the first administration or start of the administration period for the individual DMD.

[0064] In the context of the method of the invention, step a) of obtaining the absolute number can be performed by obtaining data previously generated by in vitro analysis of said first and second samples by a separate method, or can be performed by determining said absolute number as an active step of the method.

[0065] The present invention therefore also relates to a method for monitoring the response of a patient with an autoimmune disease, preferably selected from autoimmune neurological and neuroinflammatory diseases, to treatment with a disease modifying drug (DMD), preferably cladribine, comprising: b. obtaining a first biological sample from a patient; c. obtaining a second biological sample from the patient; d. In the first and second biological samples: - total PBMCs; - CD19 + B cells; - CD19 + memory B cells; - Naïve B cells; - CD4 + central memory T cells; - CD4 + effector memory T cells; - Tregs; - Teff; - Breg; - Memory B cells; - CD16 dim CD56 bright NK cells; - CD16 bright CD56 dim NK cells; - Transitional B cells: wherein: - CD19 in the second biological sample compared to the first biological sample. + Increase in absolute B cell numbers or CD19 + A decrease of 30% or less in absolute B cell numbers; - CD19 in the second biological sample compared to the first biological sample. + Increase in absolute number of memory B cells or CD19 + A decrease of 30% or less in the absolute number of memory B cells; and / or - an increase in the absolute number of naive B cells or a decrease of no more than 30% in the absolute number of naive B cells in the second biological sample compared to the first biological sample; - CD4 in the second biological sample compared to the first biological sample. + Increase in the absolute number of central memory T cells, or CD4 + A decrease of 20% or less in the absolute number of central memory T cells; - CD4 in the second biological sample compared to the first biological sample. + Increase in absolute number of effector memory T cells or CD4 + a 10% or less decrease in the absolute number of effector memory T cells; - a decrease in the ratio of the absolute number of Tregs to the absolute number of Teffs 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 to the absolute number of memory B cells in the second biological sample compared to that ratio in the first biological sample; - a ratio of CD16 in the second biological sample compared to that in the first biological sample; bright CD56 dim CD16 on absolute NK cell counts dim CD56 bright A decrease in the ratio of absolute numbers of NK cells; - the percentage of memory B cells relative to total PBMCs in the second biological sample between 0.05% and 1%; - a percentage of Breg cells relative to total PBMCs in the second biological sample of between 3% and 14%, preferably between 1% and 10%; - a ratio of transitional B cells:memory B cells greater than or equal to 1, preferably consisting of 1 to 3, in the second biological sample: Either is an indication of a suboptimal response of the patient to treatment.

[0066] Methods of monitoring patient response according to the present invention may be beneficially integrated into the course of treatment.

[0067] The present invention therefore also relates to a method of treating the response of a patient having an autoimmune disease, preferably selected from autoimmune neurological diseases and neuroinflammatory diseases, to treatment with a disease modifying drug (DMD), preferably cladribine, comprising: a. obtaining a first biological sample from a patient; b. administering treatment to a patient; c. obtaining a second biological sample from the patient; d. In the first and second biological samples: - total PBMCs; - CD19 + B cells; - CD19 + memory B cells; - Naïve B cells; - CD4 + central memory T cells; - CD4 + effector memory T cells; - Tregs; - Teff; - Breg; - Memory B cells; - CD16 dim CD56 bright NK cells; - CD16 bright CD56 dim NK cells; - Transitional B cells: wherein: - CD19 in the second biological sample compared to the first biological sample. + Increase in absolute B cell numbers or CD19 + A decrease of 30% or less in absolute B cell numbers; - CD19 in the second biological sample compared to the first biological sample. + Increase in absolute number of memory B cells or CD19 + A decrease of 30% or less in the absolute number of memory B cells; and / or - an increase in the absolute number of naive B cells or a decrease of no more than 30% in the absolute number of naive B cells in the second biological sample compared to the first biological sample; - CD4 in the second biological sample compared to the first biological sample. + Increase in the absolute number of central memory T cells, or CD4 + A decrease of 20% or less in the absolute number of central memory T cells; - CD4 in the second biological sample compared to the first biological sample. + Increase in absolute number of effector memory T cells or CD4 + a 10% or less decrease in the absolute number of effector memory T cells; - a decrease in the ratio of the absolute number of Tregs to the absolute number of Teffs 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 to the absolute number of memory B cells in the second biological sample compared to that ratio in the first biological sample; - a ratio of CD16 in the second biological sample compared to that in the first biological sample; bright CD56 dim CD16 on absolute NK cell counts dim CD56 bright A decrease in the ratio of absolute numbers of NK cells; - the percentage of memory B cells relative to total PBMCs in the second biological sample between 0.05% and 1%; - a percentage of Breg cells relative to total PBMCs in the second biological sample of between 3% and 14%, preferably between 1% and 10%; - a ratio of transitional B cells:memory B cells greater than or equal to 1, preferably consisting of 1 to 3, in the second biological sample: Either is an indication of a suboptimal response of the patient to treatment.

[0068] In the context of the present invention, an indication of a suboptimal response of a patient to the treatment is indicative of a lack of efficacy of the treatment, such as a lack of short-term and / or long-term efficacy of the treatment, and a lack of indication of a suboptimal response of a patient to the treatment is indicative of efficacy of the treatment, such as a lack of short-term and / or long-term efficacy of the treatment.

[0069] Preferably, an indication of lack of efficacy of a treatment determined by the method of the invention justifies an additional period of administration of the DMD for the patient, a switch to another DMD, and / or the application of one or more additional treatment options, preferably within 1 to 12 months after the determination of the indication of lack of efficacy, and an indication of short-term efficacy does not require an additional period of administration of the DMD for the patient, or a switch to another DMD or other treatment option. + TH1 cells and CD8 + TH17 cells) recover to levels at 12 months (M12) that prove to be indicative of the need for retreatment to achieve full cladribine efficacy within the year.

[0070] Thus, the methods described above may further include the step of administering re-treatment with cladribine, additional and / or higher doses of cladribine, or administration of a different DMD to patients with a suboptimal response to the treatment.

[0071] In the context of the present invention, re-treatment with cladribine preferably comprises or consists of the oral administration of a formulation containing cladribine, said formulation being administered by the following successive steps: (i) an induction period during which the cladribine formulation is administered, wherein the total dose of cladribine achieved 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 about 8 to about 10 months during which no cladribine preparations are administered; (iii) a maintenance period during which the cladribine formulation is administered, wherein the total cladribine dose reached at the end of the maintenance period is less than the total cladribine dose reached at the end of the lead-in period (i); and (iv) a cladribine-free period during which no cladribine preparations are administered It is administered orally according to the procedure described above.

[0072] According to the present invention, preferably, the treatment of said autoimmune disease with DMD, preferably said treatment of MS with DMD and in particular the treatment of MS with Cladribine in an individual, is achieved by the treatment of CD4 + and CD8 + Compared with effector cells, CD4 + Treg cells and CD8 + There was less effect on TEMRA cells, indicating that the increased Treg / Teff ratio suggests tolerance induction associated with long-term efficacy.

[0073] C. Use of Cladribine in Specific MS Populations Involving Plasmablastic / Cells and Other AI / Neuroinflammatory Diseases MS is a heterogeneous disease and specific population that responds poorly to treatment. Current DMDs do not target plasmablast and plasma cell subtypes, which are indicators of more severe MS disease, both directly and indirectly (Tellesford et al., 2020). The present invention addresses this need.

[0074] In accordance with the present invention, preferably, treatment of MS in an individual with Cladribine inhibits the expression of short-lived plasmablasts and CD38 +It has been shown to reduce plasma cells, and the direct effect on these plasma cell subpopulations is further supported by blood data from an MS patient who received two doses of Shingrix vaccine before initiating treatment with DMD cladribine. After the second dose of Shingrix given one week before the start of the cladribine treatment, the patient developed an acute plasma cell reaction, and the elevated plasma cell counts in the blood for the MS patient were reduced by cladribine treatment (citing vaccine patent). These findings suggest that cladribine, particularly when occurring in the cerebrospinal fluid, reduces plasmablasts and CD38 cells, which are linked to disease pathology through autoantibody production and direct cytotoxicity. + It has been shown that it can directly affect and reduce plasma cells.

[0075] The inventors' findings in MS are also important for other diseases. There are various AI and neuroinflammatory diseases in which plasma cells / plasmablasts potentially play a role in disease activity and disease course that the inventors determine would benefit from a treatment that directly affects plasma cells, preferably cladribine treatment. The list of preferred diseases involving plasmablasts and potential use of cladribine as described above includes, but is not limited to, multifocal motor neuropathy (MMN), chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), inclusion body myositis (IBM), myasthenia gravis (MG), and / or neuromyelitis optica (NMO). Furthermore, the list of diseases potentially involving plasmablasts and therefore potential use of cladribine includes, but is not limited to, giant cell arteritis (GCA), polymyositis, microscopic polyangiitis (MPA), amyotrophic lateral sclerosis (ALS), and / or CNS vasculitis (CNSV).

[0076] The inventors further found evidence that plasmablasts / cells are valuable therapeutic targets in many diseases in which B cells and autoantibodies play a role, because these cell populations have been implicated in autoantibody production and Cladribine inhibits short-lived and CD38 +Its direct depleting effect on plasma cells makes it the treatment of choice for DMD. A list of preferred diseases involving auto-antibodies and the resulting potential use of Cladribine includes, but is not limited to, acute disseminated encephalitis (ADEM), giant cell arteritis (GCA), multifocal motor neuropathy (MMN), polymyositis, adrenoleukodystrophy (ALD), anti-MAG peripheral neuropathy, Charcot-Marie-Tooth disease (CMT), diabetic neuropathy, frontotemporal dementia (FTD), Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy (HE), Graves' disease (GD), Huntington's disease (HD), inclusion body myositis (IBM), microscopic polyangiitis (MPA), myasthenia gravis (MG), and / or neuromyelitis optica (NMO). Furthermore, the list of diseases involving B cells or diseases for which B cell depletion therapy is indicated for treatment and therefore also for which Cladribine may be used preferably includes, but is not limited to, giant cell arteritis (GCA), multifocal motor neuropathy (MMN), polymyositis, chronic inflammatory demyelinating polyneuropathy (CIDP), anti-MAG peripheral neuropathy, inclusion body myositis (IBM), microscopic polyangiitis (MPA), myasthenia gravis (MG), neuromyelitis optica (NMO), neurosarcoidosis, and / or CNS vasculitis (CNSV). Accordingly, the present invention relates to a method for treating short-term plasmablasts and / or CD38 + In a patient in need of plasmablast reduction, preferably a patient with an autoimmune disease, short-term plasmablast and / or CD38 + The present invention relates to a method of reducing plasmablasts, the method comprising administering a disease modifying drug (DMD), preferably cladribine, to the patient. + In a patient in need of plasmablast reduction, preferably a patient with an autoimmune disease, short-term plasmablast and / or CD38 + It also involves disease modifying drugs (DMDs), preferably cladribine, for use in reducing plasmablasts.

[0077] In the context of the present invention, short-term plasmablasts and / or CD38 +Patients in need of reducing plasmablasts are those with an autoimmune disease, more preferably selected from the list consisting of multiple sclerosis (MS) and / or multiple sclerosis spectrum disorder (MSSD), neuromyelitis optica and / or neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis (MG), Marie-Tooth disease (CMT), adrenoleukodystrophy (ALD), acute disseminated encephalitis (ADEM), giant cell arteritis, multifocal motor neuropathy, polymyositis, diabetic neuropathy, frontotemporal dementia (FTD), Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy (HE), Graves' disease (GD), Huntington's disease (HD), inclusion body myositis (IBM), microscopic polyangiitis (MPA).

[0078] A preferred aspect of the present invention is the newly discovered role of pathological and regulatory B-cell and T-cell subtypes in MS and other AI / neuroinflammatory diseases, preferably inflammatory and / or demyelinating autoimmune diseases, and in particular MS.

[0079] MS is considered a chronic inflammatory demyelinating disease of the CNS that leads to loss of myelin and concomitant axonal and neuronal degeneration. Historically, factors thought to induce MS included pathogenic Th1, Th17, and CD8 kinases, as well as genetic and environmental factors and infections. + Involves activation of myelin auto-reactive T cells.

[0080] Immune response feedback is believed to be an important mechanism to maintain immune equilibrium. Many inflammatory diseases, including but not limited to multiple sclerosis (MS), are prominent examples of immune imbalance. As a major component of the immune system, B cells play both positive and negative roles in innate and adaptive immunity through effector molecules such as antibodies and cytokines, as well as through antigen-presentation. On the other hand, B cells mediate several potentially negative processes, such as the amplification of immune responses. Mechanistically, they can differentiate into plasmablasts that secrete effector antibodies, and regulate effector T cell responses through antigen presentation and / or the production of inflammatory cytokines. In addition, there are also subsets of B cells that control immune responses to pathogens and self-antigens. These regulatory B cells are the central targets of therapeutic treatment in autoimmune and infectious diseases, as well as cancer.

[0081] Recently, anti-CD20 therapies (rituximab, ocrelizumab, ofatumumab, and tabalumab) that target B cells and B cell cytokines, and cladribine that selectively targets B cells and T cells, have been shown to suppress MS disease activity. Furthermore, B cell depletion in MS can reduce auto-antibodies and pro-inflammatory cytokines produced by B cells, as well as impair antigen presentation. Infiltration of B cells and plasma blasts / cells has been observed in brain tissue of MS patients, and oligoclonal bands (clonal IgG) and antibody-secreting plasma cells have been found in the cerebrospinal fluid. It is clear that ectopic B cell follicles in the meninges are involved in and increase in the progression of secondary progressive MS. B cells are centrally involved in the pathogenesis of autoimmunity and exert diverse actions, including contributing to T cell activation through antibody production and antigen presentation. Antibody-secreting cells (ASCs) represent a series of antigen-activated B cells that, through differentiation into plasmablasts and mature plasma cells, commit metabolic and protein processing mechanisms to antibody production. Indeed, plasmablasts and plasma cells, as ASCs derived from antigen-experienced B cells, appear to be key drivers of both inflammatory and neurodegenerative aspects of MS pathogenesis, as well as other inflammatory and / or demyelinating autoimmune diseases. ASCs are enriched in the CSF during active gadolinium-enhancing disease, ASC-derived intrathecal IgG correlates with CNS atrophy, and IgM-producing ASCs are associated with aggressive disease course. Despite heterogeneity across the ASC spectrum, these cells can be collectively identified as brightly positive for CD27 and CD38.

[0082] B and T cells exert multiple pro-inflammatory effects, but also have regulatory functions that contribute to their role in further complicating the pathogenesis of MS and other AI diseases. In addition to antigen presentation and antibody production, B cells have also been shown to play a role in immune regulation and induction of immune tolerance through IL-10-dependent and -independent mechanisms. Various B cell subsets have been defined as regulatory B cells (Bregs), which express the CD19 + CD24 hi CD38 hiExpressing B cells and / or CD45 bright , S.S.C. low , CD3 - , CD14 - , CD56 - , CD19 + , CD24 bright , CD38 bright have been identified as the major IL-10-expressing subset ("bright" and "hi" are preferably used synonymously unless clearly specified otherwise). Bregs are normally induced and maintained in response to inflammation and participate in immune regulation and suppression of enhanced immune responses. The regulatory action of Bregs was described in various models of inflammation, autoimmune disease, transplantation reactions, and anti-tumor immunity. However, this was not explained until the late 1990s, when two independent studies revealed autoimmune diseases (experimental autoimmune encephalomyelitis (EAE) and chronic colitis) that were exacerbated in B cell-deficient groups, providing further evidence of Bregs and suggesting their function in suppressing inflammation. Bregs control the immune system by different mechanisms. The main mechanism is the production of the anti-inflammatory cytokine interleukin 10 (IL-10). IL-10 has a strong anti-inflammatory effect and it inhibits or suppresses T cell-mediated inflammatory responses, especially Th1 or TH17 type immune responses. This has been shown, for example, in models of MS (EAE, experimental encephalomyelitis), RA, CIA (collagen-induced arthritis) or contact hypersensitivity.

[0083] Another inhibitory Breg mechanism involves the production of transforming growth factor (TGF-β) and regulation of cell surface molecules, including FasL and / or PD-L1, which lead to the killing of target cells.

[0084] Transitional B cells (TrB cells) are bone marrow-derived, immature B cells that are also thought to be precursors of mature B cells. TrB cells represent a subpopulation of regulatory B cells in healthy individuals, but they do not express circulating CD24 hi CD38 hiThe frequency of TrB cells can be altered in individuals with autoimmune diseases, such as multiple sclerosis, neuromyelitis optica spectrum disorder, and systemic lupus erythematosus. TrB cells produce IL-10 and are CD4 + It controls the proliferation and differentiation of T cells into T helper (Th) effector cells. According to the current understanding of the present inventors, human CD19 + CD24 hi CD38 hi The main functions of TrB cells are as follows: First, IL-10 produced by TrB cells inhibits autoreactive CD4 + Second, TrB cells suppress T cell proliferation by inhibiting CD4 + Expansion of Th1 cells (IFN-γ and TNF-α production) and CD4 + They suppress the production of proinflammatory cytokines by limiting the expansion of Th17 cells (IL-17 production), which is dependent on IL-10, programmed cell death-ligand 1 (PD-L1), CD80, and CD86, but not on transforming growth factor-β (TGF-β). Third, TrB cells suppress the production of proinflammatory cytokines by limiting the expansion of Th17 cells (IL-17 production), which is dependent on IL-10, programmed cell death-ligand 1 (PD-L1), CD80, and CD86, but not on transforming growth factor-β (TGF-β). + Prevents T cells from differentiating into Th1 and Th17 cells and inhibits effector CD4 + CD4 on T cells + FoxP3 + They promote Treg conversion while limiting excessive proinflammatory cytokine production. Finally, TrB cells inhibit the expression of CD8 + Inhibits T cell responses and maintains invariant natural killer T (iNKT) cells.

[0085] Furthermore, significantly fewer TrB cells were detected in individuals or patients with MS / clinical isolated syndrome (CIS), preferably in body fluid samples from individuals or patients, compared to healthy controls. Similar to TrB cells, the frequency of other circulating B cell subsets (naive, MZ-like, and memory B cells) in the peripheral blood of MS / CIS cases was comparable to that of healthy controls. NMOSD is an idiopathic inflammatory demyelinating disease affecting the CNS and is clinically characterized by elongated transverse myelitis (LETM) and severe optic neuritis (ON), traditionally considered a variant of Asian MS. To date, only a few studies have been conducted to explore the role of TrB cells in NMOSD. Specifically, fewer CD19 cells were detected in NMOSD than in individuals with MS. + CD24 hi CD38 hi TrB cells were detected in individuals or patients with NMOSD, preferably in body fluid samples of individuals or patients. + CD27 + The frequencies of memory B cells and mature B cells were not significantly different between NMOSD and HC patients, but CD19 + CD5 + CD1 dhi The proportion of Bregs was decreased in NMOSD patients. The proportion of TrB cells was found to be lower in the AQP4-positive group than in the AQP4-negative group. Furthermore, it was reported that the frequency of cells expressing IL-10 among all lymphocytes was lower in individuals or patients with NMOSD, preferably in body fluid samples of individuals or patients, than in MS patients and healthy individuals. This indirectly indicates the impairment of the immunoregulatory function of TrB cells in NMOSD due to the reduction of TrB cells and / or IL-10.

[0086] Thus, administration of a disease-modifying drug (DMD), in particular cladribine, to a subject exhibiting an autoimmune disease characterized by an altered number of circulating TrB cells, in particular a decreased number of circulating TrB cells compared to healthy subjects, provides an increase in transitional B cells and / or regulatory B cells.

[0087] Thus, another aspect of the present invention is the use of a disease modifying drug (DMD), preferably cladribine, to modulate immune tolerance in a patient, preferably to improve immune tolerance in a patient in need thereof.

[0088] The present invention further relates to a method for increasing transitional B cells and / or regulatory B cells in a patient in need thereof, preferably a patient suffering from an autoimmune disease, which method comprises administering to said patient a disease modifying drug (DMD), preferably cladribine.The present invention relates to a method for improving immune tolerance in a patient in need thereof, preferably a patient suffering from an autoimmune disease, preferably by increasing transitional B cells and / or regulatory B cells, which method comprises administering to said patient a disease modifying drug (DMD), preferably cladribine.

[0089] In the context of the present invention, a patient in need of increasing transitional B cells and / or regulatory B cells is a patient with an autoimmune disease as defined herein, preferably selected from the list consisting of: multiple sclerosis (MS) and / or multiple sclerosis spectrum disorder (MSSD), neuromyelitis optica and / or neuromyelitis optica spectrum disorder (NMOSD).

[0090] Treg was originally reported in 1995 by Sakaguchi et al. as a CD4 + CD25 + They have been identified as a T cell subset. They are essential for the maintenance of self-tolerance and their impairment has been linked to autoimmunity and includes reduced numbers, loss of function and transformation into inflammatory effector cells. High expression of CD25 and low expression of CD127 are the major phenotypic markers that characterize bona fide human Tregs. Regulatory CD4 in the thymus + CD25 + The discovery of forkhead box P3 (FoxP3) as a fundamental transcription factor for T cell development has helped researchers to accurately phenotype most Tregs. Currently, Tregs are classified as CD4+ CD25 + FoxP3 + as T cells or as FoxP3, which is inversely correlated with cell surface CD127 expression + CD25 + CD127 lo / - T cells have been precisely identified as T cells. Tregs have been found to play a central role in controlling the immune system by regulating the number and function of effector cells. Thus, they play a major role in suppressing unwanted self-reactive immune responses, such as in cases of autoimmunity. Interestingly, Tregs can regulate both the adaptive and innate immune systems, and once activated, they have been noted to specifically control immune responses at multiple levels and by various mechanisms. These suppressive mechanisms can be organized into a large group, including cell-cell contact-dependent suppression, release of inhibitory cytokines (such as IL-10 and TGF-β), modulation of APC function, cell lysis, metabolic destruction, and induction of suppressor cells or "infection resistance." Recent studies suggest that the innate immune system plays a key role in both the development and progression of MS by affecting the effector functions of T and B cells.

[0091] Natural killer (NK) cells NK cells have been shown to contribute to both effector and regulatory functions of innate immunity through their cytotoxic activity and their ability to secrete pro- and anti-inflammatory cytokines and growth factors. The mechanisms by which NK cells have an influence on autoimmune responses include rapid cytokine release prior to autoreactive T cell differentiation, and modulation of interactions between autoreactive T and B lymphocytes and antigen-presenting cells. Several NK subsets responsible for different functions have been identified. Based on the surface expression of CD56, NK cells are Effector cells (CD56 dim ) or Regulatory cells (CD56 bright )Effector NK cells are characterized by the intracellular expression of perforin and granzymes, which are proteolytic enzymes involved in target cell death. They further express CD56 + CD16 + (cytotoxic effector cells) and CD56 + CD3 + (NK T cells), which produce various cytokines and regulate other immune cells. bright NK regulatory cells account for less than 10% of peripheral blood NK cells, express low levels of perforin, and can secrete large amounts of cytokines. - CD16 + NK cells are a defective NK subset with impaired cytolytic function that is increased in viremic HIV and Hepatitis C virus (HCV) infected individuals. NK cells have been suggested to play a key role at the interface of innate and adaptive responses in autoimmune diseases. Both pathogenic and protective roles in the central nervous system (CNS), depending on the cell subtype and the environment, have been attributed to various NK subpopulations in MS and its animal model experimental autoimmune encephalomyelitis (EAE). In line with this, NK cell lines are able to induce lysis of oligodendrocytes. In addition, CNS-resident NK cells can inhibit T helper type 17 (Th17) differentiation in EAE by interacting with microglial cells. MS patients have a higher prevalence of regulatory / effector (CD56 bright / CD56 dim It has been found that patients with active MS show a marked increase in the NK cell ratio (CD56). The bright / dim ratio in CSF remains relatively high in active MS [1.0 (0.57-1.89), median (25-75% interquartile range)] compared to IND patients [0.43 (0.25-1.28), P = 0.019]. Another study described an altered proportion of NK cells in the CSF of MS patients. Furthermore, the beneficial effect of daclizumab, a humanized monoclonal antibody (mAb) that blocks the binding site on the IL-2Rα chain (CD25), was observed to be related to the CD56 brightLinked to cell expansion. Regulatory CD56 bright NK cell expansion is associated with favorable responses to daclizumab and IFN-β, whereas CD56 dim The cells were reduced after immunomodulatory therapy.

[0092] Transitional B cells and Bregs B cells are centrally involved in the pathogenesis of autoimmunity and exert diverse functions, including contributing to T cell activation through antibody production and antigen presentation. B cells also regulate immunological functions by suppressing T cell proliferation and producing proinflammatory cytokines, such as interferon-gamma (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-17.

[0093] B cells develop from pre-B cells, through immature cells in the BM, to transitional cells in the periphery, and then in a linear pathway to peripheral naive cells. Transitional B cells (TrB cells) represent a crucial link between immature B cells in the bone marrow and mature B cells in the periphery. TrB cells represent a subpopulation of regulatory B cells in healthy individuals, but they do not express CD24 in circulation. hi CD38 hi The frequency of TrB cells can be altered in individuals with autoimmune diseases, such as multiple sclerosis, neuromyelitis optica spectrum disorder, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, systemic sclerosis, and juvenile dermatomyositis. TrB cells play a regulatory role under inflammatory conditions, but the consequences of their dysfunction vary depending on the autoimmune disease.

[0094] The process of B cell development in humans is similar to that in mice (B cells originating from HSCs are processed in the bone marrow where they undergo heavy and light chain rearrangement before entering the peripheral compartment). HSCs initially derive from a common lymphoid precursor and then develop into pro-B cells through rearrangement of D and J segments following flanking of variable (VH) genes by DJH elements. Pro-B cells mature into pre-B cells through rearrangement of μ-H-chain gene segments, which couple with alternative light chains. After the initial rearrangement of light chains (VL), human pre-B cells undergo one or two cell divisions and develop into immature B cells, which initially express μ / κ or κ / λ surface IgM receptors. These immature T1 B cells are thought to emigrate from the bone marrow and subsequently differentiate into T2 B cells in the periphery, and ultimately give rise to T3 B cells. However, some discrepancies regarding this process exist. TrB cells can be differentiated into T1 or T2 B cells. At any B cell stage, they can leave the bone marrow, and some T1 B cells can differentiate into T2 B cells in the bone marrow, and it is believed that similar ratios of T1 and T2 B cell subsets are noted in normal human bone marrow. Consistent with previous findings in mice, positive and negative selection can also control the development of human TrB cells. In normal human peripheral blood, approximately 40% of TrB cells exhibit autoreactive behavior; when they differentiate into mature naive B cells, their frequency is reduced to 20%. TrB cell maturation requires Btk, which reveals the possibility of B cell receptor engagement (BCR engagement), and this is accompanied by improved survival. B cell activating factor (BAFF) promotes the selection of immature B cells and the differentiation of non-autoreactive immature B cells into TrB cells, but the role of BAFF in human TrB cell development is controversial. Some previous data indicate that BAFF has no effect on human TrB cells, but it acts as an efficient pro-survival factor for mouse TrB cells. Some data suggest that BAFF may be involved in the development of human T1 They note that B cell proliferation is clearly induced by BAFF, but to a lesser extent than that of human T2 B cells.Thus, during the process of TrB cell differentiation into mature naive B cells, a selection of transitional B cells (TrB cells) are bone marrow-derived immature B cells, which are also considered precursors of mature B cells. TrB cells express all CD19 in healthy individuals. + They account for approximately 4% of B lymphocytes. They are present in human bone marrow, peripheral blood, umbilical cord blood, and secondary lymphoid tissues (i.e., spleen, tonsils, lymph nodes, and gut-associated lymphoid tissue [GALT]).

[0095] CD24 hi CD38 hi TrB cells are closely related to IL-10-producing regulatory B cells (Bregs) with respect to phenotypic and functional similarities. TrB cells also produce IL-10 and are CD4 + They can also regulate T cell proliferation and differentiation into T helper (Th) effector cells. Low frequencies of TrB cells have been noted in patients with neuroimmune diseases, including multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). As they are associated with several inflammatory diseases and are also found in the circulation and tissues of healthy individuals, TrB cells are believed to play a prominent role in immune-defense mechanisms.

[0096] Transitional B cells and their relationship to Bregs in humans Human TrB cells were first described in detail in 2005 and are CD24 hi CD38 hi It is often characterized by its phenotype. Human CD24 hi CD38 hi TrB cells express all CD19 in peripheral blood. + They account for approximately 4% of B cells and nearly 50% of B cells in umbilical cord blood, and their proportion gradually decreases during childhood. Human TrB cells are also found in the GALT. Human TrB cells are divided into three subsets, including T1 B cells, T2 B cells, and T3 B cells.

[0097] In 2016, Simon developed human CD24 hi CD38 hiWe first proposed that TrB cells contain not only one subset, but rather four subsets based on expression of CD27, IgM, IgD, CD10, CD21, and CD32. T1-T3 B cells express low levels of CD27, whereas T4-T5 B cells express high levels of CD27. + TrB cells express high levels of CD27, CD24, and CD38. In T1 B cells, expression of IgM, CD10, and CD32 is high, whereas IgD and CD21 are low. T2 B cells show intermediate expression of IgM, IgD, CD10, and CD32, and low expression of CD21. However, T3 B cells express low levels of IgM, IgD, CD10, CD21, and CD32.

[0098] Some studies have shown that CD27 + We pointed out that TrB cells act as a Breg type and not just transitional B cells. Bregs contribute to the maintenance of immune tolerance and the regulation of immune responses. Currently, human Bregs are being noted at different stages of B cell development and in various B cell subsets, namely CD24 hi CD38 hi Immature TrB cells, CD24 hi CD38 lo CD27 + Memory B cells and CD24 - CD38 + CD27 int Consists of plasmablasts that secrete IL-10. Human IL-10-producing Bregs are CD24 hi CD38 hi are enriched within immature TrB cell subsets. These findings reveal a relationship between human TrB cells and Bregs.

[0099] Key functions of transitional B cells Human TrB cells produce low amounts of Ig and exhibit less differentiation, proliferation, and chemotaxis than mature B cells in vitro. + CD24 hi CD38 hiHuman IL-10+ B cells with the phenotype are found in the peripheral blood of healthy individuals. + CD24 hi CD38 hi The main functions of TrB cells are summarized in Figure 2. First, IL-10 produced by TrB cells inhibits autoreactive CD4 + Second, TrB cells suppress T cell proliferation by inhibiting CD4 + Th1 cells (IFN-γ and TNF-α producing) and CD4 + They suppress the production of proinflammatory cytokines by limiting the expansion of Th17 cells (IL-17 production), which is mediated by IL-10, programmed cell death-ligand 1 (PD-L1), CD80, and CD86, but not by transforming growth factor-β (TGF-β). Third, TrB cells suppress the production of proinflammatory cytokines by limiting the expansion of Th17 cells (IL-17 production), which is mediated by IL-10, programmed cell death-ligand 1 (PD-L1), CD80, and CD86, but not by transforming growth factor-β (TGF-β). + Prevents T cells from differentiating into Th1 and Th17 cells and inhibits effector CD4 + T cell, CD4 + FoxP3 + They promote Treg conversion while limiting the production of excessive proinflammatory cytokines. Finally, TrB cells inhibit the expression of CD8 + Inhibits T cell responses and maintains invariant natural killer T (iNKT) cells.

[0100] Recent findings reveal distinct subpopulations of TrB cells with distinct functions. For example, T2 / T3 B cells are CD4 + It has a remarkable ability to reduce T cell proliferation (in contrast to other subpopulations), as well as CD27 + TrB cells only, CD4 + In addition to producing anti-inflammatory factors, TrB cells can also secrete pro-inflammatory cytokines, such as IL-6 and TNF-α. Data from previous studies have shown that the frequency of IL-6-producing TrB cells is elevated in SLE, SS, and other immune disorders. IL-6 is a cytotoxic inhibitor of CD4 +They limit T cell differentiation into Tregs and induce autoreactive Th1 / Th17 T cell responses. Thus, the pro- and anti-inflammatory cytokines produced by TrB cells may affect their functional stability.

[0101] 1) TrB cells express autoreactive CD4 + Inhibits T cell proliferation. 2) TrB cells are CD4 + It prevents the differentiation of T cells into Th1 and Th17 cells, thereby limiting the production of excess pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-17). 3) TrB cells are effector CD4 + CD4 on T cells + FoxP3 + Promotes conversion to Tregs. 4) TrB cells are CD8 + Inhibits T cell responses. The inhibitory function of TrB cells depends in part on the secretion of IL-10, which can downregulate CD86 expression in an autocrine manner. TrB cells can also secrete pro-inflammatory cytokines, such as IL-6 and TNF-α. An imbalance between pro- and anti-inflammatory cytokine production in TrB cells can affect their functional stability, thus participating in the development of immune diseases.

[0102] TrB cells and TrB-associated molecules in MS and EAE MS is considered a chronic inflammatory demyelinating disease of the CNS, leading to myelin loss accompanied by axonal and neuronal degeneration. The pathological features of MS are typically characterized by temporal and spatial dissemination. Histologically, factors thought to induce MS include pathogenic Th1, Th17, and CD8 deficiencies, as well as genetic and environmental factors and infections. + Contains myelin autoreactive T cells.

[0103] In support of this animal model, EAE also induces myelin-specific CD4 + T cells and CD8 +It can be induced by T cells. Recently, therapies targeting B cells and B cell cytokines (rituximab, ocrelizumab, ofatumumab, and tabalumab) have been shown to suppress MS disease activity. Furthermore, B cell depletion in MS may reduce auto-antibodies and pro-inflammatory cytokines produced by B cells, as well as impair antigen presentation. B cell infiltration has been observed in brain tissue of MS patients, and oligoclonal bands (clonal IgG) and antibody-secreting plasma cells have been found in the cerebrospinal fluid. Ectopic B cell follicles in the meninges have been formatted in secondary progressive MS. Taken together, these findings reveal the involvement of B cells in MS pathogenesis. Recent studies have shown that B cells are virtually depleted after anti-CD20 treatment of EAE, and then 6-8 weeks later, they gradually reappear in the bone marrow, spleen, lymph nodes, and blood. Surprisingly, splenic TrB and follicular B cells were found to be largely absent in anti-CD20 treated EAE at 8 weeks post-treatment.

[0104] In addition to their involvement in MS pathogenesis, B cells also exert immunoregulatory effects, either directly or indirectly, by producing anti-inflammatory cytokines. Bregs play a regulatory role in MS by helping to maintain immune tolerance. In mice, IL-10-producing CD1 dhi CD5 + B cells play a more substantial role in suppressing the initiation and development of EAE than other B cells: TrB cells represent a significant proportion of functional Breg cells. Significantly fewer TrB cells were detected in patients with MS / clinical isolated syndrome (CIS). Similar to that of TrB cells, the frequencies of other circulating B cell subsets (naive, MZ-like, and memory B cells) in the peripheral blood of MS / CIS cases were comparable to those of healthy controls.

[0105] Some data further reveal that TrB cells are capable of homing to inflammatory sites in the CNS during the early phase of MS due to upregulation of α4 and β1 integrin expression. TrB cells in the peripheral blood of MS patients treated with fingolimod produced more IL-10 and less TNF-α and expressed lower levels of CD80 than memory B cells and mature naive B cells. Related studies have demonstrated an immunoregulatory role for Bregs, mediated in part through the production of IL-10, IL-35, TGF-β, and PD-L1. In EAE, CD40 signaling is required for the induction of IL-10 competence. Subsequently, IL-21 drives the expansion of IL-10-producing B cells and the generation of effector cells. IL-6-producing B cells has been implicated in the pathogenesis of EAE / MS. More than 65% of IL-6 is produced primarily by B cells, which exacerbates EAE, enhances Th1 and Th17-type autoreactive T cell responses, and inhibits the generation of Tregs. Similar to Bregs, TrB cells play an immunoregulatory role in MS / EAE.

[0106] TrB cells and TrB-associated molecules in neuromyelitis optica spectrum disorders NMOSD is an idiopathic inflammatory demyelinating disease affecting the CNS and is clinically characterized by elongated transverse myelitis (LETM) and severe optic neuritis (ON), previously considered a variant of Asian MS. Lennon et al. first discovered a serum marker for neuromyelitis optica (NMO), known as aquaporin-4 antibody (AQP4-Ab) or NMO-IgG, which results in NMO unrelated to MS. The identification of AQP4-Ab broadened the NMO spectrum. AQP4-Ab is involved in the pathogenesis of NMOSD and depends on the presence of B cells, T cells, and complement, suggesting that NMOSD is probably mediated primarily by humoral immunity. NMOSD cases can be associated with other autoimmune diseases such as SLE, SS, and thyroiditis, implying that NMOSD may involve mechanisms similar to those of autoimmune diseases. Like SLE and other autoantibody-mediated immune disorders, NMOSD cases have a lack of integrity of central and peripheral B cell tolerance checkpoints. In addition, impaired peripheral B cell tolerance checkpoints interfere with the elimination of autoreactive B cells, leading to an increase in reservoir autoreactive de novo emigrant / transitional and mature naive B cells, and this process is associated with the production of pathogenic anti-AQP4 autoantibodies.

[0107] Currently, very few studies have explored the role of TrB cells and TrB-associated molecules in NMOSD. + CD24 hi CD38 hi TrB cells were more prevalent in NMOSD patients than in MS patients. + CD27 + The frequencies of memory B cells and mature B cells were not significantly different between NMOSD and HC cases, NMOSD In the case, CD19 + CD5 + CD1 dhi Breg The percentage of decreaseThe proportion of TrB cells was found to be lower in the AQP4-positive group than in the AQP4-negative group. IL-10-expressing B cells among all lymphocytes The frequency of It has been reported to be lower in NMOSD cases This indirectly indicates that the impaired immunoregulatory function of TrB cells in NMOSD is due to a decrease in TrB cells and / or IL-10; IL-10 production in TrB cells was not measured in most studies. Rituximab treatment in NMOSD patients significantly upregulated CD27 + Reduces memory B cell numbers and CD24 hi CD38 hi Increased the number of TrB cells. Related studies have also shown elevated levels of BAFF, IL-6, and IL-21 in both serum and CSF of NMOSD cases, although the expression of these molecules in TrB cells remains unclear. AQP4-Ab upregulates the CD27 hi CD38 hi These results suggest that IL-6-dependent B cells are likely involved in the pathogenesis of NMOSD, and are derived primarily from CD180- B cells, whose survival depends on signaling through the IL-6 receptor. + We provided evidence that IL-6 and IL-21 suppress Treg production while inducing IL-17 secretion from Th17 cells. Further studies will be required to explore the mechanism that drives IL-6 production by TrB cells in NMOSD cases. If the proportion of IL-6-producing TrB cells is elevated in NMOSD cases, IL-6 secretion may impair the immune-regulatory function of TrB cells.

[0108] Characterization of peripheral immune cell dynamics and repopulation patterns during Cladribine treatment, preferably the first 12 months of Cladribine treatment: Markers of early efficacy during and / or after DMD treatment, and preferably during and / or after Cladribine treatment (e.g., 1 and / or 2 months): % reduction from baseline CD19 + B cell depletion (>70%) Memory CD19 + B cell depletion (>70%), and / or · Reduction in both naive and memory B cells (>70%); and / or any combination thereof.

[0109] Markers of long term efficacy during and / or after DMD treatment, and preferably during and / or after Cladribine treatment (e.g., 3-6-12 months, and preferably beyond, e.g., up to 4 years, up to 6 years, up to 8 years, or up to 10 years, and preferably beyond): % reduction from baseline Memory CD19 + Sustained reduction in B cells (>70%) and / or plasma cells (>50%) Increased levels of Bregs and transitional B cells (>70%), and / or · Reduction in naive T cells, central memory and effector T cells (TBD); and / or any combination thereof.

[0110] Markers of durable efficacy during and / or after DMD treatment and preferably during and / or after Cladribine treatment: Regulatory / Effector Cell Ratio: If applicable, preferably an increase or decrease from baseline an increased NKreg / NKeff ratio compared to baseline, preferably >1.5-fold compared to baseline; Increased Breg / Beff cell ratio compared to baseline, and / or · Increased Treg / Teff cell ratio compared to baseline; and / or any combination thereof.

[0111] Markers of non-response, e.g., at 1 and / or 2 months, during and / or after DMD treatment, and preferably during and / or after Cladribine treatment: % reduction from baseline CD19 + <30% reduction in B cells Memory CD19 + <30% reduction in B cells, and / or ·<30% of naïve; and / or any combination thereof.

[0112] Markers of disease relapse during and / or after DMD treatment, and preferably during and / or after Cladribine treatment: % Deviation from Baseline memory B cells repopulating to levels greater than -40% above baseline and / or plasma cells repopulating to levels greater than -20% above baseline; T repopulating to levels greater than -40%, -20%, and -10%, respectively ナイーブ , T CM and / or T EM , - Decreased Treg / Teff and / or Breg / Bmem ratios compared to baseline, - Decreased NKreg / NKeff ratio compared to baseline; and / or any combination thereof.

[0113] Favorable characteristics of peripheral immune cell dynamics and repopulation patterns during Cladribine treatment, preferably the first 12 months of Cladribine treatment:

[0114] Preferred markers of early efficacy during and / or after DMD treatment, and preferably during and / or after Cladribine treatment (e.g., 1 and / or 2 months): % reduction from baseline CD19 + B cell depletion (>70%) Memory CD19 + B cell depletion (>70%), and / or · Reduction in both naive and memory B cells (>70%); Decrease in activated B cells (CD69+;>45%) and / or any combination thereof.

[0115] Preferred markers of long term efficacy during and / or after DMD treatment, and preferably during and / or after Cladribine treatment (e.g., 3-6-12 months, and preferably beyond, e.g., up to 4 years, up to 6 years, up to 8 years, or up to 10 years, and preferably beyond): % reduction from baseline Memory CD19 + Sustained reduction in B cells (>70%) and / or plasma cells (>50%) Increased levels of Bregs and transitional B cells (>70%), and / or · Reduction in naive T cells, central memory and effector T cells (>10%, >40%, >30%); - Reduction in TH1 and RH17 cells (35% each), and / or any combination thereof.

[0116] Preferred markers of durable efficacy during and / or after DMD treatment, and preferably during and / or after Cladribine treatment: regulatory / effector cell ratio, % of whole blood or PBMC, if applicable, preferably increase or decrease from baseline; an increased NKreg / NKeff ratio compared to baseline, preferably >1.5-fold compared to baseline; Increased Breg / Beff cell ratio compared to baseline, and / or · Increased Treg / Teff cell ratio compared to baseline; - %Breg of stable MS (preferably 14-20%, even more preferably 10-30%), and / or any combination thereof.

[0117] Markers of non-response, e.g., at 1 and / or 2 months, during and / or after DMD treatment, and preferably during and / or after Cladribine treatment: % reduction from baseline CD19 + <30% reduction in B cells Memory CD19 +<30% reduction in B cells, and / or ·<30% of naïve; and / or any combination thereof.

[0118] Preferred markers of disease relapse during and / or after DMD treatment, and preferably during and / or after Cladribine treatment: Increase in memory B cells (preferably to 0.05-1% of total PBMCs) combined with an increased transitional B cell ratio in total PBMCs - Breg% in whole blood from active or relapsing MS patients (preferably 3-14%, even more preferably 1-10%), Increase in transitional B cells to memory B cells (absolute cell number ratio: preferably >1, more preferably 1-3).

[0119] Preferred markers of disease relapse during and / or after DMD treatment, and preferably during and / or after Cladribine treatment: % Deviation from Baseline memory B cells repopulating to levels greater than -40% above baseline and / or plasma cells repopulating to levels greater than -20% above baseline; T repopulating to levels greater than -40%, -20%, and -10%, respectively ナイーブ , T CM and / or T EM , - Decreased Treg / Teff and / or Breg / Bmem ratios compared to baseline, - Decreased NKreg / NKeff ratio compared to baseline; and / or any combination thereof.

[0120] Further quantitative examples of preferably biomarker-active cells and / or cell populations are provided below: [Table 1] [Table 2]

[0121] Thus, in one aspect, the present invention relates to a method for predicting the response of a patient treated with cladribine for the treatment of an autoimmune disease, preferably multiple sclerosis, comprising: a) where the deviation of the following cell populations: - At least 70% CD19 + B cells, - At least 70% memory CD19 + B cells, - at least 70% naive B cells, - at least 70% memory B cells, - activated B cells, and / or - At least 45% activated CD69 + B cells, and / or combinations thereof; is an indication of early efficacy of the cladribine treatment; where - the cell populations are preferably determined in one or more body fluid samples from the patient at different stages of the cladribine treatment, including determining the cell populations in a body fluid sample from the patient collected before the start of treatment with cladribine, and determining the cell populations in a body fluid sample from the patient collected at least at a later time point during or after the cladribine treatment, - the deviation is presented as a decrease from a baseline level; b) deviation of the following cell populations: - At least 70% CD19 + B cells, - At least 50% CD38 + Plasma cells, - at least 50% short-lived plasma cells, - At least 70% Breg, - at least 70% transitional B cells, - at least 10% naive T cells, - at least 40% central memory T cells, - at least 30% central effector T cells, - at least 35% TH1 cells, - at least 35% TH17 cells, and / or combinations thereof; is an indicator of durable efficacy of the cladribine treatment at 1 year; where - the cell populations are preferably determined in one or more body fluid samples from the patient at different stages of the cladribine treatment, including determining the cell populations in a body fluid sample from the patient collected before the start of treatment with cladribine, and determining the cell populations in a body fluid sample from the patient collected at least at a later time point during or after the cladribine treatment, - the deviation is presented as a decrease from a baseline level; c) Where: - deviation of the memory B cell population to a level above -40%; - deviation of the plasma cell population to a level above 20%, - deviation of the naive T cell population to a level above -40%, - CD4 to levels above 20% + Deviation of central memory T cell populations, - CD4 to levels above 10% + Deviation of effector memory T cell populations, - a deviation in the cell population of the Treg / Teff cell ratio, said deviation being a decrease; - a deviation in the cell population of the Breg / memory B cell ratio, said deviation being a decrease; - a deviation in the cell population of the NKreg / NKeff cell ratio, said deviation being a decrease; and / or any combination thereof: is an indicator of risk of multiple sclerosis relapse, where: - the cell populations are preferably determined in one or more bodily fluid samples from the patient at different stages of the cladribine treatment, including determining the cell populations in a bodily fluid sample from the patient collected before the start of treatment with cladribine, and determining the cell populations in a bodily fluid sample from the patient collected at least at a later time point during or after the cladribine treatment, - The deviation is from a baseline level.

[0122] Thus, in one aspect, the present invention relates to a method for monitoring the need to adapt the treatment of an autoimmune disease, preferably multiple sclerosis, with a disease modifying drug (DMD) in a patient in need thereof, comprising: -wherein the patient is being treated with a DMD that is cladribine for the treatment of an autoimmune disease, preferably multiple sclerosis; wherein preferably, the deviation of the cell populations determined in one or more bodily fluid samples from the patient at different stages of the Cladribine treatment, comprising determining said cell populations in a bodily fluid sample from the patient collected before the start of treatment with Cladribine, and determining said cell populations in a bodily fluid sample from the patient collected at least at a later time point during or after the Cladribine treatment, is: - deviation of the memory B cell population to a level above -40%; - deviation of the plasma cell population to a level above 20%, - deviation of the naive T cell population to a level above -40%, - CD4 to levels above 20% + Deviation of central memory T cell populations, - CD4 to levels above 10% + Deviation of effector memory T cell populations, - a deviation in the cell population of the Treg / Teff cell ratio, said deviation being a decrease; - a deviation in the cell population of the Breg / memory B cell ratio, said deviation being a decrease; - a deviation in the cell population of the NKreg / NKeff cell ratio, said deviation being a decrease; - and / or combinations thereof; The deviation is now indicative of the need for re-treatment of the patient with Cladribine.

[0123] Thus, in one aspect, the present invention relates to a method for the treatment of an autoimmune disease, preferably multiple sclerosis, in a patient at risk of relapse of multiple sclerosis with a disease modifying drug (DMD), comprising: -wherein the patient is being treated with a DMD that is cladribine for the treatment of an autoimmune disease, preferably multiple sclerosis; wherein preferably, the deviation of the cell populations determined in one or more bodily fluid samples from the patient at different stages of the Cladribine treatment, comprising determining said cell populations in a bodily fluid sample from the patient collected before the start of treatment with Cladribine, and determining said cell populations in a bodily fluid sample from the patient collected at least at a later time point during or after the Cladribine treatment, is: - deviation of the memory B cell population to a level above -40%; - deviation of the plasma cell population to a level of more than 20%; - deviation of the naive T cell population to a level above -40%, - CD4 to levels above 20% + Deviation of central memory T cell populations, - CD4 to levels above 10% + Deviation of effector memory T cell populations, - a deviation in the cell population of the Treg / Teff cell ratio, said deviation being a decrease; - a deviation in the cell population of the Breg / memory B cell ratio, said deviation being a decrease; - a deviation in the cell population of the NKreg / NKeff cell ratio, said deviation being a decrease; - and / or combinations thereof; This is an indication that the patient is at risk for multiple sclerosis relapse, Here, the treatment includes administering to the patient re-treatment with cladribine, administration of additional and / or higher doses of cladribine, or administration of a different DMD.

[0124] Thus in one aspect there is preferably provided a method of treating an autoimmune disease in a patient in need thereof, the method preferably comprising orally administering Cladribine to said patient, preferably orally administering Cladribine to said patient in an amount of 0.5-1.25 mg / kg body weight of said patient, preferably orally administering Cladribine to said patient in an amount of 0.5-1.25 mg / kg body weight of said patient for a period of 4-12 weeks, wherein after said administration said patient has one or more deviations for one or more cell populations selected from the group consisting of: a) Wherein the following deviations, provided as % reduction from baseline levels, are biomarkers / corresponding to the early efficacy of the Cladribine treatment: - CD19 + B cells (>70%, preferably ±10%) - Memory CD19 + B cells (>70%, preferably ±10%) - naive B cells (>70%, preferably ±10%), - memory B cells (>70%, preferably ±10%), - activated B cells, and / or - Activated CD69 + B cells (>45, preferably ±10%) and / or combinations thereof; b) Durable efficacy (% reduction from baseline) at 1 year; - Memory CD19 + B cells (>70%, preferably ±10%) - CD38 + Plasma cells (>50%, preferably ±10%) - short-lived plasma cells (>50%, preferably ±10%), - Breg (>70%, preferably ±10%), - transitional B cells (>70%, preferably ±10%), - naive T cells (>10%, preferably ±5%), - central memory T cells (>40%, preferably ±10%), - central effector T cells (>30%, preferably ±10%), - TH1 cells (>35, preferably ±10%), - TH17 cells (>35, preferably ±10%), and / or combinations thereof; c) Durable Effectiveness: - NKreg / NKeff cell ratio (>1.5x), - Breg / Beff cell ratio (> / < times), - Treg / Teff cell ratio (> / < fold), and / or d) Disease relapse: - A decrease in memory B cells from baseline (<30%, preferably ±10%), - memory B cells (preferably up to 0.05-1% of PBMCs), - transitional B cell ratio (> / <????), preferably in total PBMCs; - Bregs (preferably 3-14%, even more preferably 1-10%), preferably in whole blood or PBMCs, preferably from active or relapsing MS patients, and / or e) Disease relapse - memory B cells (preferably repopulating to levels greater than -40% from baseline); - plasma cells (preferably repopulating to levels greater than -20% from baseline); - naive T cells (preferably repopulating to levels greater than -40% from baseline); - CD4+ central memory T cells (preferably repopulating to levels greater than -20% from baseline); - CD4+ effector memory T cells (preferably repopulating to levels greater than -10% from baseline); - Treg / Teff cell ratio (preferably decreased from baseline), and / or - Breg / memory B cell ratio (preferably decreased compared to baseline), and / or - the ratio of NKreg / NKeff cells (preferably decreased compared to baseline); - and / or any combination thereof.

[0125] In the EU and many more than 80 countries worldwide, MAVENCLAD® (cladribine tablets) is indicated for the treatment of adult patients with relapsing multiple sclerosis (MS), preferably highly active relapsing multiple sclerosis (MS), as defined by clinical or imaging features. The recommended cumulative dose of MAVENCLAD® is 3.5 mg / kg body weight over 2 years, administered as one treatment course of 1.75 mg / kg / year.

[0126] In the US, MAVENCLAD® (Cladribine Tablets), MAVENCLAD® (Cladribine) Tablets, is indicated for the treatment of relapsing forms of multiple sclerosis (MS) in adults, including relapsing-remitting disease and active secondary progressive disease. MAVENCLAD is administered in two treatment courses approximately one year apart, where the recommended cumulative dose of MAVENCLAD, administered orally, is 3.5 mg / kg body weight, preferably divided into two annual treatment courses as follows (1.75 mg / kg per treatment course): First year treatment course: Cycle 1 (Month 1): Start at any time, Cycle 2 (Month 2): Starts 23-27 days after the last dose (approximately 1 month after the start of cycle 1), Second year treatment course: Cycle 1 (month 1): Approximately 1 year after the start of the first course and at least 43 weeks after the last dose, Cycle 2 (Month 2): Starts 23-27 days after the last dose (approximately 1 month after the start of cycle 1).

[0127] Each treatment cycle consists of 4 or 5 consecutive days; the cycle dose is administered as 1 or 2 tablets once daily for 4 or 5 consecutive days.

[0128] With respect to "month(s)" (or "month(s)" in the context of times and / or periods described herein, those times and / or periods are preferably counted from the date the patient receives a first dose of the DMD, preferably cladribine, more preferably oral cladribine or cladribine tablets (e.g. Mavenclad®), and more preferably are counted from the date the patient receives a first dose of the DMD, preferably cladribine, more preferably oral cladribine or cladribine tablets (e.g. Mavenclad®), in an individual treatment period of about one year, an individual treatment year or (yearly) treatment course.

[0129] The term "baseline" as used herein is known and understood in the art. In the context of the present invention, it preferably refers to the state of an individual or patient before the start of the first treatment with an individual DMD for which the biomarker or cells to be used as biomarkers according to the present invention are to be determined. The term baseline therefore preferably reflects the "normal" state or level of the individual or patient and individual cells to be used as biomarkers according to the present invention after (i.e. after DMD treatment), but in the absence of such DMD treatment / previous DMD treatment. If the individual has already received DMD before, a washout period of at least 1-10 months, preferably at least 1-4 months, is recommended so that the cells are normal, i.e. at or sufficiently close to untreated levels.

[0130] As used herein, the term "mg / kg" preferably means "mg per kg of body weight."

[0131] More preferably, the term "mg / kg" as used herein when cladribine is administered orally to a patient, and more preferably when used in connection with a fixed dose, preferably means "milligrams" of cladribine administered "per kg body weight" (orally) to the patient.

[0132] "Total dose" or "cumulative dose" preferably refers to the total dose of cladribine administered during treatment, i.e. the dose reached at the end of treatment calculated by adding up the daily doses. For example, a total dose of cladribine corresponding to 5 days of treatment with 0.7 mg / kg of cladribine per day is 3.5 mg / kg, or a total dose of cladribine corresponding to 5 days of treatment with 0.35 mg / kg of cladribine per day is 1.7 mg / kg.

[0133] "Total effective dose" or "cumulative effective dose" preferably refers to the bioavailable dose of cladribine after a given administration period, i.e. the bioavailable dose reached at the end of treatment, calculated by adding the daily dose subtracted by the bioavailability coefficient. For example, the total effective dose of cladribine corresponding to a 5-day treatment of 0.7 mg / kg of cladribine per day when the bioavailability of cladribine is about 40% is 1.4 mg / kg, or the total effective dose of cladribine corresponding to a 5-day treatment of 0.35 mg / kg of cladribine per day when the bioavailability of cladribine is about 40% is 0.7 mg / kg.

[0134] Typically, the bioavailability of a cladribine or cladribine formulation used in the context of the present invention is about 20% to about 90%, preferably about 25% to about 80%, more preferably about 30% to about 70%, even more preferably about 35% to about 60%, and in particular about 40% to about 50%, such as about 35%, about 40%, about 45%, about 50% or about 55%. Particularly preferably, the bioavailability of a cladribine tablet is preferably 45%±25%, more preferably 45%±20%, even more preferably 45%±15%, even more preferably 45%±10%, and in particular 45%±5%, based on the total content of the active ingredient cladribine (2-CdA) in the cladribine-containing formulation, preferably the cladribine-containing oral formulation, preferably used in the method and / or treatment scheme according to the present invention.

[0135] The term "bioavailability" as used herein with respect to pharmaceutical dosage forms, including but not limited to tablets, is known and understood in the art. Similarly, methods and / or procedures for reliably determining said bioavailability are known, understood, and described in the art. These known methods and / or procedures typically conform to the requirements of "Good Manufacturing Practice (GMP)" and / or "Good Clinical Practice (GCP)" to the extent required by the FDA and / or EMA, and other health authorities.

[0136] However, bioavailability may preferably be assessed using the following methodology:

[0137] Bioavailability, usually defined as the fraction of the dose that reaches the systemic circulation, was evaluated in a randomized, two-period, two-sequence crossover study in multiple sclerosis patients after oral administration of a single 10 mg tablet of Mavenclad and after 3 mg of cladribine (leustatin 1 mg / mL) administered as a 1-h IV infusion, where cladribine plasma concentrations were measured using a validated LC / MS / MS method. Bioavailability was estimated by a nonlinear mixed-effects model (software NONMEM, version VI [newer versions also apply]) by incorporating it in the form of a logit transformation (to constrain it within the 0-1 range) as a parameter of the integrated pharmacokinetic model, developed by integrating several studies using Mavenclad under different conditions (e.g. fed / fasted) and with varying durations (see RM Savic 2017 (DOI 10.1007 / s40262-017-0516-6)). This modeling took into account several covariates, such as food intake, in relation to drug administration. In addition to parameter estimates, their precision and inter-subject variability are also included and calculated in the pharmacostatistical model. Following sequential model development, refinement and validation, the bioavailability of cladribine after administration of Mavenclad 10 mg tablets was more preferably estimated to be 45.6%, preferably about 45.6%, with a precision of 7.03% and / or a variance (on the logit scale) of 22.3%.

[0138] However, other methods and methodologies may also be successfully applied, such as, for example, those described in: "Population Pharmacokinetics of Cladribine in Patients with Multiple Sclerosis", Radojka M. Savic, Ana M. Novakovic, Marianne Ekblom, Alain Munafo and Mats O. Karlsson; Clinical Pharmacokinetics, Vol. 56, pp. 1245-1253 (2017), and / or "The Clinical Pharmacology of Cladribine Tablets for the Treatment of Relapsing Multiple Sclerosis", Robert Hermann, Mats O. Karlsson, Ana M. Novakovic, Nadia Terranova, Markus Fluck and Alain Munafo; Clinical Pharmacokinetics, Vol. 58, pp. 283-297 (2019), which are incorporated herein by reference in their entirety.

[0139] More specifically, for example, according to the approved package insert for Cladribine tablets (MAVENCLAD®), and for use assuming a bioavailability of Cladribine tablets of about 45%, the recommended cumulative effective dose to be preferably reached in the patient's body by administration of Cladribine tablets (MAVENCLAD®) is preferably about 1.58 mg / kg body weight over a period of 2 years (achieved by administering Cladribine tablets (MAVENCLAD®) in an amount of about 3.5 mg / kg body weight, based on a bioavailability of about 45% of the administered tablet as outlined above), and preferably 0.5 mg / kg body weight per year for one course of treatment. Cladribine tablets (achieved by administering MAVENCLAD®) are administered in an amount of about 1.75 mg / kg body weight, based on a bioavailability of about 45% of the tablet administered as outlined above. In accordance with the approved package insert, each treatment course preferably consists of two treatment weeks, one preferably beginning at the beginning of the first month and one preferably beginning at the beginning of the second month of a separate treatment year. Each treatment week preferably consists of 4 or 5 days, during which the patient preferably receives 10 mg or 20 mg (1 or 2 tablets), preferably as a single daily dose, preferably depending on body weight.

[0140] The term "United States Package Insert" is abbreviated as "USPI" and is known and understood in the art.Preferably, USPI presents the usage agreed by health authorities, preferably the usage agreed by the United States Food and Drug Administration (FDA), and more preferably the usage agreed by the Marketing Division of the United States Food and Drug Administration.It provides information on usage for medical professionals and is an essential part of the application for Marketing Authorization (MA) of new drugs and medicines in the United States.Preferably, the equivalent of USPI in the European Union is the European Product Summary (SmPC).

[0141] Reference to the USPI in the context of the present invention preferably refers to the USPI for oral cladribine or cladribine tablets, marketed under the trade name MAVENCLAD®, Mavenclad® or just Mavenclad. Preferably, reference to the USPI in the context of the present invention for cladribine tablets means the reference to the USPI for cladribine tablets / MAVENCLAD® valid until 2020. Preferably, reference to the USPI for cladribine tablets / MAVENCLAD® encompasses all the content of that USPI other than the content relating to immunization and / or vaccination against infectious diseases, preferably as described herein in the context of the present invention, relating to immunization, vaccination and / or infectious diseases. Preferably, the present invention provides a method of treatment that is advantageously improved over the content of the USPI relating to immunization and / or vaccination against said infectious diseases.

[0142] The term "European Product Description" is abbreviated as "EU SmPC" or "SmPC" and is known and understood in the art. Preferably, it refers to a document that describes the proprietary and officially approved conditions of use of a medicinal product. The Product Description forms the basis of information for medical professionals on how to use the medicinal product safely and effectively. More specifically, the SmPC presents the usage of the drug, preferably agreed by a health authority, preferably agreed by a European agency (or agencies), preferably more specifically agreed by the Marketing Department of the European Medicines Agency (EMA). It provides information on usage for medical professionals and is an essential part of applications for Marketing Authorization (MA) of new drugs and medicines in the European Union. Preferably, the equivalent of the SmPC in the United States is the "United States Package Insert", abbreviated as "USPI".

[0143] Reference to the EU SmPC, or preferably the SmPC, in the context of the present invention preferably refers to the EU SmPC, or preferably the SmPC, of ​​oral cladribine or cladribine tablets, preferably marketed under the trade name MAVENCLAD®, Mavenclad® or just Mavenclad. Preferably, reference to the EU SmPC, or preferably the SmPC, in the context of the present invention for cladribine tablets means reference to the EU SmPC, or preferably the SmPC, of ​​cladribine tablets / MAVENCLAD® valid until 2020. Preferably, reference to the EU SmPC, or preferably the SmPC, of ​​cladribine tablets / MAVENCLAD® encompasses all the content of that EU SmPC, or preferably the SmPC, other than the content relating to immunization and / or vaccination against infectious diseases, preferably as described herein in the context of the present invention relating to immunization, vaccination and / or infectious diseases. Preferably, the present invention provides a method of treatment which is advantageously improved over the content of the EU SmPC, or preferably the SmPC, preferably relating to immunization and / or vaccination against said infectious diseases.

[0144] "A week" preferably refers to a period of a number of days, or about 5, about 6 or about 7 days.

[0145] "A month" preferably refers to a period of a certain number of days, or about 28, about 29, about 30 or about 31 days.

[0146] The term "treatment" is known and understood in the art. In certain circumstances, the term also preferably means or includes the sequential continuation of an "induction treatment" and at least a "maintenance treatment". In this particular context, the treatment may include an "induction treatment" and about 1 or about 2 or about 3 maintenance treatments.

[0147] Typically, treatment according to the present invention will be for about 2 years (about 24 months) or about 3 years (about 36 months) or about 4 years (about 48 months), preferably for about 2 years.

[0148] Preferably, the treatment according to the invention is for about 2 years (about 24 months) or about 3 years (about 36 months) or about 4 years (about 48 months), preferably for about 2 years (about 24 months). When the treatment according to the invention is for about 2 years (about 24 months), it preferably comprises two treatment periods or two treatment courses. When the treatment according to the invention is for about 3 years (about 36 months) or about 4 years (about 48 months), it preferably comprises three treatment periods / treatment courses or four treatment periods / treatment courses.

[0149] Preferably, the treatment period or course of treatment according to the present invention comprises 1 month, 2 months or 3 months, in which cladribine or preferably cladribine tablets are administered to the subject, preferably a subject in need thereof. Preferably, the treatment period or course of treatment (causes) according to the present invention comprises 2 months, in which cladribine or preferably cladribine tablets are administered to the subject. Particularly preferably, each treatment course consists of two treatment weeks, one preferably at the beginning of the first month of the treatment course, and one preferably at the beginning of the second month of the treatment course. Preferably, the same is true for the treatment period, and these terms are preferably interchangeable in the context of the present invention.

[0150] Preferably, each such treatment week includes 4 or 5 days during which the subject receives cladribine, preferably a cladribine tablet, preferably in a daily dose of 10 or 20 mg depending on the patient's weight, to achieve the dosing of cladribine in mg / kg body weight described herein.

[0151] The term "about" as used herein with respect to numbers, figures, ranges, and / or amounts is preferably intended to mean "approximately" and / or "approximately", the meanings of which are well known in the art and preferably encompass a variance, deviation, and / or variation of the respective numbers, figures, ranges, and / or amounts of plus / minus 15%, and particularly plus / minus 10%.

[0152] In all cases, the term "about" as used herein in connection with numbers, figures, ranges, and / or amounts is preferably intended to mean "approximately" and / or "approximately," the meanings of which are well known in the art and preferably encompass a variance, deviation, and / or variation of at least plus / minus 5% of the particular number, figure, range, and / or amount.

[0153] Preferably, any range mentioned herein below includes all values ​​and sub-values ​​between the minimum and maximum limits of that range.

[0154] The terms "disorder(s)" and "disease(s)" as used herein are well known and understood in the art. In the context of the present invention, they are preferably used as synonyms and therefore preferably interchangeable unless the context in which they are used herein strongly implies otherwise.

[0155] In medical contexts, including but not limited to treatment regimes, dosing schedules and clinical trial designs, the terms "week" / "a week", "month" / "a month" and / or "year" / "a year" may be used with slight deviations from the Gregorian calendar conventions, such as for convenience and / or ease of use by patients, medical personnel and / or physicians, and reliability and / or reproducibility of results. For example, in such medical contexts, a month often refers to 28 days, and a year often refers to 48 weeks.

[0156] Thus, in the context of the present invention, the term "week" or "a week" preferably refers to a period of about 5, about 6 or about 7 days, more preferably about 7 days.

[0157] In the medical context, the term "month" or "a month" preferably refers to a period of about 28, about 29, about 30 or about 31 days, more preferably about 28, about 30 or about 31 days.

[0158] In the medical context, the term "year" or "a year" preferably refers to a period of about 12 months, or about 48, about 50, or about 52 weeks, more preferably 12 months, or about 48 or about 52 weeks.

[0159] Cladribine (2-CdA) 2-CdA and its pharma- ceutically acceptable salts may be used in the practice of the present invention.

[0160] Cladribine can be formulated in any pharmaceutical preparation suitable for oral administration. Representative oral formulations of 2-CdA are described in (WO 96 / 19230; WO 96 / 19229; US 6,194,395; US 5,506,214; WO 2004 / 087100; WO 2004 / 087101), the contents of which are incorporated herein by reference. Examples of components of oral formulations are shown below.

[0161] The process of preparing 2-CdA is well known in the art. For example, the preparation of 2-CdA is described in (EP 173,059; WO 04 / 028462; WO 04 / 028462; US 5,208,327; WO 00 / 64918) and Robins et al., J. Am. Chem. Soc., 1984, 106: 6379. Alternatively, pharmaceutical preparations of 2-CdA may be purchased from Bedford Laboratories (Bedford, Ohio).

[0162] Oral administration of cladribine may be in the form of capsules, tablets, oral suspensions, or syrups. Tablets or capsules may contain about 3 to 500 mg of cladribine. Preferably, they contain about 3 to about 10 mg of cladribine, more preferably about 3, about 5, or about 10 mg of cladribine. Capsules may be gelatin capsules and may contain, in addition to the amounts of cladribine noted above, small amounts, e.g., less than 5% by weight, of magnesium stearate or other excipients. Tablets may contain the aforementioned amounts of compound and binder, which may be gelatin liquid, starch paste in water, polyvinyl in water, polyvinyl alcohol, etc., with a typical sugar coating.

[0163] Preferably, the term "cladribine" as used in the context of the present invention, unless expressly specified otherwise, preferably means "oral cladribine", i.e. the active ingredient cladribine administered orally in accordance with the present invention. Preferably, the cladribine administered orally in accordance with the present invention is administered in the form of an oral dosage form, such as, for example, capsules filled with a cladribine solution, capsules filled with cladribine powder, cladribine-containing tablets, and tablets containing the cladribine-cyclodextrin complex, especially those commercially available under the name Mavenclad®.

[0164] Particularly preferably, the cladribine or oral cladribine for use in accordance with the present invention is orally administered as a tablet containing 10 mg of cladribine in the form of a mixture of cladribine, 2-hydroxypropyl-β-cyclodextrin, and cladribine-2-hydroxypropyl-β-cyclodextrin complex, wherein the weight ratio of cladribine to 2-hydroxypropyl-β-cyclodextrin is from about 1:10 to about 1:16, preferably from 1:13 to 1:15.

[0165] A preferred oral dosage form in the form of a tablet is described directly below:

[0166] A preferred composition of cladribine for oral use according to the invention is a tablet containing 10 mg of cladribine (2-CdA), preferably as a cladribine-cyclodextrin complex, as well as excipients, including hydroxypropyl-β-cyclodextrin in the following amounts: [Table 3]

[0167] However, similar compositions, preferably containing a cladribine-cyclodextrin complex and preferably having the same or very similar bioavailability, may also be preferably used in accordance with the present invention.

[0168] Unless expressly specified otherwise, the nomenclature of those active ingredients, active pharmaceutical ingredients (API), medicinal products or International Nonproprietary Names (INN) preferably encompasses all prodrugs, salts and solvates thereof, especially those which are considered to be functional equivalents and / or suitable substitutes from a clinical point of view.

[0169] Unless expressly specified otherwise, the terms individual, human, human being, human patient or patient are preferably used interchangeably or as synonyms herein.

[0170] Unless expressly specified otherwise, the terms individual, human (human being), human patient or (simply) patient are preferably used interchangeably or as synonyms herein.

[0171] The term "treatment period", as used herein for the time frame during which Cladribine, preferably Cladribine tablets, more preferably (Mavenclad®), is administered to a subject, preferably a human subject, more preferably a patient, and especially a patient in need thereof, preferably in accordance with the wording in the health authority approved package insert / marketing authorization, can also be interchanged with the terms "treatment course" and / or "treatment cycle". Thus, the terms "treatment period", "treatment course" and / or "treatment cycle" can preferably be used interchangeably within the context of the present invention. The same preferably applies to the terms "treatment period", "treatment course" and / or "treatment cycle". Preferably, unless expressly specified otherwise, the terms "treatment period", "treatment course" and / or "treatment cycle" can also be used in accordance with the present invention abbreviated to "period", "course" and / or "cycle", respectively. However, consistent with the USPI, the term "first cycle" preferably means "first month" of the "first-year treatment course" and / or "second-year treatment course"; similarly, "second cycle" preferably means "second month" of the "first-year treatment course" and / or "second-year treatment course".

[0172] However, in the methods of treatment described herein, where administration of cladribine, preferably oral cladribine, and in particular administration of cladribine tablets (e.g., Mabengrad) is in accordance with or substantially consistent with the dosage regimen set out in the EU SmPC / approved European package insert, a period of two or more about a year (also referred to as about 12 months, 46-54 weeks, 48-52 weeks, 48 ​​weeks or 52 weeks, etc.) at the start of administration of the cladribine, oral cladribine or cladribine tablets is preferably referred to as a "treatment year" or "treatment years", e.g. a "first treatment year", a "second treatment year" or a "subsequent treatment year". Similarly, in this context, each of the months, preferably each of two months, at the start of a "treatment year", during which cladribine, oral cladribine and cladribine tablets are administered to the patient, is preferably referred to as a "treatment course" or "treatment courses", e.g. a "first treatment course", a "second treatment course" or a "subsequent treatment course".

[0173] In contrast, in the methods of treatment described herein in which administration of cladribine, preferably oral cladribine, and particularly cladribine tablets (e.g., Mavenclad), is in accordance with or substantially consistent with the dosage regimen set forth in the USPI / approved US package insert, two or more periods lasting about one year (also referred to as about 12 months, 46-54 weeks, 48-52 weeks, 48 ​​weeks or 52 weeks, etc.) at the initiation of administration of the cladribine, oral cladribine or cladribine tablets are preferably referred to as a "treatment course" or "multiple treatment courses", e.g., a "first treatment course", a "second treatment course" or a "subsequent treatment course". Similarly, in this context, each of the periods lasting about one month, preferably each of two periods lasting about one month, at the start of a "treatment course" during which cladribine, oral cladribine and cladribine tablets are administered to the patient, is preferably referred to as a "treatment cycle" or "treatment cycles", e.g., a "first treatment cycle treatment course", a "second treatment cycle" or a "subsequent treatment cycle".

[0174] References : Black African and Latino / a identity correlates with increased plasmablasts in MS Kiel M. Telesford, PhD, Ulrike W. Kaunzner, MD, PhD, Jai Perumal, MD, Susan A. Gauthier, DO, MPH, Xian Wu, MPH, Ivan Diaz, PhD, Mason Kruse-Hoyer, MD, Casey Engel, BA, Melanie Marcille, BA, and Timothy Vartanian, MD, PhD Neurol Neuroimmunol Neuroinflamm 2020;7:e634. doi:10.1212 / NXI.0000000000000634 Immunization Responses in Rheumatoid Arthritis Patients Treated With Rituximab Clifton O. Bingham III,1 R. John Looney,2 Atul Deodhar,3 Neal Halsey,4 Maria Greenwald,5 Christine Codding,6 Benjamin Trzaskoma,7 Flavius Martin,7 Sunil Agarwal,7 and Ariella Kelman7 ARTHRITIS & RHEUMATISM Vol. 62, No. 1, January 2010, pp 64-74 DOI 10.1002 / art.25034 (c) 2010, American College of Rheumatology Dysregulation of regulatory CD56bright NK cells / T cells interactions in multiple sclerosis Alice Laroni a, b, 1, Eric Armentani a, 1, Nicole Kerlero de Rosbo a, Federico Ivaldi a, Emanuela Marcenaro b, c, Simona Sivori b, c, Roopali Gandhi d, Howard L. Weiner d, e, f, Alessandro Moretta b, c, Giovanni L. Mancardi a, b, Antonio Uccelli a, b, * Journal of Autoimmunity 72 (2016) 8-18 CD56bright Natural Killer Cells: A Possible Biomarker of Different Treatments in Multiple Sclerosis Alice Laroni 1,2,* and Antonio Uccelli 1,2 J. Clin. Med. 2020, 9, 1450; doi:10.3390 / jcm9051450 www.mdpi Increased NK Cell Count in Multiple Sclerosis Patients Treated With Dimethyl Fumarate: A 2-Year Longitudinal Study Damiano Marastoni 1†, Alessandro Buriani 2†, Anna Isabella Pisani 1, Francesco Crescenzo1, Carmela Zuco1, Stefano Fortinguerra2, Vincenzo Sorrenti 2, Bruno Marenda2, Chiara Romualdi 3, Roberta Magliozzi 1, Salvatore Monaco1 and Massimiliano Calabrese1* Frontiers in Immunology | www.frontiersin.org | July 2019 | Volume 10 | Article 1666 Martin MD and Badovinac VP (2018) Defining Memory CD8 T Cell. Front. Immunol. 9:2692. doi: 10.3389 / fimmu.2018.02692 The Increased Ratio of Blood CD56bright NK to CD56dim NK Is a Distinguishing Feature of Primary Sjogren’s Syndrome Bingxia Ming,1 Tong Wu,1 Shaozhe Cai,1 Peng Hu,1 Jungen Tang,1 Fang Zheng,2,3 Cong Ye,1 and Lingli Dong 1 1Department of Rheumatology and Immunology, Tongji Hospital, Tongji Medical College, Huazhong Hindawi Journal of Immunology Research Volume 2020, Article ID 7523914, 7 pages https: / / doi.org / 10.1155 / 2020 / 7523914 Using the Immunophenotype to Predict Response to Biologic Drugs in Rheumatoid Arthritis Ben Mulhearn 1,2,3,*, Anne Barton 2,3,4 and Sebastien Viatte 1,2,4 J. Pers. Med. 2019, 9, 46; doi:10.3390 / jpm9040046 www.mdpi.com / journal / jpm Regulatory B cells: the cutting edge of immune tolerance in kidney transplantation Bo Peng 1, Yingzi Ming1 and Cheng Yang2 Peng et al. Cell Death and Disease (2018) 9:109 DOI 10.1038 / s41419-017-0152-y CD56bright natural killer (NK) cells: an important NK cell subset Aure ́lie Poli,1 Tatiana Michel,1 Maud Theresine,1 Emmanuel Andre`s,2 Francois Hentges1 and James Zimmer1 Immunology, 126, 458-465 Effect of rituximab treatment on T and B cell subsets in lymph node biopsies of patients with rheumatoid arthritis Tamara H. Ramwadhdoebe1,2, Lisa GM van Baarsen1,2, Maria JH Boumans1, Stefan TG Bruijnen 3, Mary Safy1, Ferco H. Berger4, Johanna F. Semmelink1,2, Conny J. van der Laken3, Danielle M. Gerlag1,5, Rogier M. Thurlings1,a and Paul P. Tak1,6,7,* Rheumatology 2019;58:1075_1085 doi:10.1093 / rheumatology / key428 Advance Access publication Rituximab treatment outcomes in impaired secondary humoral immunity responsiveness Lizet E. van der Kolk, Joke W. Baars, Martin H. Prince, and Marinus HJ van Oers BLOOD, SEPTEMBER 15, 2002 _ VOLUME 100, NUMBER 6 Natural killer cell subsets in cerebrospinal fluid of patients with multiple sclerosis E. Rodriguez-Martin,*‡§ C. Picon,*‡ L. Costa-Frossard,†‡§ R. Allenda,*‡§ S. Sainz of the Mace,† E. Roldan,*‡§ M. Hawthorne,*‡§ LM Villar*‡§1 and JC Alvarez-Cermeno†‡§¶1 Clinical and Experimental Immunology, 180: 243-249 B-cell populations discriminate between pediatric- and adult-onset multiple sclerosis Alexander Schwarz, MD; Bettina Balint, MD; Mirjam Korporal-Kuhnke, MD; Sven Jarius, MD; Kathrin von Engelhardt, MD; Alexandra Furwentsches, MD; Cornelia Bussmann, MD; Friedrich Ebinger, MD ; Brigitte Wildemann, MD*; Jurgen Haas, PhD* Neurol Neuroimmunol Neuroinflamm 2017;4:e309; doi: 10.1212 / NXI.0000000000000309 Unique phenotypes and clonal expansions of human CD4 effector memory T cells re-expressing CD45RA Yuan Tian1, Mariana Babor1, Jerome Lane1, Veronique Schulten1, Veena S. Patil1, Gregory Seumois 1, Sandy L. Rosales1, Zheng Fu 2, Gaelle Picarda3, Julie Burel1, Jose Zapardiel-Gonzalo1, Rashika N. Tennekoon1,4, Aruna D. De Silva1,4, Sunil Premawansa5, Gayani Premawansa6, Ananda Wijewickra NATURE COMMUNICATIONS | 8: 1473 | DOI: 10.1038 / s41467-017-01728-5 | www.nature.com / naturecommunications Regulatory B and T lymphocytes in multiple sclerosis: friends or foes? Georgios K. Vasileiadis1 · Efthymios Dardiotis1 · Athanasios Mavropoulos2 · Zisis Tsouris1 · Vana Tsimourtou1 · Dimitrios P. Bogdanos2 · Lazaros I. Sakkas2 · Georgios M. Hadjigeorgiou1,3 Autoimmunity Highlights (2018) 9:9 https: / / doi.org / 10.1007 / s13317-018-0109-x Cladribine - an old newcomer for pulsed immune reconstitution in MS Heinz Wendl NATURE REVIEWS | NEUROLOGY VOLUME 13 | OCTOBER 2017 | 573

[0175] All references cited herein are preferably incorporated herein by reference in their entirety.

[0176] The invention is explained in more detail below by means of examples, which can be preferably carried out throughout the scope of the claims and are not limited to the examples given herein.

[0177] Additionally, the following examples are provided to assist those skilled in the art in better understanding of the present invention by way of illustration, and are not intended to limit the scope of the present invention. [Brief description of the drawings]

[0178] Drawing Legend [Figure 1] FIG. 1: B cell differentiation pathway and expression of TrB-related molecules.

[0179] [Diagram 2] Figure 2: Main functions of human CD19+CD24hiCD38hiTr B cells discovered.

[0180] [Diagram 3] Figure 3: Study design of MAGNIFY-MS, including immune cell subtype analysis performed as part of this substudy. 9HPT, 9-hole peg test; BL, baseline; CUA, combined specific activity; DMT, disease-modifying therapy; EDSS, expanded disability status scale; HIV, human immunodeficiency virus; KFS, Kurtzke functional system; MRI, magnetic resonance imaging; MS, multiple sclerosis; SDMT, signed digit modality test; T25FW, timed 25-foot walk; TB, tuberculosis.

[0181] [Figure 4] Figure 4: Median absolute B and T cell counts in patients treated with Cladribine tablets. The first treatment course of Cladribine tablets was administered at baseline and at month 1. BL, baseline; Q, quartile; TEMRA, terminally differentiated effector memory RA+; Th, helper T cells.

[0182] [Diagram 5] Figure 5: Median absolute NK cell counts in patients treated with Cladribine tablets. The first treatment course of Cladribine tablets was administered at baseline and at month 1. BL, baseline; Q, quartile.

[0183] [Figure 6] Figure 6: Median serum concentrations of immunoglobulins in patients treated with cladribine tablets. The first treatment course of cladribine tablets was administered at baseline and at month 1. BL, baseline; Ig, immunoglobulins; Q, quartiles.

[0184] [Figure 7] Figure 7: Immune cell panel MAGNIFY-MS: biomarkers for cell subset identification.

[0185] [Figure 8] Figure 8: Correlation matrix between numerical clinical attributes and B-cell deconvolution scores. The area of ​​the circle indicates the value corresponding to the Pearson correlation coefficient. Positive correlations are shown in blue and negative correlations in red. The intensity of the color (light to dark) and the size of the circle (small to large) are proportional to the correlation coefficient. Non-significant correlations (p-value <0.01) are represented by blank boxes. Only baseline EDDSS scores are shown.

[0186] [Figure 9] Figure 9: Correlation matrix between numerical clinical attributes and CD8+ T-cell deconvolution scores. The area of ​​the circle indicates the value corresponding to the Pearson correlation coefficient. Positive correlations are shown in blue and negative correlations in red. The intensity of the color (light to dark) and the size of the circle (small to large) are proportional to the correlation coefficient. Non-significant correlations (p-value <0.01) are represented by blank boxes. Only baseline EDDSS scores are shown.

[0187] [Figure 10] Figure 10: Correlation matrix between numerical clinical attributes and CD4+ T-cell deconvolution scores. The area of ​​the circle indicates the value corresponding to the Pearson correlation coefficient. Positive correlations are shown in blue and negative correlations in red. The intensity of the color (light to dark) and the size of the circle (small to large) are proportional to the correlation coefficient. Non-significant correlations (p-value < 0.01) are represented by blank boxes. Only baseline EDDSS scores are shown.

[0188] [Figure 11] Figure 11: B cell subtypes - absolute values ​​shown as cell counts / μL.

[0189] [Figure 12] FIG. 12: Absolute values ​​expressed as immunoglobulin-g / L.

[0190] [Figure 13] Figure 13: T cell subtypes - absolute values ​​shown as cell counts / μL. EXAMPLES

[0191] Working Example Example 1: Characterization of peripheral immune cell dynamics and repopulation patterns following treatment with Cladribine tablets in the MAGNIFY-MS study introduction Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) in which many immune cell subtypes play a role in the pathogenesis, with B and T lymphocytes involved in demyelination and axonal damage (Hoglund and Maghazachi, 2014; Piancone et al., 2016).

[0192] Cladribine tablets 10 mg (cumulative dose 3.5 mg / kg over 2 years) for immune reconstitution therapy (IRT) of MS, which causes a selective and transient decrease in B-cell and T-cell counts followed by a period of reconstitution (Giovannoni, 2017; Wiendl, 2017). Cladribine is a prodrug of a deoxyadenosine analogue that is sequentially phosphorylated by deoxycytidine kinase (DCK) and deoxyguanosine kinase to its biologically active form, 2-chlorodeoxyadenosine triphosphate (Cd-ATP). Dephosphorylation and inactivation of Cd-ATP is catalyzed by 5'-NTase. ◇B and T lymphocytes have a relatively high DCK / 5'-NTase ratio, making them particularly selective for cladribine (Giovannoni, 2017). Accumulation of cladribine in these immune cells leads to apoptosis through inhibition of DNA polymerase. This effect on B and T cells disrupts the cascade of immune events central to the progression of MS (Deeks, 2018).

[0193] Long-term immune cell dynamics following treatment with cladribine are being evaluated in the CLARITY, CLARITY Extension, PREMIERE and ORACLE-MS trials (Comi et al., 2019; Stuve et al., 2019). However, it remains to be fully established which immune cell subtypes are important for both the onset and durability of the therapeutic effect of cladribine in humans with MS. Against this background, MAGNIFY-MS (NCT03364036) was designed and initiated in patients with highly active relapsing MS, and here we report findings on a substudy of peripheral immune cell subset dynamics and immunoglobulin levels during the first 12 months after patients started treatment.

[0194] Materials and Methods Study design and participants -MAGNIFY-MS is an ongoing Phase IV, open-label, single-arm study with a 2-year duration (Figure 3). Patients were enrolled between May 28, 2018 and April 23, 2019. They were age ≥18 years, had an Expanded Disability Status Scale (EDSS) score ≤5, and had highly active relapsing MS. In this study, highly active relapsing MS was defined as one relapse during the previous year and at least one T1 gadolinium-enhancing (Gd+) lesion, or 9 or more T2 lesions while on treatment with another disease-modifying therapy (DMT), or two or more relapses during the previous year with or without a DMT. Patients were excluded if they had prior exposure to DMTs (fingolimod, natalizumab, alemtuzumab, mitoxantrone, or ocrelizumab), had lymphocyte counts not within normal laboratory limits, had 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 allergy or hypersensitivity to gadolinium, and / or other contraindications to undergoing magnetic resonance imaging (MRI).

[0195] Enrolled patients were scheduled to receive cladribine tablets at a cumulative dose of 3.5 mg / kg over a 2-year period, with 2 weeks of active treatment per course (weeks 1 and 5 of each year).

[0196] Peripheral blood collection This exploratory analysis of MAGNIFY-MS involved the longitudinal assessment of peripheral blood immune cell subtypes in a subgroup of patients receiving cladribine tablets. ○Immune phenotyping was performed on blood samples collected at baseline (before treatment) and at the end of months 3, 6 and 12. Further immune phenotyping was completed at months 1 and 2 for TBNK cells and B cell panels. ◇Immune cell subtypes were analyzed by flow cytometry. Absolute cell counts and % change from baseline were assessed (Table 1).

[0197] In parallel, serum levels of immunoglobulin (Ig) G and IgM were analyzed by turbidimetric assay.

[0198] statistical analysis Analysis of immune cell subtypes was an exploratory endpoint in the MAGNIFY-MS study and was therefore analyzed descriptively. All patients who received at least one dose of cladribine tablets were included in this analysis.

[0199] result Between May 28, 2018 and April 23, 2019, a total of 70 patients enrolled in the MAGNIFY-MS substudy; 57 of these patients received treatment with cladribine tablets (13 patients withdrew from the substudy prior to initiating treatment).

[0200] 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. The demographics and characteristics of patients enrolled in MAGNIFY-MS are shown in Table 2.

[0201] B cell subtypes ●CD19 + The median changes in memory and naive B cell counts were relatively rapid, showing percentage changes from baseline to month 1 of -77%, -74%, and -80%, respectively (Figure 4, Table 3).

[0202] Most B cell subtypes reached their nadir at 2 months after treatment, with CD38 + Plasmablasts and short-lived plasma cells reached a nadir at 3 months. The decline in memory B cells continued until the 12th month. Regulatory and transitional B cells recovered and increased above baseline levels by month 3. Naive B cells also recovered to near baseline levels 12 months after starting treatment.

[0203] T-cell subtypes CD4 + and CD8 + T cells showed a gradual decline in median cell counts with % changes from baseline to month 1 of -22% and -18%, respectively (Figure 4, Table 3). The nadir was reached in the second month, after which there was a slight recovery to baseline.

[0204] Other T cell subtypes reached their nadir between months 3 and 6, after which they began to slowly recover towards baseline.

[0205] Median Th17 and TEMRA T cell counts showed a decline at later time points, with Th17 reaching a nadir at month 6 (Figure 4).

[0206] Natural killer cells ●CD16 + / CD56 + Natural killer (NK) cell counts showed a decrease in the first month after treatment, reaching a nadir in the second month, representing a -40% change from baseline (Table 3, Figure 5). Recovery of these subtypes toward baseline occurred between months 3 and 12.

[0207] ●CD16 bright CD56 dim NK cell counts decreased to nadir levels 3 months after treatment, whereas CD16 low CD56 bright No effect on NK cell counts was noted.

[0208] Immunoglobulins During the first year of MAGNIFY-MS, memory B cells continued to decline and CD38 + Despite the decrease in plasma cells, IgG and IgM levels continued to remain within the normal range (Figure 6).

[0209] Intracellular cytokines

[0210] Consideration • Characterization of immune cell dynamics in response to disease-modifying treatments (DMTs) will provide a better understanding of MS pathogenesis as well as provide important information regarding the mechanism of action of such therapies. This substudy of the MAGNIFY-MS population evaluated immune cell dynamics over the first year after treatment with cladribine tablets, resulting in increased expression of CD19 +B cells, CD4 + and CD8 + T cells and CD16 + A specific focus on NK cell subtypes allowed evaluation of the kinetics of lymphopenia development and repopulation.

[0211] The kinetics of lymphopenia indicate that B cells and NK cells reach nadir most rapidly, with almost all subtypes reaching nadir in the second month. Most T cell subtypes reached their nadir in the third month, with CD8 + Central Memory, CD8 + Effector Memory, CD4 + and CD8 + Naïve and CD4 + Regulatory cells reached a nadir at 6 months. For NK cells, CD16 low CD56 bright The cytotype reached its nadir at 12 months.

[0212] Regarding repopulation, CD16 low CD56 bright With the exception of NK cells, most cell subtypes had returned to baseline by month 12. Given the decline in memory B cells, perhaps surprising was their sustained change from baseline at Month 12, with an absolute percentage change of 6% from a nadir of -93% at Month 2. However, although the median memory B cell counts remained low, the number of naive B cells recovered to baseline, which is important because these naive B cells, which are the major producers of interleukin-10, can act to suppress unwanted immune responses (Dubby et al., 2007).

[0213] ●CD56 bright The NK cell phenotype is regulatory (comprising 10% of the NK cell population) but not CD56 dim(comprising 90% of the NK cell population) are effectors. A reduction in effector cell numbers in parallel with an increase in regulatory cells would drive an anti-inflammatory environment and thus contribute to the therapeutic effectiveness of Cladribine tablets. Our results show that CD56 bright Although cell counts were maintained, CD56 dim They noted that there was a decrease in inflammatory responses, reaching a nadir at month 3. At this 3-month time point, it can be assumed that an anti-inflammatory environment is achieved, although further analysis would need to be performed to confirm this.

[0214] Our findings indicate 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).

[0215] - The inventors observed no clinically significant changes in IgG and IgM concentrations over the 12-month study period.

[0216] How do the results of this study compare with other DMTs? Alemtuzumab inhibits B cells and CD4 + and CD8 + It has been shown to reduce T cell counts followed by a period of regrowth (Wiendl et al., 2020). The effects of alemtuzumab and cladribine were compared by Baker et al. and found to be equivalent with respect to B cell depletion (Baker et al., 2017). With fingolimod, total B cell counts were observed to be reduced by treatment; the effect on naive B cell counts was less clear (Claes et al., 2014). B cell depletion after ocrelizumab treatment was rapid and CD19 + A decrease in cell counts occurred as early as 2 weeks after treatment (Montalben et al., 2016). Note that direct comparisons of peripheral immune cell subset dynamics and Ig levels across studies should be made with caution due to differences in assessment schedules and baseline patient characteristics.

[0217] ●Test strength and limits: o Small sample size and single-arm study without a control group. Measurements of immune cell counts were performed using peripheral blood only, and it remains unclear whether cladribine is distributed to tissues, such as those in the CNS, shortly after administration (Hermann et al., 2019). This raises the possibility that the agent could act to reduce lymphocyte numbers in the CNS and / or limit their recruitment to the CNS and their circulation in the periphery, justifying further testing. Note how immune cell subtype dynamics are reported separately, paralleling the early onset of efficacy for MRI findings.

[0218] conclusion The early onset and subsequent sustained therapeutic action of cladribine may be mediated through specific patterns of depletion and reconstitution of B- and T-cell subtypes. The significant effect of cladribine on B cells, particularly memory B cells, during the first 2 months of treatment suggests its contribution to the early efficacy. Sustained depletion of memory B cells and moderate reductions across T cell subtypes may contribute to the long-term therapeutic effect of cladribine tablets.

[0219] [Table 4]

[0220] [Table 5]

[0221] [Table 6]

[0222] MAGNIFY-MS Blood Biomarker 1-Year Substudy: Methods MAGNIFY-MS is an ongoing Phase IV (NCT03364036, see Figure 3), open-label, single-arm, multicenter, two-year study. Patients with highly active relapsing MS received two weeks of active treatment per course (weeks 1 and 5 of each year) of cladribine tablets. Subjects who meet the eligibility criteria during a pre-baseline screening period up to three months prior to baseline (day 1 of treatment) will receive their first treatment course in year 1 and a retreatment course in year 2. Subjects will visit for evaluation at the end of months 1, 2, 3, 6, and 12 after baseline (day 1 of treatment), and at the end of months 15, 18, and 24 of the second treatment course (month 1 of year 2), respectively. Subjects will visit for blood sampling prior to baseline and at months 2, 6, 12, 14, 18, and 24, following mandatory monitoring. The primary endpoint(s) will be assessed during the first 6 months, and secondary and tertiary endpoints will be assessed up to month 24. Subjects will be offered the opportunity to participate in optional substudies for exploratory analyses.

[0223] An exploratory substudy is planned to be completed in a subset of patients to evaluate the effect of Mavenclad® on biomarkers in blood. The goal of the blood biomarker substudy was to report peripheral immune cell subset dynamics and immunoglobulin levels during the first 12 months of cladribine tablet therapy. An important objective of conducting an exploratory study of markers of response to cladribine treatment in MS is to define responders and non-responders to therapy and to better understand disease pathology. Validated biomarkers would be of considerable value to help define optimal cladribine treatment response in MS, thus determining treatment and ensuring continued benefit from cladribine therapy. A reliable marker of cladribine treatment response in MS would correlate with cladribine bioactivity, be involved in MS pathology, and thus correlate with disease activity or severity. Given the inter-subject variability and heterogeneity of MS pathology, a combined analysis of several different markers would be most meaningful.

[0224] This blood biomarker substudy of MAGNIFY-MS involved the longitudinal evaluation of peripheral blood immune cells in patients receiving Cladribine tablets. 57 patients were treated from 70 patients enrolled. Whole blood and peripheral mononuclear cells (PBMCs) were collected at months 1, 2, 3, 6, and 12, and immune phenotyping was performed using FACS (fluorescence-activated cell sorting) technology. Absolute cell counts and percent change from baseline were assessed for each immune cell subtype. Additional first and second trial readouts are illustrated in Figure 3. In addition to the described changes in biomarkers over time, correlation of biomarkers with "traditional" MS-related assessments (e.g., relapses, MRI, EDSS) was performed. The effect of Cladribine tablets on immune cells may be important for both the onset and durability of its effect in humans with MS.

[0225] blood analysis Blood is analyzed for immune cell subtypes and intracellular cytokines using FACS technology. Blood samples are specifically drawn for the evaluation of potential immunological surrogate biomarkers (see also FIG. 8). To distinguish the different immune subtypes, the following FACS marker panel was validated and applied by Covance:

[0226] T cell panels: CD4 naive, CD4 central memory, CD4 effector memory, CD8 naive, CD8 central memory, CD8 effector memory, CD8 terminally differentiated, Th1, Treg, Th17, Th2, B cell panels: CD19 B cells, CD20 B cells, activated B cells, naive B cells, memory B cells, short-lived plasma cells, Bregs, CD38 bright Plasma cells, transitional B cells, NK cell panel: ·NKp46 NK cells, CD16 NK cells, CD16 bright CD56 dim NK cells, CD16 low CD56 bright NK cells, CD16-CD56 dim NK cells, CD16 + CD56 - NK cells, TBNK Panel: ·T cells (CD45 bright , S.C.C. low , CD3 + ), CD4 T cells (CD45 bright , S.C.C. low , CD3 + ,CD4 + ), CD8 T cells (CD45 bright , S.C.C. low , CD3 + , CD8 + ), B cells (CD45 bright , S.C.C. low , CD3 - , CD19 + ), NK cells (CD45 bright , S.C.C. low , CD3 -, CD16 + / CD56 + ).

[0227] The following intracellular cytokines may be analyzed / measured: ·T cell cytokines: IL-10, IL-17, GM-CSF, TNF-α, IFN-γ, IL-22, IL-4, · B cell cytokines: IL-10, IL-6, GM-CSF.

[0228] Example 2: Clarity Plasma Cell and T Cell Correlation with MRI Efficacy at Year 2 (De novo determined from the CLARITY 96-week study) Plasma cell correlation with MRI The plasma cell signature had weak positive but significant correlations 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 Figure 9.

[0229] MRI and CD8 + T cell correlates Naive CD8 + T cells, CD8 + T cells and CD8 + The Tem signature correlates positively 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 Figure 10.

[0230] Naive CD4 + and CD4 + Central memory T cell signature positively correlates with MRI: Most CD4 + Cell-associated signatures correlate well with MRI attributes, especially CD4 + Naive T cells. REL (relapse count) was calculated based on the naive CD4 + T cell signature and CD4+ Tcm (derived from xCell). The results are shown in FIG.

[0231] [Table 7]

[0232] [Table 8]

[0233] [Table 9]

[0234] Table Legend Percentage changes from baseline were determined for B cell (1a), T cell (1b) and NK cell subtypes. There was an early onset of action, with most B cell subtypes reaching nadir levels by month 2. CD19 + B cells, memory B cells and naive B cells showed early expression at 1 month. Short-lived plasma cells and C38 + Plasma cells declined in month 2 and reached a nadir in month 3. In contrast, transitional B cells and regulatory B cells declined by 61% and 45% at 1 month and then increased to reach levels higher than the basal values ​​measured. T cell subtypes generally showed similar profiles of decline, with CD4 + and CD8 + The nadir for the subtypes was at month 3 and month 6, respectively. + (Treg) and CD8 + (TEMRA) showed less significant decreases of 38% and 26%. ·NK cell subtypes.

Claims

1. A method for predicting a response in a patient treated with cladribine for the treatment of multiple sclerosis, the method comprising step c) and optionally step a) and / or step b), a) where the deviation of the following cell populations: - At least 70% CD19 ＋ B cells, - At least 70% of memory CD19 ＋ B cells, at least 70% naive B cells, at least 70% memory B cells, - activated B cells, and / or - at least 45% activated CD69 ＋ B cells, and / or combinations thereof; is an indication of early efficacy of the cladribine treatment; where - the cell populations are determined in one or more bodily fluid samples from the patient at different stages of the cladribine treatment, including determining the cell populations in a bodily fluid sample from the patient collected before the start of treatment with cladribine, and determining the cell populations in a bodily fluid sample from the patient collected at least at a later time point during or after the cladribine treatment, - the deviation is provided as a decrease from a baseline level; b) wherein the deviation of the following cell populations: - At least 70% CD19 ＋ B cells, At least 50% CD38 ＋ Plasma cells, - at least 50% of short-lived plasma cells, - at least 70% Breg, at least 70% transitional B cells, at least 10% naive T cells, at least 40% central memory T cells, at least 30% of central effector T cells, at least 35% TH1 cells, at least 35% TH17 cells, and / or combinations thereof; is an indication of durable efficacy of the cladribine treatment at one year; where - the cell populations are determined in one or more bodily fluid samples from the patient at different stages of the cladribine treatment, including determining the cell populations in a bodily fluid sample from the patient collected before the start of treatment with cladribine, and determining the cell populations in a bodily fluid sample from the patient collected at least at a later time point during or after the cladribine treatment, - the deviation is provided as a decrease from a baseline level; c) where: - deviation of the memory B cell population to a level above -40%, - deviation of the plasma cell population to a level above -20%, - deviation of the naive T cell population to a level above -40%, - CD4 to levels above -20% ＋ Deviation of central memory T cell populations, - CD4 to levels above -10% ＋ Deviation of effector memory T cell populations; - a deviation in the cell population of the Treg / Teff cell ratio, said deviation being a decrease, - a deviation in the cell population of the Breg / memory B cell ratio, said deviation being a decrease; - a deviation in the cell population of the NKreg / NKeff cell ratio, said deviation being a decrease, and / or combinations thereof: is an indicator of risk of multiple sclerosis relapse, Where: - the cell populations are determined in one or more bodily fluid samples from the patient at different stages of the cladribine treatment, including determining the cell populations in a bodily fluid sample from the patient collected before the start of treatment with cladribine, and determining the cell populations in a bodily fluid sample from the patient collected at least at a later time point during or after the cladribine treatment, - the deviation is from a baseline level.

2. 1. A method for monitoring the need to adapt the treatment of multiple sclerosis with a disease modifying drug (DMD) in a patient in need thereof, comprising: -wherein the patient is being treated with DMD, which is cladribine, for the treatment of multiple sclerosis; wherein deviations in the cell populations determined in one or more bodily fluid samples from the patient at different stages of the cladribine treatment, including determining the cell populations in a bodily fluid sample from the patient collected prior to the initiation of treatment with cladribine, and determining the cell populations in a bodily fluid sample from the patient collected at least at a later time point during or after the cladribine treatment, are: - deviation of the memory B cell population to a level above -40%, - deviation of the plasma cell population at a level above -20%, - deviation of the naive T cell population to a level above -40%, - CD4 to levels above -20% ＋ Deviation of central memory T cell populations, - CD4 to levels above -10% ＋ Deviation of effector memory T cell populations; - a deviation in the cell population of the Treg / Teff cell ratio, said deviation being a decrease, - a deviation in the cell population of the Breg / memory B cell ratio, said deviation being a decrease; - a deviation in the cell population of the NKreg / NKeff cell ratio, said deviation being a decrease, - and / or combinations thereof; wherein said deviation is indicative of the need for re-treatment of said patient with cladribine.

3. 1. Cladribine for use in the treatment of multiple sclerosis in a patient at risk of multiple sclerosis relapse, comprising: - wherein the patient is being treated with cladribine for the treatment of multiple sclerosis; wherein deviations in the cell populations determined in one or more bodily fluid samples from the patient at different stages of the cladribine treatment, including determining the cell populations in a bodily fluid sample from the patient collected prior to the initiation of treatment with cladribine, and determining the cell populations in a bodily fluid sample from the patient collected at least at a later time point during or after the cladribine treatment, are: - deviation of the memory B cell population to a level above -40%, - deviation of the plasma cell population at a level above -20%, - deviation of the naive T cell population to a level above -40%, - CD4 to levels above -20% ＋ Deviation of central memory T cell populations, - CD4 to levels above -10% ＋ Deviation of effector memory T cell populations; - a deviation in the cell population of the Treg / Teff cell ratio, said deviation being a decrease, - a deviation in the cell population of the Breg / memory B cell ratio, said deviation being a decrease; - a deviation in the cell population of the NKreg / NKeff cell ratio, said deviation being a decrease, - and / or combinations thereof; This is an indication that the patient is at risk for multiple sclerosis relapse. Here, the patient will receive re-treatment with cladribine, including administration of additional and / or higher doses of cladribine, or administration of a different DMD, cladribine.

4. The re-treatment with cladribine comprises or consists of oral administration of a formulation containing cladribine, the formulation comprising the following sequential steps: (i) an induction period during which the cladribine formulation is administered, wherein the total dose of cladribine achieved 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 about 8 to about 10 months during which no cladribine formulation is administered; (iii) a maintenance period during which the cladribine formulation is administered, wherein the total cladribine dose reached at the end of the maintenance period is less than the total cladribine dose reached at the end of the lead-in period (i); and (iv) a cladribine-free period during which no cladribine formulation is administered.

4. The method of claim 3, wherein said cladribine is orally administered according to the formula:

5. Cladribine for use in the treatment of multiple sclerosis in a patient in need of such treatment, wherein cladribine is orally administered to said patient in an amount of 0.5-1.25 mg / kg of body weight of said patient for a period of 4-12 weeks, wherein after said administration, said patient has one or more deviations with respect to one or more cell populations selected from the group consisting of: a) optionally wherein the following deviations, provided as % reduction from baseline levels, are biomarkers / corresponding to the early efficacy of said Cladribine treatment: - CD19 ＋ B cells (>70%), - Memory CD19 ＋ B cells (>70%), - naive B cells (>70%), - memory B cells (>70%), - activated B cells, and / or - Activate CD69 ＋ B cells (>45), and / or combinations thereof; b) optionally durable efficacy (% reduction from baseline) at one year; - Memory CD19 ＋ B cells (>70%), - CD38+ plasma cells (>50%), - short-lived plasma cells (>50%), - Breg (>70%), - transitional B cells (>70%), - naive T cells (>10%), - central memory T cells (>40%), - central effector T cells (>30%), - TH1 cells (>35), - TH17 cells (>35), and / or combinations thereof; c) Durability: - NKreg / NKeff cell ratio (>1.5x), - Breg / Beff cell ratio (> / < times), - Treg / Teff cell ratio (> / < fold), and d) Possible relapse of the disease: - A decrease (<30%) in memory B cells from baseline, - memory B cells up to 0.05-1% of PBMCs, - transitional B cell ratio in total PBMCs (> / <???), - Bregs (3-14% or 1-10%) in whole blood or PBMCs from active or relapsing MS patients, and e) Possible relapse of the disease - memory B cells repopulated to levels greater than -40% from baseline; - plasma cells repopulating to levels greater than -20% from baseline; - naive T cells repopulated to levels greater than -40% from baseline; - CD4 + central memory T cells repopulated to levels greater than -20% from baseline; - CD4 + effector memory T cells repopulated to levels greater than -10% from baseline; - a decreased Treg / Teff cell ratio from baseline, and / or - a decreased Breg / memory B cell ratio compared to baseline, and / or - a decreased NKreg / NKeff cell ratio compared to baseline; - and / or combinations thereof: Cladribine.

Images